ROLE OF THE PTEN PHOSPHATASE IN REGULATING T CELL FUNCTION

Karen Andrea Berg

A thesis submitted in conformity with the requirements for the degree of Mater of Science

Graduate Department of Immunology University of Toronto

O Copyright by Karen Andrea Berg (2001)

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Title: Role of the PTEN phosphatase in regulriting T ce11 function DegredYear: M.Sc/ 200 1 Karen Andrea Berg, Department of Imrnunology, University of Toronto

ABSTRACT

While an important role for the PTEN phosphatase in the modulation of TCR

signriling is suggested by many lines of evidence. little is known about the biochemical

mechanisms whereby PTEN impacts on TCR function. To address this issue, affinity

chromatography and mass spectrometry were used to isolate and identify T ce11 protrins

representing binding-panners for PTEN. This approach led to the identification of seven

potential ETEN ligands including the SET. PHAPI2b. PHAPI. tubulin beta-1. eta and therü

subunits of chaperonin containing T-complex protein 1, nucleolin and rnoesin proteins. with

apparent rnolecular weights of 33. 25. 30.50.55. 67 and 67 kDa, respectively.

An analysis of PTEN roles in T ce11 activation was also undertaken and the results

revealed PTEN to be inducibly tyrosine phosphorylated consequrnt to cross-linking of the T

ce11 antigen and CD28 receptors. AKT phosphorylation was also observed following

TCRICD28 costimulation and was found to be relatively increased in thymocytes lacking the

SHP- 1 tyrosine phosphatase. These results suggest that the Pi3 WAKT signaling cascade

plays a role in TCR-elicited T ceIl activation and that this pathway is regulated by SHP-1.

As PTEN plays a critical role in downregulation the coupling of TCR engagement to AKT

phosphorylation/activation, these results raise the possibility that dephosphorylation of PTEN

represents a mechanism whereby SHP-L inhibits TCR signaling.

ACKNOWLEDGEMENTS

1 am grateful to my supervisor, Dr. Katherine Siminovitch, and to the members of my

supervisory cornmittee. Dr. Michael Julius and Dr. Robert Rottapel. for their valuable advice

and construciive criticism through these studies. 1 would also like to thank Dr. Jinyi Zhang

for al1 the help and encouragement and for his friendship. I am also grateful to several

individuals who contri buted with reagents for the experiments descn bed herein and provided

me with trernendous help and invaluable technical advice and who are acknowledged in the

relevant chapters.

Finally, 1 would like to thank al1 my friends and family for their continuous

encouragement and support. In particular, 1 would like to thank my parents and my brother

for their unconditional love and immeasunble patience and encouragement. which made this

endeavor possible.

TABLE OF CONTENTS

Abstrm ................................................................................................................................... ... 11

Acknowledgements .............................................................................................................. 111

Table of Contents............ ..................................................................................................... i v List of Tables ....................................................................................................................... v i List of Figures ..................................................................................................................... vii ... List of Abbreviations .......................................................................................................... V l l l

CHM"rER 1: INTRODUCTION .............................................................................................. 1 btroductory ovel-view and rationi-lle ...................................................................................... 1 T ce11 signüling ..................................................................................................................... 2

Role of phosphatases in regulating T ce11 function ..................................................... 4 n'EN is a phosphatase and a umor suppressor ...................................................................... 7

Initial charactuktion of m'EN ................................................................................ 7 Gennline PTEN mutations in cancer predisposition syndromes ................................. 8 EyrJ2N is inactivated in multiple h.lrmn hmlors .......................................................... 3 F"n3 is inactivated by multiple mechanisms ............................................................ 9

d e f i c h mice ................................................................................................. 9 SmJcture and catalytic properties of l'T'EN .......................................................................... 11

Overd1 !n-ucture of F'mN ....................................................................................... 11 Catalytic properties of P'I'EN .................................................................................. 13 Structure of the PTEN phosphatase domriin... implications ...................................... 14 s tmmre of the PTEN C:! dormin ........................................................................... 17 The pm-d~main binding site ................................................................................. 17 Effect of I-llutations on P n N tumor suppressor f'nction ......................................... 18

The PI3K pathw3y ............................................................................................................... 19 The PI3K family of ~roteins.~ .................................................................................. 19 Activity and regulation of Class IA PI3Ks ................................................................ 20 A m is 3 ~ownsueam target of PI3K... .................................................................... 31

' 5 bas multiple physiological fmxions ......................................................................... ..- Inhibition of apoptosis ............................................................................................ 25 Gene expression ...................................................................................................... 26

P'EN down-regulates the FWUAKT pathway .................................................................... 30 ........................ ETEN suppresses ceIl growth by inhibiting the PI3WAKT pathway 30

I'-'rEN and ~aenorhabditis elesans .......................................................................... 33 A rote for P7-m in T ceIl signal@ ..................................................................................... 34

A mXhanism for regulation ........................................................................ 36 Role of PTEN protein phosphatase rictivity in tumor suppression ......................................... 37 s u m w ........................................................................................................................... 39

Idencification of novel ligands/substrates for PTEN ............................................................. 51 p~rification of l'''EN for affinity c h o ~ t ~ g w h y ................................................... 51 Affinity chromtography ......................................................................................... 52 Identification of potential PTEN ligands by mass spectrornetry ............................... 55

De finition of E"TEN effects on T ce11 function ...................................................................... 59 PTEN is tyrosine-phosphorylated in thymocytes upon TCR engagement ................. 59 TCR-induced activation of AKT is increased in SHP-l-deficient T cells ................. 60

Menti fication of novel l'.EN liganddsubstrates ................................................................... 63 analy sis of the potential PTEN ligands .............................................................. 64

Affinity chromatography/mass spectrometry is a powerful approach ....................... 67 A d e for in T cd1 sipaling ..................................................................................... 71

LIST OF FIGURES

Figure 1-6.. Promotes ceIl survivd though multiple substrates -...---...---.---------.--.-. .-- ..-........-. - ------.- 29

Figure 1-7, antagonizes the P I 3 K A m pathway in C- e l e w w --------- * .---.-.--+----.------**.-*.***...* * 33

Figure 3- 1. GST-k'-rEN fusion protein*.**-** --*-*-*.*..*.-.-**.-..------ * * -.---...-..--. * ..--.---.-------.-. ...*- *.**** ...*.----------- -U

Figure 2-3- Sequence of PTEN c w k i ----.-*-*....*+.*.* * * .-.**..... * ...- **-+** - - * * m---------...----....----.-.*..-...** * -.*...... ..---- 45

Figure 1-3. Affinity chromWF~phy... ..----------.-.....-....-.----- - - - - - - . -....a - ....... - ............................................. 46

Figure 3- 1. Expression md purification of cN=pT'EN ..*.-..---------.----.-.---.--* * - - - - - - - - * -**.....--... * -.... * *... 53

Figure 3-3. Potential binding-partners for F"rEN ..*.*...*.-* * * *..- *.* .***.*....- - -------------..------.---- *-..*.* -..*-..... .. .. - - - 54

Figure 3-3- Y E N is tyrosine-phosphor~lated v o n TCR engagement - -.-...---.....---- - .--... -..-.*- .......-.------ 6 1

Figure 3 4 . TCR-induced activation of AKT is increased in SHP- 1 deficient T cells -_------------....--..*+..... 62

Figure 4- 1 - Schematic of a ~ALDI-ToF mass spectrometer -------.-------.-...........+-........ .. .-..---------------------- 70

Figure 4-2. A rnechanisrn for PTEN regulation: a role for SHP- 1 in the PI3K,AKT/PTEN pathway,-----.74

vii

LIST OF ABBREVIATIONS

aa ACB AICD AP- 1 Apaf- 1 MC ATP BCR B rdU b.v. BZS CBP CD cDNA c-LAP CREB C-terminus DMEM DNA DSP DTT EDTA rlF2B ES FAK FasL FBS FH FPLC Grb2 GSK-3 GST GTP HEPES HRP 12PP2A IGFB P 1 IgG IKK IL-3 IL-2R L K IPTG IRS ITAM It k D a

Arnino acids Affinity chromatognphy buffer Activation induced ce11 death Activating protein 1 Apoptosis protease-activating factor 1 Antigen presenting ce11 Adenosine triphosphate B ce11 receptor 5-bromo-2' deoxyuridine Bed volume Bannayan-Zonana syndrome CREB binding protein Cowden's disease CompIementary DNA Cellular inhibitor of apoptosis CAMP response elrment binding protein Carboxy-terminal Dulbecco's Modified Eagle Medium Deoxyribonucleic acid Dual-speci ficity phosphatase Dithiothreitol Ethy lenediamine trtraacetic acid Eukaryotic initiation factor 2B Embryonic stem Focal adhesion kinase Fris ligand Fetal bovine serum Forkhead homoIogy Fast Performance Liquid Chromatography Growth-factor receptor bound 2 GI ycogrn synthase kinase-3 Glutathione S-transferase Guanidy l triphosphate N[Z-hydroxyethyllpiperazine-N'-(2-ethanesulfonic acid] Horseradish peroxide Protein phosphatase 2A inhibitor lnsulin-like growth factor binding protein 1 Imrnunoglobulin G 1 kappa B kinase tnterleukin-2 Interieukin-2 receptor Integrin-linked kinase Isopropyl fi-D-thiogalactoside Insulin-responsive sequence [mmunoreceptor tyrosine-based activation motif Inducible T ceil tyrosine kinase Kilo Dalton

LAT LB LOH rnAb MAGI MAGUK MALDI-ToF MAPK Me Mev MHC MMAC 1 mRNA MS MW NCBI NF-KB NF-AT N-terminus ORF p l30Cas PA^ PAGE PBS PCR PDGF PDK 1 PDZ PH P W Pf3K PIF PKA PKB PKC PLCy PMSF PP2A PRK2 PtdIns PtdIns(3)P Ptdhs(3.4)P2 PtdIns(3.4,5)P3 PtdIns(3 S)PZ Ptdhs (4) P PTEN PTK m P-Tyr RAC RNA

Linker for activation of T cells Luria broth Loss of heterozygosity Monoclonal antibod y Membrane-associated guan ylate kinase wi t h inverted orientation Membrane-associated guanylate kinases Matrix-assisted laser desorption ionization-time of flight Mitogen-activated protein kinase Motheriten Motheaten viable Major histocompatibility complex Mutated in multiple advance cancers I Messenger RNA blass spectrometry iMolecular weight National center for biotechnology information Nuclear factor kappa B Nuclear factor of activated T ceIls Amino-terminal Open reading frrirne p 130 Crk-associated substrrite Pol yclonal rintibody Polyricrylamide gei electrophoresis Phosphate-buffered saline Polyrnerast: chah reaction Piatelet derived growth factor 3 '-phosp hoinositidedependent kinase- 1 PSD95 DiscsLarge ZO 1 Pleckstrin homology Putative HLA-DR associated protein Phosphatidyl inositol 3-kinase PDK 1 -interacting fragment Protein kinase A Protein kinase B Protein kinase C Phospholipase C g a m Pheny lmethy Isul fonyl fluoride Protein phosphatase 2A Protein kinase C-related protein kinase 3 Phosphatidylinositol Phosphatid y linositol 3-monophosphate Phosphatidylinositol 3.4-biphosphate f hosphatidylinositol 3.4.5-triphosphate Phosphatidylinositol 3.5-biphosphate Phosphatidylinositol4-monnphosphate Phosphatase and tensin homologue deleted on chromosome ten Protein tyrosine kinase Protein tyrosine phosphatase Phosphotyrosine ReIated to A and C protein kinases Ribonucleic acid

rpm SDS Sm SH3 Sm- 1 SLP-76 TBB 1 TCP 1 TCR TEP1 TGF-B TRIM w ZAP-70

Revolutions per minute Sodium dodecyl sulphate Src homology 2 Src homoIogy 3 SH2 domain-containing ETP 1 Src-hornology domin SHî-containing leukocyte protein 76 Tubulin beta- 1 chah T-complex protein 1 T ce 11 receptor TGF-p-regulated and epithelial crll-rrnrichrd phosphatase Transforming growth factor beta T ce11 receptor interacting rnokcule Ultraviolet 6-associated protein of 70 D a

(The one- and the-letter abbreviation system is used for amino acids. X refen to any amino acid)

Chapter 1: Introduction

INTRODUCTORY OVERVIEW AND RATIONALE

The recently discovered PTEN tumor suppressor gene is one of the most cornmon

sites of mutation in human cancers. Gemline mutations of PTEN give rise to a related set of

disorders, including Cowden's disease and Bannayan-Zonana syndrome, which are

characterized by the development of numerous benign tumors and an increased incidence for

developing cancer. The PTEN phosphatase has been implicated as an important modulator

of ce11 survival and apoptosis. rnainly by regulating the PI3WAKT pathway. but it is now

becoming clear that PTEN also plays a significant role in other aspects of ce11 physiology,

including the regulation of ce11 adhesion, migration and differentiation. At present many

lines of evidence indicate a key role for PTEN in regulating T cell function and TCR

signaling. including the finding that heteroz ygous PTEN mice exhibi t an increased incidence

of T-cell lymphomas. Similarly. TCR engagement has been shown to evoke AKT activation.

the activity of which is down-regulated by PTEN. Since its discovery, PTEN has been

extensively studied and despite several important advances many aspects of PTEN remain to

be clarified.

In this context, my research has focused on elucidating the effects of PTEN on T ce11

signaling, characterizhg biochemical mechanisms whereby PTEN might exert its function,

and finding whether additional PTEN substrates exist. Addressing these questions would not

only provide new insights into the function of PTEN and clarify the extent to which the

PTEN and PI3 WAKT pathways intersect. but could ultimately provide dues relevant to

developing novel strategies for cancer therapy.

T CELL SIGNALING

To participate in an adaptive immune response, niiive T cells must be activated in an

antigen-specific manner in order to differentiate into effector T cells capable of recognizing

and responding to foreign antigenic peptides. T ce11 activation requires a minimum of two

distinct signals. The first signal is delivered when the T cell antigen receptor (TCR)ICD3

complex recognizes specific antigen peptide within the context of the Major

Histocompatibility Cornplex (MHC) on antigen presenting cells (APCs) (1). Costirnulatory

molecules present on the surface of T cells provide the second signal. referred to as the

costimulatory signal. The best characterized costimulatory signal is delivered when the T

ceil surface rnolecule CD28 binds to B7 ligands on @Cs. This signal plays a critical role in

augmenting and sustaining ü T cell response initiated through TCR engagement. leading to T

ceIl clonal expansion and initiation of effector functions such as IL-2 production. Activated

T cells express a second receptor for B7 molecules on their surface. CTLAJ, which plays a

critical role as a negative regulator of lymphocyte homeostasis (2).

The ability of the TCR to transduce signais ücross the membrane is mediated by the

cytoplasmic domains of the CD3 subunits and TCRC chahs (3), which contain conserved

signaling motifs known as the ITAMs ammunoreceptor tyrosine-&ed gtivation motifs) (4,

5). These motifs are crucial for coupling to intracellulu tyrosine kinases and therefore

absolutely required for al1 subsequent TCR sipaling responses (6,7).

The earliest biochemical response elicited upon TCR engagement is the activation of

protein tyrosine kinases (FilCs), which regulate the function and interaction of a divergent

array of signaling molecules (8- 10). The Src-family tymsine kinases Lck and Fyn are the

fint PT& to be activated (3. 9). Once activated, these enzymes phosphorylate the ïïAMs of

the CD3 and chains allowing them to associate with ZAP-701Syk protein tyrosine kinase

(11-13). ITAM-bound ZAP-70 is tyrosine phosphorylated and activated by Lck, which in

tum associates with phosphorylated ZAP-70 by means of its S R domain (14). The Tec-

family tyrosine kinase Itk is also tyrosine-phosphorylated and activated by TCR signaling

(LS), and is required for efficient signaling through the TCR (16). Activation of PTKs

following TCR engagement initiates a cascade of sipnaling pathways; the key events are the

activation of PLCy, pZlras and PI3K (6). The signaling molecules implicated in TCR

signaling and that are relevant to the work presented herein are presented in Figure 1-1.

PLCy @hospho!ipase C gamma) controls the production of inositol polyphosphates and

diacylgl ycerols. which induce intracellular calcium mobi lization and acii vûte the

serinelthreonine protein kinase C (PKC) family. respectively. Active GTP-bound Ras

couples the TCR to multiple biochemical effector signaling pathways includin; the MAPK

(mitogen-gctivated protein kinase) pathway. TCR-engagement also controls inositol lipid

metabolism through the activity of phosphatidyl inositol 3-kinase (PI3K). The activation of

P13K is necessary for the synthesis of PtdIns(3,4,S)P3 and PtdIns(3,4)Pr required for the

recruitment of pleckstrin homology (PH) domain-containing proteins to the plasma

membrane and for the activation of proteins such as the serinelthreonine kinase AKT (11).

As it will be discussed later. the PI3WAKT signaling pathway has been implicated in

numerous cellular functions such as the regulation of ce11 sumival, apoptosis and cell-cycle.

to name a few.

In summary. T ceIl sipaling is initiated by the action of tyrosine kinases and

propagated by the combined action of multiple signaling pathways involving numerous

downstream adapter and effector molecules (17). These signaling events. summarized in

Figure 1-2, culminate in the reorganization of the cytoskeleton (18) as well as the

transcriptional activation of multiple genes leading to T ce11 proliferation, differentiation

andor effector function (19). The induction of the U--2 gene, for example. requires the

coordinate action of several signaling pathways that integrate at the level of multiple

transcription factors that include NFAT, AP- 1, NF-KB. and Oct-1 (19). Among the proteins

implicated in T cell signaling, PI3K is most relevant with respect to the role of F E N in

regulating T cell function. as i t is the PI3K signaling pathway that is negatively regulated by

PTEN.

Role of phosphatases in regulating T cell function

The activation of PTKs and subsequent tyrosine phosphorylation of downstream

signaling components is crucial to normal T cell function. and is therefore, tightly regulated

by the opposing action of protein tyrosine phosphatases (PTPs) (9). The disruption of this

balance has been shown to be an important hallmark of cellular transformation (70.21).

Figure 1-1. Signaling molecules involved in TCR signaling

Lc k/ fyn

ZAP-7OISy k

f LCy

PI3K(p85)

S hc

Grb2

[t k

S T

SHP- L

- SH3 - - S H 2 - Kinase - - S H 2 - SH2 - Kinase - - PH - PLC - SH2 - SH2 - SH3 - PLC -

. -- L-

- SHZ - Si42 - Phosphatase -

SH2- Src homology 2 SH3- Src hornology 3 PH- Piecksuin homology TH- Tec homology Pro- Proline-rich

Figure 1-2. Signaling pathways involved in T ce11 activation

TCR CD4/CD8

CD38

Protein Tyrosine Kinases

Protein Tyrosine Phosp hatases

Ras PI3K PLCy + J k MAPK Ip3 DAG

I I [ca2+] PKC

Cytoskeletal change Transcriptional activation

Tyrosine phosphorylation is crucial for the initiation of TCR-mediated signaling and is regulated by the opposing actions of PTKs and PT'Ps. At les t three signalhg pathways are involved in TCR-rnediated sipaling; those mediated by pZlRas, PLCy and PI3K. The nuclear iarget of each signahg pathway is a transcription factor. The coordinate action of multiple transcription factors is required for the transcriptional activation of cytokine genes such as IL-2.

PTEN IS A PHOSPHATASE AND A TUMOR SUPPRESSOR

While sevenl protein kinases have been implicated as oncogenes. i t has long been

postulated that some phosphatases may act as tumor suppressors. The identification of the

PTEN phosphatase as the tumor suppressor residing on 10q73 generated a great deal of

interest. While initial reports suggested that PTEN might act as a protein phosphatase. more

recent evidence by Maehama et al. demonstrated that PTEN can also function as a lipid

phosphatase that dephosphorylates by-products of PDK and thereby functionally antagonizes

signaling pathways that rely on PI3K activity (22). The catalytic properties of PTEN will be

discussed in more detail in another section.

Four lines of evidence (discussed below) have established PTEN as 3 turnor

suppressor gene. First. germline mutations in PTEN are associated wi th autosomal dominant

cancer predisposition syndromes. Second. the PTEN gene is homozygously inactivated in a

variety of sporadic human cancers. Third, the introduction of PTEN into cancer cell lines

leads to turnor suppression. and finally, germline heterozygous mutations in ETEN

predisposes mice to a variety of different tumors.

Initial characterization of PTEN

Loss of heterozygosity (LOH) at chromosome lOq23 occurs at a high frequency in a

variety of human cancers, reaching 60-809 in the case of prostate cancer, endometrial

carcinoma, and advanced glial tumon. By means of representational difference analysis and

positional cloning, PTEN (zhosphatase and m s i n homologue deleted on chromosome

m/MMACl (mutated in multiple gcivanced Gncers 1) was identified by two groups almost

simuItaneously as a candidate tumor suppressor gene located at lOq23.3 (33, 24). Another

group (25) identified the same gene in a search for new dual-specificity phosphatases (DSPs)

7

and named it TEP1 (TGF-P-regulated and gpithelial cell-enriched phosphatase) because its

transcription was modulated by TGF-P.

Germline PTEN mutations in cancer predisposition syndromes

Germline mutations of the PTEN gene are responsible for at least two rare autosornal

dominant inherited cancer predisposition syndromes: Cowden's disease (CD) (26-28) and

Bannayan-Zonwa syndrome (BZS) (58,79). These findings verified that PTEN functions as

a tumor suppressor. These disorders share similar pathological features. such as the

formation of multiple hamartomas (non-malignant growths) in multiple organs and a higher

incidence of developing malignant tumors. The predominant phenotype of CD is

hamartomas of the skin. Other organ si tes developing hamanomas include breast, th yroid,

endornetnum. gastrointestinal tract. and central nervous system, with breast cancers

developing in 25%-50% of affected women and thyroid cancer in 38-106 of affected

individuals (27. 30). BZS is characterized by mental retardation, macrocephaly. benign

neoplasms (lipomas, intestinal polyps), speckled penis and vascular malformations (29, 3 1).

The non-neoplastic features of these syndromes suggest that PTEN plays a role in the normal

development and formation of certain tissues.

PTEN is inactivated in multiple human tumors

In addition to germline mutations, sornatic mutations in the PTEN gene are frequently

found in a variety of sporadic human tumors. For example. in glioblastomas (the most

malignant form of glioma) the F I E N gene is inactivated with approxirnately 24% frequency

(23. 24). with mutations detected at a much lower frequency in many other types of bnin

tumon (32, 33). Several reports show that PTEN is the most frequently mutated gene

identified yet in endometrial cancers (30-504) and in endometrioid ovarian tumors PTEN

mutations have been found with a 26% frequency (34-36). An important observation is that

endometrial as well as ovarian tumon of endometrioid histology that contain PTEN gene

mutations are well or moderately differentiated, suggesting the involvement of the tumor

suppressor in the initiation of disease (37). Other tumors where ETEN mutations are

commonly found include advanced prostate cancers (3840). melanomas (>XI%) (4 I . 12). and lymphomas (4345). A detailed analysis of the nature and frequency of PTEN gene

mutations in different cancers may provide insights into the underlying genetic mechanisms

of tumongenesis in specific tissue types.

PTEN is inactivated by multiple mechanisms

The PTEN g n e is inactivated by multiple mechanisms, including homozygous

genomic deletions. In tumors with hemizygous deletions at chromosome 10q23. frameshift

or nonsense mutations, or mutations resulting in splice variants. prernaturely terminate the

ORF of the remaining copy of the gene. thus producing a truncated and nonfunctional

protein. Altematively. the remaining copy of the gene is altered by missense mutations that

are predicted to severely impair the function of the protein (37). Inactivation of the PTEN

gene may also occur by mechanisms other than deletions and mutations. Loss of expression

or reduced expression of PTEN. both at the mRNA and pmtein levels, was recently

demonstrated in xenografred prostate tumors (38). Although this mechanism requires further

characterization, restoration of PTEN expression by demethylation in cells derived from the

xenogafts suggested loss of expression by promoter methylation (38).

PTEN deficient mice

Further evidence that strongly implicated PTEN as a tumor suppressor carne from

several studies using knockout mice. A number of PTEN mutant mice have been descnbed

(46-48). Although the spectrum of disorders affecting these mice differed drarnatically

arnong the different strains, al1 the homozygous PTEN mutants manifested early embryonic

lethality (E7.5-E9.5) whereas the heterozygous mice exhi bited a predisposi tion to developing

tumon. These results supponed the notion that PTEN is a tumor suppressor essential for

embryonic development. The YIEN+'- mice and chimenc mice genented by eliminating

exons 4-6 (48) exhibited a high incidence of tumor formation involving the prostate, skin and

colon. which are characteristic of CD and BZS. They also spontaneously developed germ

cell, gonadostromal. rhyroid and colon tumors. The PTEN heterozygotes generated through

deletion of exons 4 and 5 displayed a high incidence of lymph node hyperplasia with

consequent disrupiion of lymphoid (B and T cells) organization (46). A high incidence of T

cell lymphomûs was observed in the heterozygous mice generated by eliminating exons 3-5

( 7 ) The appeannce of these turnors wlis markedly enhanced by irradiation and exhibited

elevated levels of phosphorylated AKT, a crucial reguiator of cell survival (see section on

PI3K). Moreover. Fas-mediated apoptosis was impaired in the PTEN"' mice lacking exons

4-6 and the responsiveness to Fas could be restored with PI3K inhibitors. Similarly, T

lymphocytes from these mice showed reduced activation-induced ce11 death (AICD) and

increased proliferation upon activation (49). These results not only indicated that PTEN is an

essential mediator of the Fas response but also implicated the PDWAKT pathway in Fas-

mediated apoptosis.

STRUCTURE AND CATALYTIC PROPERTIES OF PTEN

Overall stmcture of PTEN

The PTEN gene has 9 exons and its product is a 403-mino acid protein (55 kDa) that

is ubiquitously expressed. The structure of PTEN is illustrated in Figure 1-3. PTEN has

been described mostly as a cytosolic protein although one study daims that PTEN is

expressed in both the nucleus and the cytoplasm dunng neuronal differentiation (50). PTEN

contains the signature motif HCXXGXXRSrï present in the active sites of protein tyrosine

phosphatases (PTPs) and duai-specifici ty phosphatases (DSPs). but i t has little sequence

similarity to these protein fimilies outside this motif. Rather. homology searches reveal that

the amino-terminal 190-ürnino acid region of FTEN encompassing the signature motif has

sequence similarity to chicken tensin and bovine auxillin (33. 24). Tensin is an actin-binding

protein locaiized to focal adhesions complexes and auxillin is involved in the uncoating of

clathnn-coated vesicles. Recent crystallographic studies revealed that the carboxy-terminal

region of PTEN contains a C 2 domain. a structure that interacts with phospholipid

membranes in vitro (51). The C3 and phosphatase domains associate across an extensive

interface that is adjacent to the phosphatase active site. The last three amino acids of PTEN.

TKV. fom the consensus sequence (T-X-V-COOH) for binding PDZ domains. PDZ

domains are protein-protein interaction domains that bind to consensus motifs (SîTXV) in the

C terminus of partner proteins or. altematively, to other PDZ domains (52). In addition to

these features, ETEN has two putative consensus binding sites for the SHP-1 tyrosine

phosphatase SH2 domains (MYFEF at residues 239-243 and RYFSP at residues 335-339),

one for the Crk S H 2 domain (RYFSP at residues 335-339) and one for Shc (EYLVL at

residues 3 14-3 1 8).

Figure 1-3. Structure of PTEN

PDZ-BD

HCKAGKGRT

t t TKV EYLVL

Shc binding site MY FEF

SW-1 SH2 domain binding site

f RYFSP

SHP- 1 , Crk SH2 binding site

1 Potencial Tyrosine 1 Phos~horvlrition sites

The PTEN phosphatase is a 403 aa protein and contains the signature motif that defines PTPs and DSPs. Overlapping the phosphatase domain there is a region of sequence homology to the cytoskeletal proteins tensin and auxillin. The carboxy-terminal region contains a C3 domain and a consensus sequence (TKV) for binding PDZ domains. In addition, there are putative consensus sites, which if phosphorylated can potentially bind the SH2 domains of SHP- 1 , S hc and Crk.

Catalytic properties of PTEN

In early reports by Myen et al., PTEN was originally described as a dual-specificity

phosphatase (DSP) capable of dephosphorylating tyrosine. serine and threonine residues in

vitro. However, unlike other DSPs ETEN would not dephosphorylate members of the

MAPK famil y and showed preference towards extremel y acidic substrates (53).

Interestingly, a Cowden's disease-denved PTEN mutation (Glyl29Glu) was observed to

retain phosphatase acti vi ty against the substrates used in these studies. Because PTEN

mutations derived from CD samples rnust be. by definition. biologically inactive. this

observation implied that the G179E mutation might interfere with the ability of PTEN to

dephosphorylate its physiologie targets. Consequently. a later report by Maehama et al.

demonstrated that PTEN dephosphorylates the position D3 of phosphatidylinositol (PtdIns)

phosphates both in vivo and Ni vitro (22). Analysis of the various mutations found in tumors

and patients with CD. including the G119E mutation, revealed that they specifically ablate

the abili ty of PTEN to dephosphory late 3'-phosphorylated PtdIns (54. 55 ) . These results

provided strong evidence that it is the lipid phosphatase activity of PTEN that is required for

its tumor suppressor activity. In agreement with this idea. PTEN-deficient cell lines (56) and

immonalized fïbroblasts from ETEN'" mice (57) have elevated levels of intracellular

PtdIns(3,4,5)P3.

Although the protein phosphatase activity of PTEN seems not to be critical for its role

as a tumor suppressor, it may still be of physiological importance. At present. only two

proteins. the focal adhesion kinase (FAK) and Shc have been identified as targets for ETEN

tyrosine phosphatase activity. PTEN has been shown to directly associate with and

dephosphorylate both proteins thereb y inhi biting integrin-mediated ce11 spreading, migration.

invasion and cytoskeletal organization (58-60). The role of PTEN in the signaling pathways

mediated by the signaiing molecules aforementioned is discussed in more detail in another

section.

Structure of the PTEN phosphatase domain---implications

The phosphatase domain of PTEN consists of a central five-stranded sheet that

packs with two u helices on one side and four on the other (51). The signature motif

HCXXGXXR at residues 113-130, forms a loop (P loop) located at the bottom of the active

site pocket. in comrnon with DSPs and PTPs. In this motif (see Figure 1-4)' the Cys-174 and

Arg- 130 residues are essential for catalysis whereas the His-123 and Gly- 127 residues are

imponant for the conformation of the P loop (6 1.62). The C ys- 1% is absolutel y required for

catalysis. as it executes a nucleophilic attack upon the phosphate moiety of the substrate.

forming a thiol-phosphate intemediate (63). Mutation of this residue to senne or alanine

results in the complete loss of phosphatase activity (64). Another catalytically important and

conserved residue of PTEN is the aspartic acid at residue 97 (Asp-92). It is located on a

mobile loop and it acts as a general acid to facilitate protonation of the phenolic oxygen atom

of the tyrosyl leaving group (65.66). Mutation of this residue to alanine in PTEN results in a

700-fold reduction in catal ytic activit y toward PtdIns(3.4,5)P3 (5 1 ).

The P loop also has two basic residues in its center. This feature gives the PTEN

pocket a unique highly basic character. which is consistent with PTEN's preference for

highly acidic polypeptide substrates as well for the negative charge of PtdIns(3.4,5)P3 (53).

Furthemore, the active pocket is both deep and wide enough to accommodate the bulky and

polyphosphorylated sugar moiety of PtdIns(3.4.5)P3 (5 1). The crystal structure of PTEN

reveded that Gly-129 is at the bottom of the pocket and suggested that the mutation of this

residue to glutamic acid would reduce the size of the ETEN pocket, preventing

PtdIns(3,4,5)Pj from binding but allowing the smaller phosphorylated protein substrates to

bind (51). This provided the structural basis for an explanation of the lack of lipid

phosphatase activit y of the tumor-derived G L29E mutation w hich retained protein

phosphatase activity. However. further characterization is required since the mutation of the

sarne residue to arginine (found in glioblastoma) abolishes both the lipid and the protein

phosphatase activity (53).

The homology of PTEN to tensin and auxillin maps primarily to the hydrophobic core

and to residues that pack with the C2 domain. These residues are highly conserved

suggesting that tensin and auxillin may also contain a C2 domain. an idea thüt is supponed

by the fact that rnost of the PTEN C2 residues that interact with the phosphatase domain are

similar in both proteins (5 1 ).

Figure 1-4. Prirnary structure of the signature motif of PTEN phosphatase domain

1 . 1 - 1 - 1 Lipid Phosphatase

r 1 - 1 + 1 Protein Phosphatase

PTP signature motif

PTEN signature motif

The active-site sequence motif that characterizes the catalytic domains of PTPs and DSPs is illustrated dong with that of PTEN. Cys-124 and Arg-130 are essential for catalysis (in bold) whereas His-123 and Gly-117 are required for the conformation of the P loop (underlined). Asp-93 (double line), dthough not located within the signature motif, is important for catalytic activity (see text). The catalytic consequences of important mutations within the signature motif are also indicated.

Structure of the PTEN C2 domain

The C2 domain (170 amino acids) folds into a sandwich structure consisting of two

antiparallel sheets with two shon u helices intervening between the strands. Its structure is

similar to the C2 domains of PLCy, PKC6 and phospholipaseA7, but i t differs to these in that

it does not require calcium to bind the cell membrane as i t lacks al1 but one of the ca2'

ligands (5 1). The C? domain of PTEN was shown to have affinity to phospholipid

membranes in vitro. Moreover, the C2 domain is tightly associated with the ETEN

phosphatase domain. suggesting that i t may also serve to position the catal ytic site correctl y

with respect to its substrate. Consistent with this hypothesis, mutagenesis of residues in the

C? dornain reduced the tumor-suppressive iictivity of PTEN without interfering with its

enzymatic activity in vitro ( 5 1 ).

The PDZ domain-binding site

PDZ domains mediate protein-protein interactions that can result in the formation of

localized rnulti-protein complexes (67. 68). One study found that deletion of the PDZ-

binding motif of PTEN did not have a detectable effect on its ability to reduce the P13K-

dependent activation of AKT when overexpressed in U87-MG cells (69). On the other hand,

the effects of PTEN on membrane ruffling. in response to stimulation of Swiss 3T3 cells with

PDGF, were completel y blocked by the deletion. Membrane ruffiing due to PDGF is thought

to involve the PDK-dependent but AKT-independent activation of Rac. This effect reflects

the possibility that a PDZ-domain based interaction, although not required for AKT down-

regulation. may facilitate the regulation of other lipid signaiing responses. In a recent report,

PTEN was found to

MAGI3 (70, 7 1).

interact with a specific PDZ domain of MAGE and of the novel protein

These proteins belong to a family of proteins known as MAGUKs

17

(membrane-gsociated guanylate kinases), which appear to function as scaffold proteins to

assemble multiprotein signaling complexes and enhance their stability. Both proteins.

through a PDZ domain-mediated interaction with PTEN, were shown to enhance the

efficiency of PTEN to inhibit AKT. Furthetmore. the PTEN PDZ domain-binding motif was

shown to be important for contriburing to its association to the membrane and for

rnaintaining PTEN protein stability (70). The latter observation is in agreement to other

studies (69. 72) assigning a role for the C-terminus of PTEN in maintaining its protein

stability. More studies need to focus on determining whether PTEN-PD2 domain

interactions may be pathologically significant.

Effect of mutations on PTEN tumor suppressor function

Germline mutations in the PTEN gene detected in CD and BZS as well as somatic

mutations in various tumon are distributed over the entire gene with a clustering in three

regions. the phosphatase signature motif in exon 5 and phosphorylation sites in exons 7 and

8. A great majority of these mutations. 67-838. result in premature temination of the ORF

producing a truncated protein (53) (73). Moreover, the C-terminal region of PTEN contains

predicted secondary structure elements that are ais0 essential for the tumor suppressor

function of PTEN. Furthemore, the majority of residues that make interdomain hydrogen

bonds are found mutated in cancer. Two of them are among the eight most frequently

mutated residues in PTEN. indicatîng that the integity of the interface is also important to

the function of PTEN (51). In support of this contention, a tumor-derived mutation

(Asp252Tyr) which disrupts an interdomain hydrogen bond causes an 85% reduction in

PtdIns(3,4.5)P3 phosphatase activity ( 5 1 ).

THE PI3K PATHWAY

The finding that PTEN dephosphorylates 3'-phosphorylated PtdIns led to a mode1 for

how PTEN acts as a tumor suppressor, linking FTEN to the control of at least two known

cellular protooncogenes, P13K and AKT. PTEN inhibits PDK-dependent activation of AKT.

and deletion or inactivation of PTEN results in constitutive activation of AKT. An overview

of the PDK and AKT family of kinases is provided in the following sections.

The PI3K family of proteins

PI3Ks are enzymes that phosphorylate the position D3 of the inositol ring of PtdIns

and thus give rise to lipid molecules that function as second rnessengers. including

PtdIns(3)P. PtdIns(3.4)P2, PtdIns(3.5)P2, and PtdIns(3,J.5)P3. Three classes of PI3Ks (types

1. II and III) have been identified based upon substrate preference (74, 75,75). Class 1 PI3Ks

consist of heterodimeric enzymes composed of an 85kDa SH2 domain-containing regulatory

subuni t and a 1 lOkDa catalytic subunit (76). There are two subclasses: IA and tB. Subclass

IA (pl LOU, pl IOP. and pl 106) enzymes are regulated by tyrosine-phosphorylated proteins or

by proteins containing proline-rich domains. There are three known regulatory subunits

(p85a, p85P and p55y) (75. 77), as well as sevenl additional isofonns of p85a generated

through differential splicing of the gene (78.79). Both p85u and p8@ contain two carboxy-

terminal S E domains that are separated by an inter-SHZ region that serves as the docking

site for the catalytic subunit. Class IB (pl lOy) transduces signals from G protein-coupled

receptors by binding the fly subunit and does not interact with p85 proteins (80). Class II

PUKs are defined by a carboxy-terminal C2 domain which may confer phospholipid-binding

ability. They seem to preferentially phosphorylate PtdIns and Ptdns(4)P (75). The class Kt

PI3Ks phosphorylate only PtdIns. The yeast homologue VPS34 for this class seems to be

essential for accurate transport of newly synthesized proteins from the Golgi to the vacuole

(75).

For the purposes of this thesis, 1 will focus on class 1 PI3Ks. which regulate a

multitude of cellular functions including apoptosis, cellular proliferation, vesicular

trafficking, cytoskeletal structure and cellular morphology, glucose utilization. protein

biosynthesis. and lipid metabolism. Although this class of PI3Ks has been the focus of the

majonty of studies in signal transduction, the possible effects of the other family members

cannot be disregarded.

Activity and regulation of Class IA PI3K.s

The activation of PI3K clin be accomplished through receptor tyrosine kinase

activation. the activation of non-receptor tyrosine kinases and through interaction with the

activared form of Ras. Receptor activation followed b y dimerization. triggers receptor

autophosphorylation on tyrosine residues creating docking sites for the S H 2 domains of p85

8 8 ) . Non-receptor tyrosine kinases have been implicated in the activation of PI3Ks by

B- and T-cell antigen receptors. many cytokine receptors and CO-stimulatory molecules (such

as CD28). as well as by cell-ce11 and cell-rnatrix adhesion (74). The non-receptor tyrosine

kinase Lck, for example, may recruit p85 via an SH3-mediated interaction (83-85). p85 thus

brings the PDK into proximity with the ce11 membrane where its lipid substrates are located.

GTP-bound Ras also leads to the recruitment and activation of the catalytic subunit (pl 10) of

PDK (86). Because Ras is also a membrane-associated protein, its interaction with PDK

also helps recruit PUK to the membrane where it c m it interact with its substrates. Although

the activation of PDK has been shown to be necessary for TCR signaling, the biochemical

events linking its activation to TCR engagement remain elusive. Recent data has implicated

the adapter molecules TRiM (T ce11 receptor interacting ~olecule) and LAT Uinker for

activation of 1 cells) in the recruitment of PI3K to the ce11 membrane following TCR -

engagement (17. 87, 88). These proteins become phosphorylatrd in multiple tyrosine

residues after T ce11 activation allowing their association with many important signaling

molecules such as p85-PI3K in an SHZdomain dependent manner. Once activated and

brought to the membrane. PI3K specifically phosphorylates the D3 position of the inositol

ring of PtdIns to generate PtdIns(3,4)P2 and PtdIns(3,4.5)P3 (80. 8 1). These localized second

messengen recruit certain cytosolic proteins to the membrane. an event mediated by a lipid-

protein interaction domain known as the Pleckstrin homology (PH) domain. The class IA

PI3Ks. hereafter referred to as PI3Ks. possesses an intrinsic protein kinase activity in virro

directed towards senne residues within the catalytic subunit itself andfor its associated

regulatory subunit. The phosphorylntion of a senne in p85 by the pl LO catalytic subunit has

been documented to result in the down-regulation of PI3K lipid kinase activity (89).

AKT is a downstream target of PI3K

Several molecular targets for Ptd1ns(3.4.5)Pj and PtdIns(3.4)P2 have been identified.

which are tnnslocated and activated upon interaction with PtdIns. A well documented

example of a PDK downstrearn target is the AKT family of serinekhreonine kinases. AKT

(also known as PKB or RAC-PK) is the cellular homologue of the transfoming v-akt

oncogene and has been shown to deliver cell survival sipals in many systems involving a

variety of stimuli including growth factor withdnwal, loss of ce11 adhesion and DNA damage

(90-93).

AKT proteins are distinguished by the presence of an arnino-terminal PH domain that

preferentially binds PtdIns(3,4,5)P3 and PtdIns(3.4)P2 (94). PH domains are conserved lipid-

protein interaction domains that are found in a large variety of proteins involved in cellular

signaiing or cytoskeletal functions (95, 96). Different PH domains exhibit different

phosphoinositide-binding specificity.

There are two major PI3K-dependent events that are required for the full activation of

AKT: membrane localization (97) and senne/threonine phosphorylation (98. 99). The

translocation of AKT from the cytosol to the plasma membrane is accomplished by the

binding of the PH domain of AKT to PtdIns(3,4)P2 (100, 101), which in tum causes a

conformational change in the activation loop of AKT. In addition. two specific sites in AKT

need to be phosphorylated for full activation, one in the kinase domain (Thr-308) and the

other in the C-terminal reguiatory region (Ser-473). The enzyme thiit phosphorylates Thr-

308 was shown to be absolutely dependent on the presence of synthetic PtdIns(3.4.5)P3 in

vitro and therefore it was termed 3'-phosphoinositide-dependent kinase- 1 (PDK1) ( 102- 104).

Like AKT. PDKl also contains a PH domain that binds PDK-phosphorylated lipids. The

identity of the kinase that phosphorylates Ser-473. tentatively terrned PDKî. has remained

elusive. It has been claimed that integrin-linked kinase (LK) is capable of phosphorylating

Ser-473 in vitro and when overexpressed in cells ( 105). A recent study demonstrates that the

activity of ILK c m be regulated by PTEN (106). L K is constitutively activated in human

prostate carcinoma cells lacking PTEN expression presumably due to increased

PtdIns(3,4.5)P3 levels. The activity of L K in these cells was inhibited upon restoration of

PTEN expression by tnnsfection. In addition. selective inhibitors of ILK activity resulted in

inhibition of AKT-Ser-473 phosphorylation (106). These data not only places PTEN

upstream of ILK, but also demonstrates that inhibition of ILK affects the phosphorylation of

Ser-473. Moreover, recent findings have shown that PDKl can interact with a frqgnent of

the C-terminus of PRK2 (107), which w u termed PDK1-interacting fragment (Pm. Remarkably. this interaction converts PDKl to an enzyme that can phosphorylate both Thr-

308 and Ser-473 residues of AKT. Therefore, the activity of PD= might. in fact, be PDKl

itself or, alternative1 y. L K . Important1 y, the phosphorylation of both Thr-308 and Ser-473

and subsequent activation of AKT are abolished if the cells are incubated with PI3K

inhibiton pnor to stimulation with agonists (98). A schematic representation of growth

factor-induced activation of AKT is presented in Figure 1-5.

Figure 1-5. Growth factor-induced activation of the PDKfAKT signaling pathway

'1'

Thr-308 ' Inactive

Ser- 473 AKT

The growth factor-mediated activation of PUK results in increases in 3'-phosphorylated Ptduis. which allows for the translocation of AKT to the plasma membrane followed by a conformational change that renden the phosphorylation sites Thr-308 and Ser-473 available to PDKs. Once activated, AKT is released from the membrane to phosphorylate specific protein targets. PTEN negatively regulates AKT-mediated signaling by its ability to specificaily dephosphorylate PtdIns(3,4.5)P3.

AKT HAS MULTIPLE PHYSIOLOGICAL FUNCTIONS

When activated. AKT transduces signals that regulate multiple biological processes

including apoptosis, gene expression. and cellular proliferation by phosphorylating proteins

on senne and threonine residues. The wide range of biological functions of activated AKT

suggests that it may phosphorylate several targets. The definition of the AKT consensus

phosphorylation sequence (RXRXXSn) has aided the identification of a large number of

proteins that müy represent targets of AKT. many of which are components of the apoptotic

rnachinery. Below is a description of some of the proteins that have been reported to be

targets of AKT and a summary of sorne of rhe sipaling events that are regulated by these

proteins that are likely to be relevant to T ce11 signaling. The role of PTEN as an antagonist

to some of these signais is discussed in a later section.

Inhibition of apoptosis

The fint component of the apoptotic machinery found to be phosphorylated by AKT

was the Bcl-2 fmily member B A D (for a review on Bcl-2 family see 108). This protein

forms a heterodimer with the anti-apoptotic proteins Bcl-2 or Bcl-XL and thereby prevents

(hem from exening their anti-apoptotic function. When BAD is phosphorylated on Ser-112

or Ser- 136 ( log), it associates with cytoplasmic 14-3-3 proteins ( 1 10) and thus can no longer

interaci with Bcl-2 or Bcl-XL. AKT can phosphorylate BAD on Ser- 136 (1 1 1. 1 12) which

inactivates its ability to cause ceIl death and thereby promotes ce11 survival. However. the

fact that BAD is not ubiquitously expressed and that cell survival has been shown to be

regulated independently of both AKT activation and BAD phosphorylation (113, 114).

suggests that BAD is unlikely to be the major mechanism by which AKT blocks apoptosis.

In addition, BAD cm be phosphorylated by other kinases including PKA and kinases

activated through the MAPK pathway (1 15- 1 17).

Another mechanism whereby AKT can promote ce11 survival is by phosphorylating

the ceIl death protease caspase-9. The phosphorylation of human caspase-9 at Ser-196 by

AKT inhibits its proteolytic activation, although the exact basis for this inhibition is not clear

yet (1 18). Dunng the course of apoptosis, mitochondna release cytochrome c from the

intermembrane space into the c ytosol. Cytochrome c binds to Apaf- 1 (apoptosis grotease-

activating factor iJ in a dATP-dependent fashion, recruiting caspase-9 into a complex known -

as the apoptosome. These events activare caspase-9 (1 19, 170) which in turn cleaves and

activates other caspases including caspase-3. thereby initiating an apoptotic protease cascade

that ultimately leads to the cleavage of various key substrates and subsequently to the death

of the cell. Interestingly. the phosphorylation of caspase-9 by AKT does not appear to be a

generalized mechanism for its inactivation ricross species. Not only are AKT

phosphorylation sites and motifs found in humm caspase-9 absent in the mouse, rat and

monkey homologues. but mouse caspase-9 is not phosphorylated by AKT in vitro (121).

Gene expression

The observation that activated AKT translocates to the nucleus (97) led to examining

the possibility that AKT might regulate the activiiy of transcription factors that control genes

involved in cell death. Genetic studies done on C. elegans (discussed in another section)

implicated the PI3WAKT signaling pathway in the suppression of the activity of the

transcription factor DAF16, a member of the Forkhead (FH) family ( 122). Three memben of

the FH family of transcriptional activaton have been identified in mammalian cells, FKHR,

FKHRLl and AFX. XKT modulates the FH farnily members pnmarily though regulation of

their subcellular localization (133). Upon phosphorylation by AKT, the FH family is

exponed from the nucleus to the cytoplasm where they are retained by means of their

association with 11-3-3 proteins (123, 124). When not phosphorylated, members of the Ri

family are localized to the nucleus where they bind specific DNA elements such as IRS

within the IGFBPl promoter (125- 128). FKHRLl has also been found to bind to DNA

sequences found within the regulatory region of the Fas ligand (FüsL) sene (1 23). Thus,

when FKHRLl is phosphorylated. FasL is not expressed allowing the cells to survive.

AKT can also phosphorylate the transcriptional activator CREB. which increases the

binding of CREB to the coxtivator CBP and thus enhances CREB-mediated transcription

( 129). A study using dominant-negative CREB transgenic thymocytes, shows that CREB

might be lictivated upon T cell activation and required for the normal induction of the

transcription factor AP- 1 and subsequent IL-2 production ( 130). Furthemore. recent data

has irnplicated AKT in the modulation of NF-KB-dependen t expression of survi val genes

including the anti-apoptotic Bcl-2 family member Bfl-1/Al and the caspase inhibiton c-iAP I

and c-IAP2 (13 1). The induction of NF-KB by AKT has also been implicated in transcription

from the IL-2 promoter thereby suggesting an important role for the control of T ceII growth

and survival (132). NF-KB is sequestered in the cytoplasm by its cytosolic inhibitor IKB.

Phosphorylation of IKB by MKs (IKB kinases) targets i t for degradation, which allows NF-

KB to move to the nucleus and activate transcription. AKT activates NF-icB by enhancing

the degndation of IKB through phosphorylation and activation of IKKs (133).

Finally, another major downstream target of AKT is glycogen synthase kinase-3

(GSK-3), a serinekhreonine kinase whose activity is inhibited by AKT in response to growth

factor stimulation. GSK-3 negatively regulates a broad range of substrates, including several

transcription factors and the translation initiation factor eIF2B (80, 134). and is involved in

multiple cellular processes, such as metabolism (135, 136) and the regulation of apoptosis

(137). GSK-3 has also been shown to phosphorylate cyclin D l resulting in its degradation

(80). suggesting a role for AKT in the regulation of the cell-cycle partly through its ability to

inactivate GSK-3. In addition. GSK-3 has been identified as the kinase that phosphorylates

NF-AT (nuclear u t o r of ~ t i v a t e d cells) resulting in its expon from the nucleus (138). In

B cells, the engagement of the BCR has been shown to induce the PI3K-dependent

phosphorylation and inactivation of GSK-3 by AKT causing NF-AT to accumulate and

translocate to the nucleus where i t can promote the transcription of MAT-dependent genes

(139). Figure 1-6 provides a sumrnary of some of the biological functions associated with

the activity of AKT and that are discussed in this section.

Figure 1-6. AKT promotes ce11 survival through multiple substrates

Transcriptional activation Metabolisrn

.-.A....CI.- . .. Cell-cycle

death g e n s 1 -"uu

I

APOPTOS lS

I I survival genes I I I 1 I I 1 1 1 1 1 1 1 1 1 I I I

SURVIVAL

M T regulates ceIl survival through the phosphorylation of multiple substntes. AKT can block apoptosis by regulating the activity of transcription factors for death-inducing or survival-promoting genes. AKT c m also prevent apoptosis by phosphorylating and inactivating members of the apoptotic machinery. The phosphorylation and inactivation of GSK-3 by AKT can regulate other cellular functions neccesary for cell survival such as metabolism and ce11 cycle.

PTEN DOWN-REGULATES THE P13WAKT PATHWAY

As previously discussed, PTEN c m specifically dephosphorylate PI(3,1,5)P3 (22.57).

an important second messenger generated by PI3K. which in turn activates AKT-mediated

signals. Overexpression of PTEN or the catalytically inactive mutant (C124S) of PTEN in

393 cells reduces the Ievels of insulin-induced PI(3,4.5)Pi in a dose-dependent manner (22).

Conversely, F'TEN-deficient mouse embryonic fibroblasts display elevated levels of

PI(3.4,5)P3 when compared to their heterozygous counterparts. accompanied by increased

activity of AKT CO a peptide substrate (57). In addition. the CD-derived G129E PTEN

mutation retains protein phosphatase activi ty but lacks PTEN lipid phosphatase activity (54).

This indicates that the ability of F I E N to function as a tumor suppressor is due, in part, to its

ability to dephosphorylate PI(3.4.5)P; and thus affect the downstream target of PI3K. AKT.

PTEN suppresses ce11 growth by inhibiting the PI3WAKT pathway

Interestingly. PTEN appears to suppress cell growth by distinct mechanisms. For

example, while PTEN rnediates a GI cell-cycle arrest in glioblastoma cells ( 5 3 , i t induces

apoptosis/anoikis in other ce11 types (57. 140. 141). PTEN has been demonstrated to induce a

G l block when reconstituted in the PTEN-deficient U87 MG and U178 glioma ceIl lines and

the 786-0 rend carcinoma ce11 line ( 55 . 142-144). The ability of PTEN to induce a G1 block

was correlated with its lipid phosphatase activity since the G l29E PTEN mutant failed to

alter growth suppression. PTEN efficiently down-regulated the activity of AKT, whereas the

G129E mutant did not. Moreover, the coexpression of a mynstoylated form of AKT, but not

wild-type AKT, was able to ovemide the ETEN-mediated cell-cycle block (142). These

resulrs suggest that PTEN-mediated cell-cycle inhibition depends upon the inhibition of the

PUWAKT signaling pathway. In addition. the ability of PIW to block cell-cycle

30

progression correlated with a significant increase of the cell-cycle kinase inhibitor p27KIPl

and a concomitant decrease in the activities of the Gl cyclin-dependent kinases after

restoring PTEN expression (143). The levels of p27KIPl are also found to be reduced in

PTEN-deficient embryonic stem (ES) cells. which correlated with increased phosphorylation

of AKT (145). Consistent with a role of ectopic ETEN in suppressing ce11 growth is the

observation that ETEN-deficient mouse embryos display regions of increased proliferation.

confirmed by high rates of BrdU incorporation (57).

Numerous studies have implicated PTEN as an important regulator of ceil survival by

inducing apoptosis through the inhibition of the PI3WAKT pathway (57. 146). PTEN-

deficient immonalized mouse embryonic fibroblasts display decreased sensitivity to

apoptosis in response to apoptotic stimuli such as UV irradiation. a defect that is restored

upon expression of exognous PTEN (57). Similürly, expression of ETEN in a PTEN-

deficient prostate carcinoma ceIl line induced apoptosis ( 1-16). Interestingl y. enhanced

apoptosis was also observed in mN-deficient prostate carcinoma cells upon transfection of

a kinase-inactive. dominant-negative LK. which inhibited AKT activity (106). As explained

earlier. ILK has been shown to phosphorylate AKT on Ser-473 on a PDK-dependent manner

and its activity was demonstrated to be regulated by PTEN. The dominant-negative ILK also

induced a G1 phase cell-cycle arrest in ihese cells (106). The implication of these findings is

that another mechanism whereby PTEN might suppress ce11 growth is by negatively

regulating the activity of ILK. It is important to mention. however. that ILK may also

regulate cell-cycle arrest via an AKT-independent mechanism involving GSK-3 (105).

Finally, Ci25 1 glioma cells with infected PTEN were induced to undergo anoikis (140).

which is a form of apoptosis that occurs when cells dissociate from the extracellular matrix

and thus plays an important role in preventing transformed cells from becoming metastatic or

invasive. This data in addition to the implication of the PUWAKT pathway in the inhibition

of anoikis (92) strongly suggests an important role for PTEN in mediating this survival

pathway. In conclusion. PTEN-mediated growth suppression. either through the induction of

cell-cycle arrest or apoptosis. requires the inhibition of the PI3 WAKT pathway.

PTEN and Caenorhabditis elegans

Genetic evidence that PTEN acts as a negative regulator of PDK-regulated pathways

has been recently obtained through genetic analysis of a PI3K pathway in the nematode C.

elegans that controls dauer formation, a developmental stage which ensures survival under

stress conditions by suppressing feeding and metabolism. In this state. the animal displays a

prolonged life-span (147). The charxterization of mutants defective in dauer formation led

to the identification of the genes involved. called daf genes. Mutations in the genes daf-2

(insulin-receptor homologue). age-1 (PI3K homologue). pdk-1 (PDKl homologue) and

AKT-1 and AKT-2 (AKT homologues) result in a dramatic increase in longevity which can

be suppressed by mutations in daf-16 (homologue of the marnmalian transcription factors

FKHR, AFX and FKHRLl). Under normal reproductive growth conditions. the DAF-16

protein is phosphorylated and inactivated to allow the expression of metabolic genes

necessary for reproductive development and short lifespan ( 148). D M - 18, the C. elegans

homologue of PTEN. functions as a negative regulator of the DAF-2 and AGE-1 signaiing

pathways. Loss of DAF-18 rescues the extended longevity associated with loss of AGE-1

(149-152). Figure 1-7 illustrates the insulin receptor-like metabolic signaling pathway in C.

Figure 1-7. PTEN antagonizes the P13WAKT pathway in C. elegnns

AGE- I A Insulin Receptor

PTEN

PDK 1

important genetic evidence implicating PTEN as a negative regulator of the PDWAKT pathway has been derived from studies in C. eiegans. This pathway is important for the regulation of dauer formation (explained in text). The PTEN homologue in the nematode, DAF- 18, negativel y regulates the signals mediated by the PI3K homologue AGE- 1 .

A ROLE FOR PTEN IN T CELL SIGNALING

Many lines of evidence indicate a B y role for PTEN in regulating TCR signaling and

T cell function. For example, PTEN knockout mice can-ying one copy of the nul1 allele

develop a high frequency of T cell lymphomas (47). T cells from heterozygous PTEN mice

generated by another group (49) display reduced activation-induced ce11 death (AICD) and

increased proliferation upon activation. indicating that PTEN may act to suppress the

growthlproliferation of T lymphocytes. P13K. whose activity is antagonized by the action of

F E N , has been implicated in the mediation of signals transduced through sevenl surface

molecules involved in T ceIl activation and proliferation such as the TCR. CD28, and the IL-

2R (153).

Insight into the involvement of P13K in T ceIl function in vivo and its role in

tumorigenesis has been recently provided through transgenic mice expressing an active form

of P13K (p65PI3K) in T cells (154). These mice displayed an increased AKT kinase activity

and developed an infiltrating lymphoprolifentive disorder as well as autoimmune rend

disease. Interestingly, a similar phenotype was described for heterozygous PTEN mice (49).

suggesting that it is the lipid substrates of ETEN that are responsible for the

l yrnphoproli ferative and autoimmune disorders seen in these mice. PI3K has also been

shown to play important roles in BCR signaling. Both PI3K activity and the products of this

activity have been shown to increase following BCR engagement (155). Genetic evidence

demonstrating the importance of PDK for B cells has been provided by examination of the

phenotype of mice lacking p85-PI3K expression. which showed defects in B ceIl function

and development ( 156).

While work for this thesis was carried out, a report demonstrated that PTEN

antagonizes the PDK pathway in Jurkat T cells. such that cells transfected with PTEN

displayed an increased rate of apoptosis which was reversed when a constitutively active

membrane-bound form of AKT was coexpressed (157). Similady. the activation of AKT has

been assessed in T cells, revealing that AKT is activated upon TCR engagement or upon

expression of an active form of PI3K in T cells. The TCR-mediated activation of AKT could

in tum be inhibited by exposure to PI3K inhibiton (158). The function of AKT in T ceil

survival was elucidated further by generating mice expressing a constitutively active fom of

AKT in the T cell lineage (159). The active AKT increased resistance to apoptosis to various

stimuli. a property that was correlated to the increased levels of the anti-apoptotic molecule

Bcl-XL. In contrast to other studies (160). AKT did not have an effect on the

phosphorylation of BAD, which in tum was found to be poorly expressed in T cells. Thus

BAD may not be a relevant substnte for AKT in T cells. However, nuclear NF-KB was

elevated in the active AKT transgenic T cells upon TCR engagement, which correlated with

the accelerated degradation of the NF-KB inhibitory protein IKB. This finding suggested that

AKT might enhance the nuclear translocation of NF-KB upon TCR cross-linking.

AKT is constitutively phosphorylated in resting ETEN-deficient Jurkat T cells. a

reflection of the increased levels of PUK products in the cell membrane (161). The

dysregulation of PtdIns in this ce11 line results in the constitutive membrane association of the

PH domain-containing kinase Itk (161). The expression of PTEN or the use of PI3K

inhibiton was shown to not only block the constitutive activation of AKT, but also io induce

the redistribution of Itk to the cytosol.

In summary, these observations pertaining PTEN provide evidence that PTEN plays

an important role in the modulation of TCR signaling and the growth/proliferation of T cells,

primarily through its ability to regulate the PDKIAKT pathway.

A mechanism for PTEN regulation

Very little is known about the regulation of PTEN expression, an event that could

have a dramatic impact on ceIl survival and tumorigenesis. Expression of PTEN mRNA is

rapidly down-regulated upon TGF-8 treatment of a human keratinocyte tumor ce11 Iine (25),

but the biological signi ficance of this regulntory pathway is still unclear. In collaboration

with Our group, Dr. G. Mills found that PTEN is inducibly tyrosine-phosphorylated in BCR-

stimulated CH 12 (B Iymphoma) cells (G. Mills, persona1 communication). Moreover,

phosphorylated PTEN is capable of associating with a catal ytically inactive form of the SHP-

1 tyrosine phosphatase (C435S) when both proteins are expressed in COS7 cells with a

constitutively active form of Lck, capable of phosphorylating PTEN as well as SHP- 1. SHP-

1 (SH2 domain-containing phosphütase I) is a cytosolic protein tyrosine phosphatase highiy

expressed in T cells. SHP-1 has been shown to exert a predorninantly negative effect on the

sigrialing events linking TCR engagement to prolifention (162). This inhibitory effect is

evidenced b y the increased TCR-induced proli ferative response of th ymoc ytes and T cells

from SHP-l-deficient motheaten (me) and viable motheaten (me') mice upon TCR

stimulation (163). The association between PTEN and SHP-1, however, was not observed in

cells expressing wild-type SHP- I instead of the catal ytically inactive SHP- 1. This

observation suggested that &N rnight represent a substrate for SHP- 1. In agreement. the

lipid phosphatase activity of PTEN was found to be down-regulated when PTEN is

phosphorylated (G. Mills, penonal communication).

Although none of these findings have been investigated in T cells yet, the above

observations suggest that the ability of SHP-1 to interact with phosphorylated PTEN may

provide for a mechanism to modulnte the enzymatic activity of PTEN as well as its capacity

to interact with other targets.

ROLE OF K E N PROTEIN PHOSPHATASE ACTIVITY IN TUMOR

SUPPRESSION

As stated earlier, PTEN can dephosphoryiate protein substrates. In particular

overexpression of PTEN leads to the dephosphorylation of focal adhesion kinase (FAK)

resulting in a decrease in ceIl spreading. cell motility and the formation of focal ûdhesions

(58. 164). The dephosphoryliition of FAK ût tyrosine 397 by PTEN also seems to decrease

the tyrosine phosphorylation of its potential downstream effector. pl30Cas (58). FAK is a

non-receptor tyrosine kinase that colocalizes to cellular adhesions with integrins and is an

important element in some integrin-regulated pathways (165). FAK is expressed in most

tissues. and becomes tyrosine phosphorylated and activated after the stimulation of various

receptors and it has been linked to signaling pathways that regulate MAPKs (166). There

exists evidence irnplicating FAK in receptor-initiated activation of T cells showing that FAK

becomes phosphorylated after TCR ligation (166-168). Moreover. the expression of FAK is

increased in invasive and metastatic turnors (166). FAIS Iacks SH3 or SH3 binding domains

so its phosphorylation on tyrosine residues is critical for its interaction with the S H 2 domains

of other signaling molecuIes such as the adapter molecule Grb3 and p85-PI3K (164. 166,

169). The association of PUK and FAK occurs upon attachment of cells to the matrix and

results in a rapid increase of PI3K lipid products and AKT and protection from apoptosis

( 164. 166). Thus PTEN seems to regulate the extracellular rnatrix-dependent PI3 WAKT ce11

survival pathway by a mechanism that may involve FAK. An anti-apoptotic role for F M

has also been recently demonstrated against oxidative stress-induced apoptosis in HL-60

cells and a glioblastoma ce11 line (170). In addition. ETEN has been observed to inhibit

integrin- and growth factor-mediated MAPK signaling pathways. This inhibition was

associated with the ability of PTEN to dephosphorylate Shc. which in tum inhibits the

interaction of Shc with the adapter protein Grb2 and the subsequent activation of the

RaslMAPK pathway (60, 171). These results indicate that PTEN can suppress ceIl motility

by modulating two independent pathways involving Shc and FAK (60).

Importantly, ü PTEN mutation (G119E) that lacks lipid phosphatase activity but

retains protein phosphatase activity (51) is still capable of inhibiting ceIl spreading and

motility (58). This finding indicates that the inhibitory activity of PTEN in these cellular

processes is independent of PI3K regulation. Although the tumor-suppressive functions of

PTEN appear to depend only on its lipid phosphatase activity. the tyrosine phosphatase

activity of ETEN might still be necessary for other aspects of tumorigenesis. However. it is

important to keep in mind that one group only has reponed these findings. and that the direct

interaction between PTEN and FAK. and FAK dephosphorylation upon PTEN transfection.

have not been reproduced by other groups. Therefore. this issue remains rather controversial

and requires a more thorough analysis. Cenainly, the availability of a mutant that retains

only lipid phosphatase activity would allow a better understanding as to whether the lipid

phosphatase activity of PTEN is sufficient for tumor suppression and whether the protein

phosphatase activity is a necessary function.

SUMMARY

One of the challenging questions that remain to be addressed in the future is whether

additional PTEN substrates exist. The evidence indicating that PTEN down-regulates PI3K-

dependent pathways does not rule out that PTEN might control other pathways irnplicated in

functions other than the control of cell growth and survival. Given the importance of PTEN

in regulating cell survival pathways. it is critical to target future experiments into

undentanding the mechanism or mechanisms involved in the regulation of PTEN expression

and function.

In this context, my research has focused on elucidating mechanisms whereby PTEN

influences some of the signaling pathways involved in the activation and proliferation of T

cells. Part of my work focused on finding new substriites for PTEN. by coupling the

technologies of affini ty chrornatograph y wi th the sub-picomolar protein identification

capabilities of rnass spectrometry. These powerful techniques are discussed in more detail in

Chapter 4. In addition. based on preliminary data 1 proposed a novel mechanism for the

regulation of PTEN activity in T lymphocytes.

Chapter 2: Materials and methods

CELL LINES

The human T lymphoblastic leukemia cell line Sup-Tl was kindly provided by Dr. D.

Branch (Canadian Blood Services, Toronto, ON). Cells were cultivated in Dulbecco's

Modi fied Eagle Medium (DMEM) supplemented with 10% heat-inactivated fetal bovine

semm (FBS) (Sigma), 1mM L-glutamine (GibcoBRL), 5x lo-' M B-mercaptoethanol

(GibcoBRL) and 100 UImL ampicillin, at 37°C in a humidified atrnosphere containing 5%

CO..

GENERATION AND PURIFICATION OF GST-PTEN FUSION PROTEINS

The cDNA encoding human PTEN (C124S) was PCR amplitïed with the forward

primer Y-TAGGGATCCATGACAGCCATCATC-3' and the reverse primer 5'-

GGCGAAM'CTCAGACTMTGTAAT-3' from the plasmid MMAClPGEM3 (C124S). a

generous gift from Dr. G. Mills (Houston. Texas). The fonvard and reverse primers contain

an added BamHl site and an EcoR 1 site respectively (underlined). The PCR reaction was

cmied out for 25 cycles (denaturing for 1 min at 94°C: annealing for 1 min at 55°C;

extension For 2.5 min at 72OC) using a Perkin Elmer Cetus DNA thermal cycler. The DNA

polymerase pfu (Stratagene) was used to minimize the introduction of mutations. The 1230

base pair PCR product was isolated from a 1% agarose gel using a QIAquick Gel Extraction

Kit (Qiagen), digested with BarnHl and EcoRl (Amersharn Pharmacia Biotech) and cioned

into the pGEX-2T vector (Amersham Pharmacia Biotech), which had been linearized using

the same enzymes (Figure 2-1). The sequence of PTEN (C124S) once in the pGEX-3T

plasmid was verified by using the PGEX forward and reverse pnmers (Amersham Pharmacia

Biotech) and sequencing facilities at Samuel Lunenfeld Research Institute (S W). Mount

Sinai Hospital. The sequence of PTEN (C 124s) cDNA is provided in Figure 2-2.

Ovemight cultures of E. coli strain BL21 cells (Stratagene) transfomed with the

pGEX plasmid or the recombinant PTEN (C124S)-pGEX plasmid, were diluted 1 5 in 2L of

fresh LB medium + ampicillin (50 pg/rnL) and grown at 37°C to an between 1 and 1.5.

To induce the expression of the GST protein or the GST-ETEN fusion protein. 0.2mM IPTG

was added to the bacterial cultures for an additional 2-3 hours. Cells were harvested by

centrifugation for 20 min at 12.000 rpm in a Sorvall SLA-3000 rotor at 4OC. The cells were

resuspended in 20mL of ice-cold sonication buffer (1% PBS. 150rnM NaCI. lOOrnM EDTA.

2mM PMSF, Zpg/rnL aprotinin. IpglmL leupeptin. Zpg/mL pepstatin. 5mM DTT) and

sonicated. After adding Triton X-LOO (Sigma) to a final concentration of 1%. the lysates

were centrifuged for an additional 10 min at 15.000 rpm and 4"C in a Sorvall SS-34 rotor.

To funher clarify the supernatant from cellular debris. the lysates were centrifuged a second

time under the same conditions. Al1 subsequent steps were performed at 4°C. The lysates

were mixed with 3mL of 75% slurry of Glutathione Sepharose 4B beads (Amenham

Pharmacia Biotech) in a 50mL polypropylene tube on a rotating platform for lh. The beads

were packed into a disposable 5mL PD- IO column (Amersham Pharmacia Biotech) and

washed three times with 4OmL of ice-cold sonication buffer with 1% Triton X-100 and two

times with ice-cold 1X PBS. The GST or GST-&N fusion proteins were eluted off the

beads by incubating with 1OrnM reduced glutathione (Amersharn Pharmacia Biotech). 25mM

Tris-HCI (pH8.0) and 1rnM DTT, as recommended by the manufacturer. Cleavage of the

GST affinity tag was accomplished by incubating the colurnn with the site-specific thrombin

protease instead of reduced glutathione (according to manufacturer's instructions). The flow-

through containing purified PTEN or GST-PTEN were loaded ont0 a lm1 HI Tnp Q

Sepharose column (Arnersham Pharmacia Biotech) equilibrated wi th Buffer A (20mM Tris-

HCI (pH 8.0) and ImM D'TT) and at an approximate rate of 1 mumin. The proteins were

eluted with a linem gradient of Buffer B (500mM KCI, 25mM Tris-HCI pH 8.0 and 1mM

DTT) at a rate of 0.5 Wrnin. Ten fractions of 1mL each were collected. Al1 this was done

using an FPLC system (Amersham Phmacia Biotech). The punty and relative amount of

purified PTEN or YTEN fusion proteins in each fraction were analyzed by coomasie blue-

staining of an 10% SDS-PAGE gel. The fractions containing the most amount of

PTEN/GST-PTEN were pooled and immediately dialyzed against ACB (20rnM HEPES pH

7.5. 10% glycerol. 1mM DTT. 1mM EDTA. 1miM PMSF and 2 pM of each leupeptin.

aprotinin. pepstatin) containing lOOmM NaCl. The amount of K E N present after dialysis

was estimated by the BCA Protein Assay system (Pierce).

AFFTMTY CHROMATOGRAPHY

Sup-Tl ce11 entract was prepared as follows. The cells were grown and collected by

centrifugation at 500 x g at 4'C in a Sorvall SLA-3000 rotor. A cell pellet of approximately

3g was lysed in ice-cold ACB containing J O O m M NaCl and subjected to mild sonication.

Cellular debris was removed by centrifugation for 3h at 4OC and 37.000 rpm in a Beckman

70Ti rotor. The supemantant was dialysed against ACB containing lOOrnM NaCl and

centnfuged at 4°C at 14,000 in a table-top microcentrifuge for 15 min. The foiiowing steps

were performed at the laboratory of Dr. J. Greenblatt (The Banting institute. University of

Toronto) and under the supervision of Dr. D. Awrey. A series of 4 columns were prepared

by coupling each with an increasing arnount of GST, GST-PTEN. or PTEN (0.1.0.5, 1,7 pg)

to the column matrix (Affigel 10, Bio Rad). The matrix was then blocked at both specific

and non-specific binding sites by incubating with ethanolamine and BSA, respectively. A

column containing no protein was used as a control column for identifying background

proteins that would interact with the column matnx under these conditions. The ce11 extract

from Sup-T 1 cells was loaded onto the columns, washed. and eluted sequentially with ACB

containing 1% Triton X- 100. LM NaCl or 1% SDS. Another control column containing 2 pg

of cross-linked PTEN. but ro which no ce11 extract was added. was used to identify

components of the cross-linked protein that may dissociate from the rnatrix during washing

and elution. The eluates (90pL) from the column washings were collected and the proteins

separated by an 12.5% SDS-PAGE gel ( U m m thick) at 30-35 mArnps for 4.5 hrs. The

proteins were visuaiized by silver staining safe for MS analysis. as follows.

Silver staining

The gel was fixed in 50% methanol and 10% acetic acid for 20 min. This step was

repeated once. after which the gel was rinsed in 10% ethanool for 10 min followed by a rinse

in water for 10 min. The gel was reduced with sodium thiosulfate (O.?@) for 1 min and

rinsed twice with water for 20 sec. The ge1 was then incubated in silver nitrate (2@) for 30

min and washed once with water for 20 sec. To develop the staining. the gel was incubated

with developing solution (30zA sodium carbonate, formaldehyde ( 1 A d of 37% solution/L)

and LOmgL sodium thiosulfate) until the desired intensity was attained. The reaction was

immediately stopped by replacing the developing solution with 1% acetic acid and incubated

for a minimum of 20 min. The stained gel was photogaphed using a digital canera. Figure

2-3 summarizes the various steps involved in the affi ni ty chromatograph y expenmen ts.

Proteins that specifically interacted with cross-Iinked ETEN were seen to increase in quantity

with increasing amounts of bound F E N , whereas background proteins remained constant

regardless of the arnount of cross-linked PTEN.

MASS SPECTROMETRY AND IDENTIFICATION OF PROTEINS

Candidate protein bands were excised from the ge1 and digested ovemight with

trypsin. This step and the preparation of the sample were done by B. Cox of Dr. J.

Greenblatt's Iaboratory. The analysis of the samples were performed using a PerSeptives

Biosystems Voyager STR MALDI-ToF in reflector positive ion mode, using the following

settings; ücceierating voltage: 10000. Grid voltage: 72.000%. Guide wire voltage: 0.050%

and Delay: 200 ON. The protein identification by peptide mapping was done using

ProFound search engine from ProteoMetrics (at www.proteometrics.com).

Figure 2-1. GST-PTEN fusion protein

Thrombin cleavage-site

H3N

The GST-PTEN fusion protein was generated by subcloning the cDNA of human PTEN into the pGEX-2T vector containing the GST affinity tag. The BamHl and EcoR 1 sites were introduced into PTEN by PCR. The pGEX-7T protein expression vectcr contains an accessible thrombin recognition sequence, which allowed the site-specific cleavage of the GST affinity tag from the PTEN protein once expressed and purified.

GST COOH

t- F t- w w W t - W O @ln 4 t J U l m LIVU & n u m w

" r 2 " " C "Z 1') C l x

E; 3 y

O 'jiï

m $

W C > U C J

1: K " E " f

< L i X 3 ' r! " ; "' :l xl: 2 ' i

*4 Pl

L-l L I R " il z*

" 5 Y

I d la tnrn u o cnw u i w

Figure 2-3. Affinity chromatography

Ce11 extract ACB

O O. 1 0.5 1 2 - 3 (pg of cross-linked PTEN) y-.. ,-. , - - y - ',-. .-. 5 4 :-- 1, \ --q +----j \

+ protein elution

Increasing amounts of purified PTEN (CIZJS) protein were cross-linked to a series of columns. Proteins from ce11 extract that associated with PTEN were eluted from the columns, resolved by SDS-PAGE and silver-stained. Proteins that were found to interact with FIEN were recognized as bands that increased in quantity with increasing amounts of cross-linked PTEN. These bands were excised from the gel and digested with trypsin. The resulting peptides were analysed by mass spectrometry.

MlCE

Motheaten mice for these studies were obtained by mliting heterozygous motheaten

mice (mec'-) in C57BU6 background breeding pairs from stock maintained at SLRI and have

been previously described (172). Fernale mice 3 4 weeks old were used in the experiments.

~ e " ' mice used in these experiments were littermates of the me"' mice.

ANTIBODIES

The monoclonal CD3 and CD28 mouse antibodies were purchased from Pharmingen

and anti-hamster IgG from Jackson Laboratones. Goat anti-mouse PTEN (N-19) pAb and

rnouse PTEN (AIB 1) mAb and IgG mAb were purchased from Santa Cruz Biotechnology.

Mouse phosphotyrosine (G410) mAb was purchased from Upstate Biotechnology and

phospho-AKT (Ser.173) nbbit pAb and AKT rabbit pAb were purchased from New England

BioLabs. The secondüry antibodies used were honeradish peroxide (HRP)-conjugated rabbit

ünti-goat IgG (Santa Cruz Biotechnology), HRP-conjugated goat anti-rnouse IgG (BioRad)

and HRP-conjugated Protein A ( Bio-Rad).

ACTIVATION OF THYMOCYTES

Freshly isolated thymocytes (3 x 10') from rnotheaten (meime) and their littermate

(+/+) rnice were resuspended in cold serum-free RPMI-1640 media. After 2 hours on ice, the

cells were spun down briefly and resuspended in 0.2mL serum-free RPMI-1640 media in

1.5rnL Eppendorf tubes. Anti-CD3 and anti-CD28 were added to each sample alone or

together to a final concentration of 25pdmL and 15pdrnL respectively. The cells were

incubated on ice for 20 minutes followed by the rernoval of the antibody-containing media

and resuspension in fresh media. Anti-hamster IgG was added to the sarnples to a final

47

concentration of 25pg/pl in order to crosslink the bound antibodies. The sarnples were

incubated at 37OC water bath for 2 , 5 and 10 min. time after which the cells were quickly

spun down and resuspended in 0.2m.L cold lysis buffer (1% Nonidet P-40, 50mM HEPES

(pH 7.4), l5OrnM NaCI2, 50mM NaF. 50mM sodium phosphate. 3mM EDTA, 3mM sodium

onhovanadate, 2mM PMSF and 2 pM of each leupeptin. aprotinin. pepstatin). The O min

group was lysed immediately after the 20 min incubation on ice. Following lysis, the

samples were transferred to ice for an additional 10 min and centrifuged at 14,000 rpm at 4°C

for 15 min to remove cellular debns. The total protein concentration of the supernatant was

determined by using BCA protein detemination system (Pierce). In order to check whether

thymocyte activation was successful, 50pg of activated and non-activated lysate were loaded

onto a 12% SDS-PAGE gel. transferred ont0 a nitrocellulose membrane and blotted using an

antibody against phosphorylated tyrosine residues. The membrane was re-blotted with p-

actin antibody to check for equal loadin;. Details on western blotting are explained in the

next page.

IMMUNOPRECIPITATION OF PTEN A M ) ELECTROPHORESIS

The lysate frorn activated or non-activated thymocytes (500pg total protein) was pre-

cleared with 15pL of Protein G sepharose 1FF beads (Arnersharn Pharmacia Biotech) for 30

min at 4OC. PTEN was immunoprecipitated by incubating the pre-cleared lysates with anti-

PTEN (A2B1) conjugated to Protein G sepharose beads for 2 4 hours at 4OC. The beads

were washed three tirnes with cold lysis buffer and three times with PBS. and then boiled for

5 minutes in Laemmli buffer (2%SDS, l O O m M Tris-HCI (pH 6.8). 20% fhnercaptoethanol,

0.0 1 % bromophenol blue).

The proteins were resolved by SDS-PAGE (10%) at LOO V and

electroblotted ont0 a nitrocellulose membrane (Xyrnotech Biosysterns) for 2

Amps. Purified PTEN obtained frorn the purification procedures described ear

i mmedi atel y

hours at 0.5

*lier was used

as a positive control for the PTEN blots. The anti-PTEN (A2B1) antibody was covaalently

coupled to Protein G sepharose beads as follows. Protein G sepharose 4FF (0.2rnL of slurry)

was washed twice with 3 bed volumes (b-v.) of 20mM sodium phosphate (pH 8.2). The

beads were bnetly spun down and the buffer removed carefully by suction. The beads were

then incubated with O.5rnL of sodium phosphate buffer and anti-PTEN antibody (O.lmg/rnL)

with gentle rotation for 2 h at room temperature, time after which the beads were spun down

and the buffer removed. This was followed by washing the beads with 6 b.v. of 30mM of

sodium phosphate buffer. The antibody was then crosslinked by adding 1mL of the

crosslinking solution (8mg of Dimethyl pimelirnidate. Sigma. dissolved in Z m L of O.2M

triethanolamine pH 8.1) and rotated for 1 h at room temperature. After removing the

crosslinking buffer. the beads were wüshed sequentially with 6 b.v. of 0.2M triethanolamine

(pH 8.2), 2 b.v. of 0. iM ethanolamine (pH 8.2) for 15 min. 3 b.v of O.iM glycine (pH 2.8)

and 6 b.v. of 20mM sodium phosphate (pH 8.2).

WESTERN BLOTTING

For detection of phosphorylated PTEN or phosphoryiated proteins following

thyrnocyte activation. the membranes were blocked for 1 h at room temperature with 3%

(w/v) of gelatin in TBS-T (20mM Tris-HCi (pH 7.6). 150mM NaCl. 0.05% Tween-20). The

membranes were rinsed three times with TBS-T before ovemight incubation at 4OC with the

phosphotyrosine antibody ( 1:5000). The membranes were washed three times for 5 min with

TBS-T and then incubated for 1 h at room temperature with HRP-conjugated goat anti-mouse

IgG (1:10,000) in TBS-T containing 5% (wlv) nonfat dry milk powder. The membranes

were washed three times for 10 min with TBS-Tl and immunoreactive bands were visualized

by enhanced cherniluminescence detection (Amersham Phmacia Biotech). To reprobe these

blots, bound Abs were eluted by incubating the blots for 30 min at 55 OC with stripping buffer

(62.5rnM Tris-HCI (pH 6.7). 2% SDS, lOOmM P-mercaptoethanol). The membranes were

blocked with 5% (wlv) nonfat dry milk for 1 h at room temperature followed by overnight

incubation with with anti-PTEN (N-19) Ab (diluted 1:200 in TBS-T) at 4*C. The KRP-

conjugated rabbi t anti-goat was used at a dilution 1 :3000 in TBS-T.

For the detection of phosphorylated AKT. 50yg of total protein lysate from activated

or non-activated lysates were sepanted on a 12% SDS-PAGE gel and transferred ont0 a

nitrocellulose membrane. The membranes were blotted using an antibody specific to Ser-473

and in ;i similar manner as described above with the following three exceptions. Fint, the

membranes were blocked with 5 '70 nonfat dry milk in TBS-T. the pnmary Ab was diluted

1:1000 in TBS-T plus 5% BSA, and visualized using HRP-conjugated Protein A (1:3000).

The membranes were reprobed with an antibody to AKT (1: 1000) to check for equal Ioading.

Chapter 3: Results

IDENTIFICATION OF NOVEL LIGANDSISUBSTRATES FOR PTEN

As mentioned in Chapter 1. the spectmm of proteins which physically associate with

PTEN remain elusive. In order to identify proteins that may represent substrates or

interacting proteins of F I E N in vivo, the technique of affinity chromatography was used to

Rnd potential protein ligands/substrates for PTEN followed by the identification of such

proteins by mass spectrometry. In an attempt to maximize the likelihood of obtaining

potential ETEN-interacting proteins, the catalytically inactive fom of PTEN containing the

C 124s mutation was used in these expenments. The alteration of the nucleophilic Cys to Ser

has been shown to allow some PTPs to be isolated in a complex with their target substntes

(64).

Purification of PTEN for affinity chrornatography

The purification of GST-PTEN was initially performed using the E. coZi competent

cells of the strain DH5u instead of BLIl to express the fusion proteins. However. numerous

contaminating bands were obtained in the final product. To resolve this problem and thus

minimize the chance of acquiring unwanted and irrelevant proteins. the PTEN fusion proteins

were expressed in B L l l E. coli strain instead (Figure 3-1). BL21 cells are deficient in

certain bacterial proteases that may potentially cleave the protein of interest thus generating

multiple bands. To funher clean the purified fusion PTEN to be used in subsequent steps,

FPLC (Mt performance liquid bromatography) was carried out using an anion-exchange

column.

Affinity Chromatography

Affinity chromatography was tint canied out by coupling purified GST-PTEN

protein to a column matrix. As a control, this experirnent was done in parallel to another

where GST protein was coupled to the column matnx. The protein elution steps revealed

that no proteins from the ce11 extract bound to the cross-linked GST-PTEN. In view of these

results. the GST portion of the GST-PTEN was cleaved off (Figures 3-LB). as explained in

Chapter 2. This was done under the assumption that the GST affinity tag might be

responsible for this negative result. After cleavage of the GST moiety. the experirnent was

repeated by cross-linking PTEN to the column rnütrix. The cell extract was loaded ont0 the

columns and the proteins eluted as explained in Materials and Methods. The eluted proteins

were separated on an SDS-PAGE gel and subjected to silver-staining, which revealed 8

bands. These bands were seen to increase in quantity as the amount of cross-linked PTEN

increased, and were absent in the control colurnns. These bands were then selected 3s

potential ligands for PTEN. Figure 3-2 shows a picture of the silver-stained gel containing

the proteins eluted after the LM salt and 1% SDS wash. The bands identified were of

approximate sizes 23. 28. 30. 45, 50. 55. 67 and 110 kDa. and were eluted off the columns

after washing with 1M salt. The purified PTEN that was cross-linked to the columns was

seen to corne off with the l%SDS wash. The 8 bands identified were then excised from the

silver-stained gel and trypsinized. as explained in Chapter 2. The choice of Sup-Tl cells as

the source of cell extract from which PTEN ligands were to be found was subjective. This

ce11 was shown to express PTEN, and thus favored, in a screen of various T ce11 lines (cell

Iysates were provided by Dr. D. Branch). as seen in a western blot using monoclonal PTEN

antibodies (Figure 3-IC). The implications of using this ce11 line in these experiments are

discussed in Chapter 4.

Figure 3-1. Expression and purification of GST-PTEN

B GST kDri kI GST PTEN PTEN -

Jurkrit SiT Sup-Tl Molt-l

(A) Expression of GST-PTEN in BL21 E. d i cells after 2 hr induction with IPTG visualized by coornasie blue staining after separation of cell lysate by SDS-PAGE. (B) After induction of protein expression, GST. GST-FIEN and PTEN were purified from bacterial ce11 lysate as explained in Materials and Methods. The purified proteins were resolved by SDS-PAGE and stained with coomasie blue to check for purification quality. (C) 50pg of protein lysate from the human T ce11 lines indicated were immunoblotted using PïEN-specific antibodies.

Figure 3-2. Potential binding-partnen for PTEN

Salt SDS

kDa O O.! 0.5 1 2 ACB O 0.1 0.5 1 7 ACB - (pg of cross-linked PTEN)

+ PTEN

A series of 5 columns were prepared by cross-linking 0, 0.1. 0.5. 1 and 2 pg of puified PTEN to column matrix. Cell extract derived from a human T ce11 line was loaded onto the columns. A sixth colurnn with I pg of FTEN was loaded with buffer instead of cell extnct. This colurnn was used as a control for determining components of the cross-Iinked PTEN that rnight corne off with the various protein elution steps. Proteins that associated with the bound F E N were eluted sequeniially with 1M NaCI, 1% Triton X-100 and 1% SDS. The protein eluates collected were separated by SDS-PAGE and visualized by silver staining. Because the 1% Triton X-100 wash did not provide any novel bands, only the proteins eluted with 1M NaCl and 1% SDS are shown in this figure. The position of the proteins that were identified as potential PTEN ligands are indicated by short arrows. These proteins were seen to increase in levels with increasing amount of cross-linked PTEN and were absent from the column to which buffer was added instead of ce11 extract, whereas Ievels of irrelevant proteins remained constant throughout the various columns. Cross-linked PTEN carne of the columns after washing with 1% SDS, shown with a long m o w .

Identification of potential PTEN ligands by mass spectrometry

Protein digestion of the 8 bands generated numerous specific peptide fragments,

whic h were subjected for anal ysis b y MALDI-ToF (Matrix-bsisted @er Bsorption

bnization-Iime of Elight) mass spectrometry. This technique is explained in more detail in

Chapter 4. In brief, MALDI-ToF determines the mass of each digest fragment, generating

unique spectra for each protein. These data are compiued, by means of a cornputer program.

to theoretical data that would have been generated if the protein had been digested by trypsin.

The proteins are thus identified by the masses of their proteolytic peptides. From the 8

protein bands initially isolated in this experiment. only 6 provided spectra with the masses of

their peptides. which allowed their identification. The names of these proteins and their

approximate molecular size. as derived frorn the silver-stained gel. are summarized in Table

1. The matching of pruteins was perfonned with +/- 0.1 Da error. The measured mass and

the calculated mass of each peptide measured by mass spectrometry are indicated in Table II

dong with their sequence and their position in the stipulated protein. It remains to be

mentioned that these results do not. by any meüns. assert that the proteins found to interact

with PTEN represent physiologie ligands for PTEN. Whether these proteins may be relevant

to the role of PTEN in T ceIl function. is discussed in Chapter 4. Given the complexity of

these experiments and time limitations for my thesis work. this part of my research was

discontinued at this stage.

Table 1. Summary of proteins identified by MALDI-ToF mass spectrometry* after

performing affinity chromatogruphy using purified FFEN

Protein name (as identified by MAI,I>I-ToF MS) NCBI protein accession number

Hurnün SET protein (Protein phosphütüse 2A inhibi tor-I2PPîA) (Putative HLA-DR Associüted Protein ILPHAPII)

Humun PHAPI2b protein

PHAPI (putative humün HLA clüss I I associated proicin 1 ) (humiin potent heitt- stable prolein

Humün chüperonin contüining T-complex protein 1 (TCPI ), eta subunit Hurnün T-complcx pmtein 1 (TCP 1 ), thetü subunir

Humün tubulin bela- 1 chuin (TBB I )

Humün nucleolin Human moesin (membrine-orgünizing extension spike protein)

135448

* The protein mutching wiis donc wi th ü +/- 0.1 Dn error

Table II . Information data on the proteolytic peptides by Mass Spect rometry (Continued)

Name of Protein (MW)

'I'CP 1 (55 )

Nuclcolin (67)

Mocsin

- Nurnbcr of rneasurcd peptides

35

Number of niatchcd peptides I

1 O

Covcrrrgc of protcin sequencc

2O'Xl

M casurcd Mnss (M)

Coniputed M a s (M)

- Hcsidues

44 1-447 309-375 44-53

43 1-440 358-366 388-397 370-387 3 19-230 345-357 107- 133

Peptide Scquence

AIXIIPK r ~ s c r ~ ~ ~ LU GMDK1,IVI)GR QQLLIGAY AK Y NI;I;RGC13K SLI IIIAIMIVK GGAEQFMEI'I'EK TFSY AGFEMQPK CQVIWITQIGGER QVKI'Y VEEGLI-1PQIIIK

1,lIL.QGPK GGFGGKGGGK GGCiRGGIGGK NDLAVVDVK Ai RL1il.QGPK 'I'I,I,AKNI,PY K SISLY YTGEK HVFEDAAEIR VIGNEIKLEKPK G1:GFVDGNSIIIIDAK GY AFIEFASFEDAK FGY VDGESAEDLEK KFGY VDESAEDLEK SlSLY YTGEKGQNQDYR G1,SED'I"I'EETLKESl:IlGSVR

IGFPWSEIR APDFVFY APR LNKDQWEER IQVWHEEHR ESEAVEWQQK SGY LAGDKLLPQR

DEFINITION OF PTEN EFFECTS ON T CELL FUNCTION

PTEN is tyrosine-phosphorylated in thymocytes upon TCR engagement

As mentioned earlier, Dr. G. Mills showed. in collaboration with our group. that

PTEN is inducibly phosphorylated upon BCR engagement in a B ce11 line and that its

phosphorylation. in vitro, resulted in a reduction of its lipid phosphatase activity (G. Mills,

persona1 communication). Consequently. we wanted to examine these findings in T cells and

thus specifically determine if the phosphorylation of PTEN could be induced in normal

thymocytes upon TCR ligation.

In order to examine this possibility. thymocytes from normal mice were stimulated

with monoclonal anti-CD3- and -CD28 antibodies followed by crosslinking with anti-

hamster IgG for 2 minutes and lysed. Lysates were resolved by SDS-PAGE and

immunoblotted wi th antibodies speci fic to phosphorylated-tyrosine residues (p-Tyr). to

ascenain that the stimulation procedure resulted in an increase of phosphorylated proteins

compared to lysate from non-stimulated cells, a characteristic result of the ligation of the

TCR. PTEN was then immunoprecipitated from the remaining activated lysate with a mouse

monoclonal anti body that had been previousl y chemicall y cross-linked to beads. which was

required to prevent phosphorylated IgG heav y-chains. w tiose size approxirnate that of PTEN.

from masking the PTEN band in the p-Tyr biots. Similady. the immunoprecipitated proteins

were resolved by SDS-PAGE for an extended period of time to maximize the sepantion of

proteins and facilitate the identification of PTEN. Figure 3-3A shows that TCRKD28

engagement results in the tyrosine phosphorylation of PTEN. The identity of the

phosphorylated band was confirmed to be PTEN by re-blotting the membrane with

antibodies specific to PTEN (Figure 3-3A. bottom panel). In addition. the

59

irnmunoprecipitation of ETEN with the aforementioned antibodies is specific, since no bands

are detected when mouse IgG is used instead (Figure 3-3B). The conditions for the

activation of thymocytes were worked out pnor to PTEN immunoprecipitation. Thymocytes

were stimulated for various induction times (2. 5, 10. 30, 60 min). Stimulation using

CD3+CD28 anti bodies for 1 minutes, resulted in the most protein phosphory lation, and thus

was chosen for immunoprecipitating PTEN (Figure 3-43).

TCR-induced activation of AKT is increased in SHP-1 deficient T ceIls

As explained earlier the phosphorylation of AKT on Ser-473 is essential for its

activation. To determine whether TCR engagement might activate AKT. the thymocytes

from both motheaten and wild type mice were stimulated using CD3+CD28 antibodies for 2.

5 and 10 min and then anti-phospho-AKT immunoblotting was performed on the lysates

using an antibody specific for AKT phosphorylated at Ser-473. Fig 3 4 A shows that AKT

was not phosphorylated to a significant extent in unstimulated cells, but that TCR ligation

caused a substantial increase in the phosphorylation of AKT on Ser-473. The

phosphorylation of AKT peaked at 2 min and progressively declined to lower levels at 5-10

min.

In order to examine the possibility that SHP-1 does indeed play a role in modulating

the activity of PTEN in T cells in vivo. we took advantage of the availability of naturally

occumng SHP- 1-deficient motheaten (Me) mice and examined the phosphorylation state of

AKT. Figure 3 4 A also shows that the lack of SHP-1 in Me thymocytes clearly has an effect

on the phosphorylation of AKT on Ser-473. The level of phosphorylated AKT is elevated in

SKP-1-deficient cells compared to control ceils throughout the various stimulation time

points. Tu confirm that the protein being recognized by the anti-phospho-AKT antibody was

in fact AKT. the membrane was re-blotted with an antibody specific to AKT. shown in

Figure 3-4 (lower panel). This figure also demonstrates that the totai levels of AKT in the

cells are not affected by TCR ligation or SHP- 1 expression.

Figure 3-3. PTEN is tyrosine-phosphorylated upon TCR engagement

(min)

Blot: mi-pTyr

B Iot: rtnti-REN

Thymocytes from wild-type mice were isolated and either stimulated with CD3- and CD28- specific antibodies followed by cross-linking with anti-hamster IgG for 2 min or left unstimulated and lysed. Purified PTEN was used as a positive control for the PTEN antibody. (A) PTEN-specific antibodies covalently linked to Protein G Sepharose beads were used to immunoprecipitate P E N , the resulting protein complexes were analysed by Western blotting using phosphotyrosine-specific antibodies. The bottorn panel was immunoblotted with anti-ETEN. (B) The experiment in A was repeated. including a non-specific Ig conuol.

Figure 3-4. TCR-induced activation of AKT is increased in SHP-1 deficient T cells

W ild-type Me

O 2 5 10 O 2 5 10 Stimulation (min)

O 2 5 LO Stimulation (min) -

Thymocytes from Me and wild-type mice were isolated and stimulated with ûnti-CD3 and anti-CD28 antibodies followed by cross-linking with anti-hamster IgG for 0, 2. 5, and 10 min and lysed. The cells were lysed and 50pg of lysate (total protein) was sepanted by SDS- PAGE, (A) Western blot analysis using an antibody specific for the phosphorylated Ser-473 residue of AKT is shown. The bottom panel was immunoblotted with anti-AKT to check for equal loading. These data represent one of two sirnilar experiments. (B) The same as above, except an antibody against phosphotyrosine residues was used.

Chapter 4: Discussion Signais delivered through the TCR and CD28 play an integral role in T ceIl functions

such as proliferation. survival and apoptosis. Given the importance of these signals for T cell

homeostasis. the elucidation of how these signals are regulated seems critical. In this context

the recent discovery of the tumor suppressor PTEN, a lipid/protein phosphatase. has

stimulated new ideas and challenged others. Many lines of evidence implicate PTEN as an

important regulator of some of the sipals originating frorn the TCR. in particular those

mediated by the PUWAKT pathway. This pathway affects many different cellular functions

uitimately responsible for the survival of the cell and thus represents an imponant

mechanism for the regulation of T cell homeostasis during immune responses. However.

many aspects of PTEN function remain to be elucidated. Although it is clear that the

function of PTEN as a tumor suppressor is due in part ro its ability to down-regulate the

PDWAKT pathway, the possibility that it may play a role in other signaling pathways cannot

be excluded. In this context. I set out to unravel some questions. Does PTEN have other

protein substrates aside from FAK and Shc? Does PTEN interact with other signahg

molecules? Does PTEN function downstream of the TCR? How is PTEN activity regulated?

IDENTIFICATION OF NOVEL PTEN LIGANDS/SUBSTRATES

One of the goals of the work presented in this thesis was to identify additional

proteins that may bind PTEN and might represent substrates of its protein phosphatase

activity. This objective is derived from the observation that the GL29E mutation. which is

lipid phosphatase inactive. behaves like ETEN in some biological assays (57). This finding

immediately suggested that the protein phosphatase activity of PTEN might be relevant to

sorne of its physiologie functions. The approach 1 used to identify novel substrates for PTEN

63

involved the use of two techniques, affinity chromatography and mass spectrometry. These

techniques constitute a powerful combination for chancterizing protein biochemical

pathways through the definition of protein-protein interactions. The results presented in

Table I demonstrate that these techniques can be used successfully to identify potential

binding panners for PTEN. However, to establish w hether these proteins represent PTEN

binding partners in vivo requires additional experiments. Therefore. whether these findings

may be of physiologic relevance will be eaamined in future experirnents. Figure 4-1 shows a

schematic of a MALDI-ToF mus spectrometer and brie fl y summarizes how this technology

operates.

An analysis of the potential PTEN ligands

Table 1 shows a summary of the proteins that were identified. In the following

section, I will introduce these proteins and their functions and attempt to propose when

appropriate whether they might represent relevant PTEN substrates.

One of the proteins positively identified is the beta-subunit of tubulin. Tubulin is a

major cytosolic protein whose assembly into microtubules is criticai to many cellular

processes, such as ceIl mobiiity. intracellular transport and T ceil polarization (173. 174).

Microtubules are heterodimers of two soluble proteins, alpha- and beta-tubulin. Tubulins or

microtubules might function in pan to promote the formation of signaling complexes or

perhaps aid in the intracellula. localization of these signaling molecules. In support of this

idea. tubulin has been found to form protein-protein interactions with a wide variety of

signaiing components, including Zap-70, Fyn. CD?. Vav, and P13K (173, 175. 176). In some

cases, the stimulation of T cells affects these interactions suggesting that they may play a role

in T ce11 activation ( 175). Through the experiments 1 performed, 1 was only able to detect the

beta subunit. It has been proposed that polymerization of tubulin heterodimers rnay be

negatively regulated by tyrosine phosphorylation of the alpha-subunit during T cell activation

(177). Cell lines by definition are not normal and thus expected to have an increased level of

activation. This might explain why the alpha-subunit was not copurified with the beta-

subunit. In the event thüt the interaction between PTEN and tubulin is physiologically

relevant. it can be speculated that tubulin rnight be acting to place PTEN properly in the

context of other sipnaling molecules or complexes nearby the cytoskeleton machinery.

Altematively, PTEN might be acting directly to regulate the polymerization of tubulin by

affecting its phosphorylation state or indirectly by affecting the phosphorylation state of a

protein required for tubulin stabilization.

In addition to tubulin. two subunits (eta and theta) of a chaperonin complex required

for the proper folding of tubulins were also copurified. These subunits form part of the T-

cornplex polypeptide (TCPI), the only known chaperonin in the cytosol of eukaryotes. It is

usuall y composed of eight di fferent but homologous subuni ts assembled into a hexadecamer

of two double rings, but recent studies show that they can also exist in the cell as individual

subunits or smaller oligomers (173), explaining in part why only two subunits were identified

herein. Subunits of TCPl do frequently copunfy with microtubules, providing a likely

explanation for their presence in these experiments (178).

Affinity chmmatography also provided a band of approximately 67 kDa. Based on

the information collected by mass spectrornetry two proteins with different functions were

identified. nucleolin and moesin. Nucleolin is an acidic phosphoprotein abundantly

expressed in exponentially growing cells and is located mainly in dense fibrillar regions of

the nucleolus. It is a multifaceted protein involved either directly or indirectly in modulating

transcriptional processes. cytokinesis. nucleogenesis, signal transduction. apoptosis.

induction of chromatin decondensation. and replication. In addition to several functions in

the nucleolus, nucleolin functions cytoplasrnically and on the ce11 surface to provide a

shuttling mechanism for cytoplasmic and extracellular regulation of nuclear activities (179.

180). For these reasons nucleolin is fundamental to the survival and proliferation of cells, yet

many of its functions rernain to be elucidated.

However. nucleolin is a 110 kDa protein, and the protein derived by affinity

chromatography in these expenments identified a protein of approximately 67 kDa. By

analyzing the position of the peptides measured by the mass spectrometer with respect to the

707 aa nucleolin protein. it can be seen that the peptides cover a stretch of 333 an situated

towards the C-terminus of the protein. This stretch corresponds to approximately 47% of the

complete nucleolin sequence. Given these observations, it is likely that the protein found

might represent a fragment of nucleolin derived from its own degradation or proteolysis. It

would be of interest to determine if PTEN binds to the cornpiete nucleolin, and subsequently

determine if this interaction might be physiologically relevant. However. given that this

protein is involved in so many functions and that it seems to act more as a housekeeping

protein, it is most likely that that the interaction found herein is not physiologically relevant.

On the other hand, moesin (for membrane-grganizing -tension spike pmten) is

widely expressed in different tissues in cells, where it is localized to filopodia and other

membranous protrusions. It is a critical regulator of cytoskeletal-plasma membrane

interactions, especially in polarized cells. as well as important components of signal

transduction pathways. During activation, T lymphocytes become motile cells, switching

from a spherical to a polarized shape with the formation of a uropod in the rear pole. Moesin

seems to be important for redistribution of adhesion molecules to this cellular structure (18 1.

182). As in the case of nucleolin, moesin is highly expressed in malignant ce11 types. which

might have increased the chances for copurification with PTEN. More detailed analysis of

these interactions needs to be performed.

Finally, the low-molecular weight proteins identified seem to be the same protein.

The putative HLA class II-associated proteins PHAP-1 and PHAP-II were purified and

cloned on the basis of their ability to bind to the cytoplasmic domain of the HLA DR-alpha

chah. and may be components of the transmembrane sipaling pathway activated üfter

extracellular binding of ligands durin; the immune response. Sequence identity

demonstrates that PHAP-U is identicai to the protein named SET (1 83). In addition, PHAP-1

has been shown to be equivalent to I2PP2A (potent heat-stable protein inhibitor of protein

phosphatase 2A) ( 184) and crin inhibit PP3-A. a major mammalian serinefthreonine

phosphatase involved in the regulation of most major metaboiic pathways. PP2A may

function as either a tumor promoter or tumor suppressor. depending on the cell type or the

tnnsforming agent (184). It would be interesting to examine whether these proteins rnight

function in regulating the function of PTEN.

Affinity chromatography/Mass spectrometry is a powerful approach

Much of what is known about protein-protein interactions and protei n biochemical

pathways has been denved through the use of protein affinity chromatography and

immunoprecipitation techniques. 1 have used affinity chromatography as a rneans to find

proteins that may interacr with PTEN in vivo. followed by mass spectrometry (MS) for the

identification of these proteins. However, together with these techniques are a number of

factors and variables that need to be taken into consideration for their success. MALDI-ToF

MS c m detect as little as -100 fmol of protein (the limit of silver stain sensitivity), which

means that most procedures have to be performed carefully in order to minimize the presence

of contaminating proteins. However. aside from these obvious considentions. other

decisions need to be made such as the source of ce11 extract to be used. In my expenrnents 1

used a human T cell line because we were interested in defining biochemical pathways for

PTEN in T cells. The cell line 1 used expresses PTEN, which rnight have implications in

itself. Given that PTEN is commonly mutated in ce11 lines. it is not implausible that the

protein expressed might be a truncated or a substr~te-trap mutant (64) which might "trap"

PTEN substrates thereby preventing their association with PTEN cross-linked to the affinity

chromatopph y column. Altemati vel y. wild-type PTEN may be expressed in these cells

thereby impeding the isolation of phosphorylated target substrates.

The purified PTEN to be cross-linked to the column needs to be preferably void of

contarninating proteins which may be minimized by choosing an appropriate expression

system. The stringency and pH of lysis buffen and the use of protease inhibitors are just a

few examples of parameters that can be examined when trying to minimize degradation of

proteins and contamination with unwanted proteins. It is also important to consider which

affinity tag to use for protein purification purposes and whether it should be cleaved or not. I

found that when GST-PTEN was used. no detectable F IEN binding proteins were revealed.

GST is a relatively big protein. -27 kDa and as such it might have interfered with the binding

of proteins perhaps by blocking a binding area or by interfering with the proper folding of

ETEN. Another factor affecting the folding of the protein in the column is the nature of the

matrix used to cross-link it. The matnx used in my expenments cross-links FTEN at pnmary

amino acids. As mentioned earlier. the crystal structure of PTW reveals that the

phosphatase domain and the C2 domain are tightly associated and mutations that disrupt this

structure are tumorigenic (5 1). These observations suggest that the structural state of PTEN

in the column influences the way it binds to candidate ligands or, altematively, rnight

promote the binding of proteins that are not physiologically relevant. As a consequence, in

future experiments we will experiment with other affinity tags such as 6XHIS located either

at the C- or N-terminus as well as with other column matrix. The purpose of the latter is to

avoid disturbing the conformation of PTEN and as a consequence promote the binding of

physiologically relevant substrates. In addition. a more efficient PTEN substrate-tnpping

mutant, D92A, will be used to rnaximize the likelihood of finding PTEN protein substraies.

The wild-type PTEN will be used in parallel as a control for detemining whether these

interactions are mediated through the phosphatase domain.

Figure 4-1. Schematic of a MALDI-ToF mass spectrometer

Accelenting field

Detectors

Proteins to be identified are isolated from the silver-stained gel and trypsinized. The generated peptides are then mixed with a suitable matrix. applied to the sample plate and dned. The plate is inserted into the source where the laser strikes the sample. When the laser hits the plate. some of the sample and matrix are ejected into a chamber where ionization takes place. This means that protons and other cations attach to the sample molecules. When the ions are formed. a voltage pulse is applied sending the ions on their flight. They continue to travel until they hit the plate detector producing an electncal signal. The ions disperse as a function of time. with lighter ions traveling faster than heavier ones. Al1 ions of the same m a s hit the detector at nearly the same kinetic energy. The collection of al1 the signais generates a spectmm with the mass of each digest fragment ion.

To identify the protein, the empirical data from the MALDI analysis is compared to data calculated from a virtual digestion. In the virtual digestion. a cornputer program analyses the sequence of a known protein and calculates the mass values of the fragments that would be genented by trypsin digestion. These calculated results are compared with experimental results. During the database search, any protein containing a specific number of identical fragments is selected as a potential match.

A ROLE FOR PTEN IN T CELL SIGNALING

Our data (Figure 3-3) demonstrated that signals transduced through the TCR in

combination with CD28 c m induce the tyrosine-phosphoryiation of PTEN. These results are

consistent with those by another group. (G. Mills, personal communication) which showed

that BCR engagement could also induce the tyrosine-phosphorylation of PTEN and

suggested that this biochemical modification obsewed following TCWCD28 ligation might

represent an important event relevant to the function of PTEN. The tyrosine phosphorylation

of PTEN might allow its association with SH2-dornain containing molecules resulting in

their dephosphorylation by PTEN and thereby affecting their association with other signaling

molecules. Alternative1 y. the tyrosine phosphorylation of PTEN might be necessary for its

own activity. This possibility 1s very attractive since there is no current understanding of

how PTEN is regulated.

In collaboration with Our lab. Dr. G. Mills recently overexpressed a catalytically

inactive form of PTEN (C121S) in COS7 cells and examined the capacity of tyrosine

phosphorylated PTEN to interact with other proteins (G. Mills. unpublished data). In view of

the presence in PTEN of a consensus motif for binding to the SH2 domains of the SHP-1

cytosolic tyrosine phosphatase, the fint experirnent cmied out in this system involved

investigation as to whether SHP- L interacts with phosphorylated K E N . To address this

issue. a consti tutivel y active form of Lck (Lck Y505F). previousl y shown to phosphorylate

both ETEN and SHP-1. were also expressed in these cells. The results of this study revealed

that PTEN is capable of binding the inert form of SHP-1 (C435S) if PTEN was tyrosine-

phosphorylated. This association was also observed in cells expressing wild-type PTEN. but

not in cells expressing wild-type rather than catalytically inert SHP-1. suggesting that PTEN

might be a substrate of SHP-1. In addition. the lipid phosphatase activity of PTEN was

diminished in vitro when PTEN was tyrosine-phosphorylated (G. Mills. unpublished data).

These findings led us to investigate funher whether the association of PTEN and

SHP-1 was relevant and necessary for TCR signding. Specifically, we set out to explore

whether SHP-1 may modulate PTEN activity and/or its capacity to interact with downstream

targets relevant to TCR signaling. To explore this idea, 1 took advantage of our availability

of motheaten mice that lack SHP-1 protein and examined the levels of phosphorylated AKT

in freshly isolated thymocytes following TCR engagement. The data 1 obtained from these

expenments (Figure 3-4A) demonstrate that the levels of phosphorylated AKT. detected by

using an antibody specific to phosphorylated Ser-473 residue. were elevated in rnotheaten

mice following TCR/CD?8 ligation when compared to thymocytes from wild-type mice

activated in the same fashion. The overall expression of AKT was not affected by

TCWCD28 ligation or by SHP-1 deficiency (Figure 3-4. lower panel), an indication that the

lack of SKP-1 affects the phosphorylation of AKT only. These findings are therefore

consistent with a role for SHP-I in the regulation of PTEN lipid phosphatase activity.

A recent study by Cuevas ri al. that is relevant to the data presented in this thesis has

shown that SHP-1 associates with P13K in TCR-stimulated Jurkat T cells (185). This

association is mediated through binding of a p85 S E domain to phosphotyrosine residue(s)

within the SHP-1 carboxy terminus and appean to reduce PI3K activation. This study

suggested that SHP-1 might work in vivo to down-regulate the activity of the PI3WAKT

pathway.

The data that 1 present herein provide an alternative mechanism by which SHP-1

might regulate the PI3WAKT pathway and in tum inhibit TCR signaling. 1 propose that

TCR signaling induces the tyrosine-phosphorylation of PTEN as a means of enhancing the

activation of AKT. an event required for many cellular functions necessary for the ultimaie

activation of T cells. SHP-1 is an important protein tyrosine-phosphatase in T cells required

for the down-regulation of T ceIl signal transduction. Consistent with its role as a down-

regulator of T ce11 signaling. 1 propose that SHP-1 dephosphorylates PTEN resulting in its

activation or enhanced catalytic activity, which in tum leads to the down-regulation of the

PDWAKT pathway. A schematic representation of this model is presented in Figure 4-2.

Although many aspects of this model remain to be clarified, these preliminary data

offer an exciting direction of study. In future experiments. we will attempt to elucidate this

mechanism further. We will confirrn the association of PTEN with SHP-1 and determine

whether it is dependent on TCR-induced PTEN tyrosine phosphorylation. The

phosphorylation stüte of F E N will be analyzed following longer stimulation times to

detemine the extent and duration of PTEN tyrosine phosphorylation. It is also important to

detemine whether the phosphorylation of PTEN is indeed dependent on PI3K activity. This

can easily shown by pre-treating thymocytes with PDK inhibitors prior to lysis and

immunoprecipi tation.

We will also examine SHP-1-deficient thymocytes in more detail. If the model

presented here is correct, we should be able to detect higher levels of phosphorylated PTEN

in these cells upon TCR engagement. It is also of interest to determine which kinase(s)

phosphorylates PTEN. We will also transfect T ce11 lines with various catalytic foms of

PTEN to further chmcterize some of these findings Ni vitro. For example. tnnsfection of

PTEN protein with a mutated SKP-1 binding site will also help confirm these findings.

Figure 4-2. A mechanism for PTEN regulation: a role For SHP-1 in the

PIJWAKTlPTEN pathway

i INACTIVE)

1 AKT 1

FIEN plays an important role in T ce11 signaling, that of down-regulating the PI3WAKT pathway. T ceIl Iigation leads to a series of signaling events including the activation of PDWAKT. which is required along other signaling molecules for the activation of T cells. The mode1 presented in this thesis suggests that the engagement of the TCR leads to tyrosine- phosphorylation of PTEN resulting in the suppression of its lipid phosphatase activity. This inhibition relieves the inhibition of AKT by PTEN. Similarly, the tyrosine phosphatase SHP- 1, an important down-modulator of TCR signaling, helps down-regulate the augmentation of AKT activity following TCR ligation by dephosphorylating PTEN and PDK. In this manner. the inhibitory effects of SHP- I on TCR signaihg are mediated via different mechanisms, by restonng the negative and positive influence of PTEN and PI3K on the activity of AKT, respective1 y.

The work presented in this thesis has focused on answering some important questions

regarding the function of PTEN. I attempted to identify novel PTEN binding partners

through the combination of affinity chromatography and mass spectrometry. 1 showed that

through this approach it is possible to identify potential PTEN ligands. Future experiments

will attempt to find if these proteins are relevant to PTEN function in T cells. In addition. the

results obtained through this work provide a framework for the characterization of a

mechanism for PTEN regulation. The elucidation of this mode1 will allow for better

understanding of the involvement of PTEN in TCR signaling.

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