Protein acyl thioesterases (Review)

Running title: Protein acyl thioesterases

Ruth Zeidman, Caroline S. Jackson and Anthony I. Magee

Address:

Molecular Medicine

National Heart & Lung Institute

Sir Alexander Fleming Building

South Kensington

Imperial College London

London SW7 2AZ

UK

Corresponding author: Anthony I. Magee

E-mail: t.magee@imperial.ac.uk

Telephone: +44 20 7594 3135

Keywords

APT1

Palmitoylation

Protein acyl thioesterase

Protein acyl transferase

S-acylation

1

Abstract

Many proteins are S-acylated, affecting their localization and function. Dynamic S-

acylation in response to various stimuli has been seen for several proteins in vivo. The

regulation of S-acylation is beginning to be elucidated. Proteins can autoacylate or be S-

acylated by protein acyl transferases (PATs). Deacylation, on the other hand, is an

enzymatic process catalyzed by protein thioesterases (APT1 and PPT1) but only APT1

appears to be involved in the regulation of the reversible S-acylation of cytoplasmic

proteins seen in vivo. PPT1, on the other hand, is involved in the lysosomal degradation

of S-acylated proteins and PPT1 deficiency causes the disease infant neuronal ceroid

lipofuscinosis.

S-acylation

Proteins are modified in many ways as a means to control their localisation and

function. Many proteins that exert their function at or near a cellular membrane are

modified by the covalent attachment of lipids. S-acylation is the post-translational

attachment of fatty acids to proteins through a thioester bond, usually on a cysteine

residue. Palmitic acid, a 16-carbon saturated fatty acid, is the most common fatty acid to

be linked to proteins in this manner, explaining why S-acylation is often referred to as

palmitoylation, although proteins can also be modified in this manner by many other

fatty acids, both saturated and unsaturated, including myristic, oleic, arachidonic and

stearic acids.

A wide variety of proteins are acylated, including transmembrane proteins like G-

protein coupled receptors, T cell co-receptors CD4 and CD8 and ion channels, cytosolic

2

and membrane-associated proteins like members of the Ras and Src families,

heterotrimeric G-proteins, endothelial nitric oxide synthase (eNOS), SNARE vesicle

fusion proteins, as well as secreted and viral proteins. Addition of fatty acids to a

protein has various effects on signalling, trafficking, protein stability, protein-protein

interaction and membrane and membrane subdomain association [1-3].

In the cases where the acylation takes place on an N-terminal cysteine, the fatty acid is

often found linked to the protein through an amide bond to the N-terminus. This type of

acylation is referred to as N-acylation and often occurs in secreted proteins, for example

Hedgehog/Sonic hedgehog [4], but also in Gαs [5]. This may well occur in two steps; S-

acylation via a thioester bond between the fatty acid and the sulphur in the cysteine,

followed by an internal S to N acyl shift to the α-amino group.

S-acylation is a reversible modification

One interesting difference between S-acylation and N-acylation is the reversibility of

the S-acylation. In fact, S-acylation is the only lipid modification of proteins that is

readily reversible, seen as the half time of the attachment of the fatty acid side chain

being significantly shorter than the life time of the protein. Many proteins have been

shown to be dynamically S-acylated in vivo with different turnover rates. For instance,

S-acylation of Fyn, a member of the Src kinase family, is required for its plasma

membrane localisation and has been shown to be reversible, with a half life of 1.5-2 h

[6]. The unpalmitoylated pool of Fyn was found in intracellular membranes. Another

Src family kinase, Lck, has also been demonstrated to be reversibly palmitoylated in

pervanadate-stimulated human T blasts, with a half life of 15-30 minutes (Fig. 1B;

unpublished work, C.S. Jackson and A.I. Magee). Ras proteins were one of the first

3

proteins to be reported to have dynamic S-acylation [7] and different H-Ras variants are

also deacylated at variable rates. Oncogenic H-Ras mutants, which are GTP-bound to a

higher proportion than wild-type H-Ras, were determined to have shorter palmitate half

lives than the wild type H-Ras, even though the steady state levels of palmitoylation did

not differ [8].

(Insert Fig 1 near here)

For some proteins, the increased acyl turnover seen on many proteins in vivo is in

response to various stimuli. One such example is the acylation turnover of the neuronal

protein PSD-95, which is increased upon neuronal activity and causes a clustering of

PSD-95 at the synapse [9]. eNOS deacylation is more efficient in the presence of Ca2+-

calmodulin, an eNOS activator [10]. Many G-protein coupled receptors - including the

β2-adrenergic, α2A-adrenergic, m2 muscarinic acetylcholine, 5-HT4a serotonin, V1a

vasopressin and δ opioid receptors - are also dynamically acylated after agonist binding

[11-16]. The effects of increased fatty acyl turnover on the receptors are not clear. The

steady state levels of acylation will depend on the relative reacylation and deacylation

rates. Even if the steady state levels are unchanged, under conditions with of fatty acyl

turnover there will be a proportion of transiently deacylated receptors. The relative size

of this pool and the time the receptors stay deacylated might be a mechanism for

controlling signalling through the receptors. In fact, receptor desensitization by

phosphorylation is increased in depalmitoylated receptors compared to their

palmitoylated counterparts [17-19].

4

The turnover of palmitate on Gα subunits is also regulated by stimulation of their

associated receptors [20-24] as activation of the receptor leads to the dissociation of the

α subunit from the βγ subunits, making the cysteine on the α subunit available for

depalmitoylation by the thioesterase APT1 [25].

Autoacylation

Even though proteins have been known to be modified by fatty acids and the effects of

acylation on localization and signalling have been known for many years, the

mechanism behind the actual process of attaching the acyl chains has not been very

clear. Many proteins and peptides can autoacylate when incubated with the appropriate

acyl-CoA in vitro. Myristoylated Giα1 is autopalmitoylated in the presence of palmitoyl-

CoA at the same cysteine resdidue that is acylated in vivo [26]. Similarly, the

autoacylation of other proteins have been reported, including rhodopsin [27], SNAP-25

[28], carbamoyl-phosphate synthetase 1 [29], Bet3 [30], β2-adrenergic receptor [31] and

c-Yes [32]. Autoacylation does not seem to be a universal mechanism as, for example,

GAP-43 and Fyn do not autoacylate under the same conditions as Giα1 does [26]. In

addition, not all of these in vitro reactions take place at physiological pH and/or acyl-

CoA concentrations. In some cases the reaction times are also too long to accommodate

the fast palmitate cycling seen on many proteins in vivo.

DHHC PATs

It seems more likely that, at least for most proteins, S-acylation is an enzymatic process.

In recent years there has been much progress in identifying protein-acyl transferases

(PATs). Two families of these enzymes are the subjects of other reviews in this issue.

5

Briefly, one such group of PATs is the DHHC family, consisting of multi-spanning

transmembrane proteins that have a cysteine-rich domain containing a conserved

asparatate-histidine-histidine-cysteine (DHHC) motif, with is essential for the PAT

activity [33, 34]. The PAT activity of the DHHC proteins was originally discovered in

yeast, where the DHHC protein Erf2, together with Erf4, mediates the acylation of yeast

Ras [33]; another DHHC protein, Akr1, is a PAT for yeast casein kinase 2 [34]. The

palmitoylation of specific proteins could not be detected in yeast strains deficient in

different combinations of the yeast DHHC proteins, confirming the in vivo acyl-

transferase activity of the DHHC proteins [35]. In a screen on potential substrates, the

23 DHHC proteins predicted in the mammalian genome had variable substrate

specificities, with some overlap [36]. The DHHC PAT substrates are intracellular

proteins [1, 37, 38] including: H-and N-Ras which are S-acylated by DHHC9 together

with a Golgi-associated protein GCP16, echoing the yeast Erf2/Erf4 complex [39]; the

DHHC21 substrate eNOS [40]; huntingtin which is acylated by HIP14/DHHC17 [41,

42]; the γ2 subunit of the GABAA receptor which is acylated by GODZ/DHHC3 [43]

and the neuronal PSD-95 protein, which in slightly contradictory reports has been

suggested to be the substrate of DHHC2, 3, 7 and 15 (but not DHHC17) [36] or of

HIP14/DHHC17, in addition to other neuronal substrates [41].

MBOAT PATs

A different group of PATs appear to have secreted proteins and peptides as their

substrates. Hedgehog (Hh) proteins e.g. Sonic hedgehog (Shh), Spitz, Wnts and ghrelin

require acylation for their function and are acylated by members of the MBOAT

(membrane-bound-O-acyltransferase) family [44]. A common feature of this group of

proteins is that they have several membrane-spanning domains and that they transfer

6

organic acids, typically fatty acids, onto hydroxyl groups of membrane-embedded

targets [45]. Only a small subset of members is known to transfer fatty acids and other

lipids to proteins. Rasp/Skinny hedgehog/Hedgehog acyl transferase (Hhat) is the PAT

for Hh/Shh [46] and Spitz [47], Porcupine (Porc) is the PAT for Wnts [48] and GOAT

is the PAT for ghrelin [49]. The acylation of secreted proteins is reviewed elsewhere in

this issue.

Ykt6-type PATs

A third mechanism for fatty acid attachment to proteins incorporates elements of both

autoacylation and enzymatic acyl transfer. The yeast SNARE protein Ykt6 binds

palmitoyl-CoA at an N-terminal longin domain and autoacylates cysteines at its C-

terminus [50] in addition to mediating the palmitoylation of Vac8, presumably in a non-

enzymatic way as equimolar amounts of Ykt6 and Vac8 are needed [51]. The exact

mechanism behind Vac8 palmitoylation is not clear, and further complicated by the fact

that the DHHC protein Pfa3 can palmitoylate both Vac8 [52] and Ykt6 [53]. One

possibility is that Ykt6 could act as a co-factor for Pfa3 in the way that Erf4 does for

Erf2, although this remains to be elucidated.

Regulation of deacylation

It is thus not completely clear if all fatty acylations are enzymatic and if they are

regulated. This raises the possibility that the level of regulation of acylation lies on the

deacylation step, especially for those proteins where autoacylation has been suggested.

Deacylation of proteins appears to be an enzymatic process. Despite the fast progress

that has been made, and is continously being made, in identifying and characterizing

PATs, much less is known about the thioesterases that deacylate proteins. So far, only

7

two thioesterases have definitively been shown to catalyze the removal of fatty acids

from proteins, and out of those two, only one appears to be involved in regulation of the

dynamic acylation cycles seen for many intracellular proteins.

Acylprotein thioesterases

Acyl protein thioesterase 1 (APT1) was originally isolated from rat liver as a

lysophospholipase [54], but was later shown to have a preferred substrate specificity for

palmitoylated proteins over lysophosphatidylcholine and palmitoyl-CoA [25]. It is a

cytosolic protein [25] with a widespread tissue distribution [55]. Several proteins have

been identified as APT1 substrates in in vitro assays, like Ras [25], various

heterotrimeric G protein α subunits [25, 56], eNOS [10], RGS4 [25] and SNAP-23 [57].

Viral proteins, like the spike glycoprotein E2 from the Semliki Forest virus, HEF and

HA from influenza viruses and the G protein from vesicular stomatitis virus (VSV) are

also deacylated by APT1 in in vitro assays [58], although whether this has any

biological significance remains to be elucidated.

Cell-based experiments have validated APT1 as a thioesterase. In yeast cells deficient in

Apt1p, the yeast homologue of APT1, no Gαi1 deacylation takes place [56], confirming

its status as an APT1 substrate. In permeabilized platelets, infusion of APT1 protein

causes an almost 50% reduction of the steady state levels of acylation and, more

specifically, a reduction of the acylation of Gαq and concomitant reduction of membrane

association [59]. Co-expression of APT1 and eNOS increases the level of eNOS

depalmitoylation compared with expressing eNOS alone [10]. Furthermore, the

dislocation of the APT1 substrates SNAP-23 and a mutant Gsα from the plasma

membrane has also been seen on elevating APT1 levels in cells [59, 60].

8

There is no specific consensus sequence for S-acylation, although some patterns of S-

acylation exist, for instance N-terminal dual S-acylation and myristoylation, C-terminal

dual S-acylation and prenylation, and multiple S-acylation at cysteine string motifs [1].

Following on from the lack of distinct S-acylation motifs, there is also no clear

consensus for the sequences surrounding the thioacyl group recognized by APT1. In

fact, APT1 appears very promiscuous in its substrate specificity. In vitro, APT1 can

depalmitoylate structurally different proteins, both soluble intracellular proteins, like

eNOS and Gαi1, which are S-acylated on cysteines in proximity to an N-terminal

myristoylation site [61, 62] and Ras, which is modified at its C-terminus by both

acylation and prenylation [63], as well as integral membrane proteins. SNAP- 23 is S-

acylated at multiple cysteines located in the transmembrane domain [64, 65] and the

viral proteins E2, HEF, HA and VSV G protein are S-acylated at the interface between

the transmembrane domain and the intracellular part of the proteins [66, 67].

APT1 does not, however, deacylate proteins without any discrimination. Caveolin, for

instance, an integral membrane protein which is acylated on cysteine residues in the

intracellular C-terminal domain [68] is not deacylated by recombinant APT1 under

conditions where eNOS deacylation is readily seen [10]. Moreover, not all substrates

are deacylated with the same efficiency. Rat APT1 is 10-fold more efficient in

deacylating Gαi1 compared to Ras. This preference for Gα subunits over Ras is even

more pronounced in yeast APT1, where the difference is 70-fold [56]. Also, the

activational status of the substrates may influence the catalytic efficiency displayed by

APT1 towards it; free Gαi1 is a more readily deacylated by APT1 than heterotrimeric

Gαi1 [56].

9

Another explanation for the variable substrate preference could be the possibility that

APT1 could have selectivity for different fatty acids linked to its substrates. This is,

however, contradicted by the fact that two good viral APT1 substrates, the Semliki

Forest virus (SFV) protein E2 and the influenza virus protein HEF, are acylated almost

exclusively with palmitic and stearic acid, respectively, whereas the SFV E1 protein,

which is mainly palmitoylated [69], is a poor APT1 substrate [58].

The crystal structure of human APT1 has been solved and shows that the enzyme is a

member of the large enzyme group α/β hydrolases, which also includes lipases,

esterases and dehalogenases, and has a classical catalytic triad made up of Ser-114, His-

203, and Asp-169 in APT1 [70]. A Blast search of the APT1 sequence reveals

homologues in a wide variety of species, including humans and other mammals as well

as lower organisms such as Drosophila melanogaster, Saccharomyces cerevisiae,

Caenorhabditis elegans, Arabidopsis thaliana and Mycobacterium leprae, implying an

essential role for ATP1 as it is so well conserved throughout evolution.

APT1 inhibitors have been developed based on the structure of the H-Ras C-terminus

and in vitro assays show promise for some of the compounds, with IC50 values in the

low nanomolar ranges [71]. Their effects in vivo have not yet been determined but they

could provide a useful tool for studying the role of APT1 in cells. In general, the field

of protein acylation has suffered from a lack of specific potent inhibitors so these

compounds could be an important addition to the toolbox.

10

A homologue of APT1, called lysophospholipase II or APT2 with 64% identity to

APT1, has been cloned [55]. It was reported to have activity against several lipid

substrates, with varying efficiencies. APT2 mRNA transcripts can be found in a wide

variety of tissues, suggesting it is ubiquitously expressed [55]. An interesting question

that has not yet been answered is whether APT2, just like APT1, is a cytosolic protein

thioesterase involved in protein acylation regulation. In fact, in extracts from yeast

strains where the APT1 gene is disrupted, the thioesterase activity against acylated H-

Ras is similar to that of extracts from wild-type yeast, despite deacylation of Gαi1 being

almost completely abolished [56]. It is tempting to speculate that APT2 could be

responsible for this residual thioesterase activity. However, Blast searches using either

yeast APT1 or human APT2 sequences do not suggest that there is an APT2 protein in

yeast (R. Zeidman and A.I. Magee). Instead, the Ras deacylation could be caused by

another still unidentified thioesterase activity.

A Blast search of the human APT1 sequence reveals another homologue of APT1, with

31% identity to APT1 and with the catalytic triad conserved (R. Zeidman and A.I.

Magee). This protein was named lysophospholipase-like 1 when its gene was

discovered during the sequencing of chromosome 1 [72]. As of yet there are no reports

of this protein’s biological activity and substrates, but if any thioesterase activity of this

protein will be identified, we suggest that it should be called acyl protein thioesterase

like 1 (APTL1) in accordance with the naming of APT1.

APT1 was purified from the soluble S100 fraction of a rat liver homogenate [25] and it

does not contain any predicted transmembrane domains or obvious sites for lipid

modification. The substrates of APT1 are, however, membrane-associated, which raises

11

the question of how APT1 can access its substrates efficiently. One answer could be

that APT1 might interact with membrane proteins, either its direct substrates or other

proteins that bring APT1 in close proximity to its substrates. Our own data suggest that

a significant proportion of APT1 and APT2 sediment with cellular membranes from

cultured cells (R. Zeidman and A.I. Magee, unpublished work), supporting this

speculation.

Protein-palmitoyl thioesterases

Palmitoyl-protein thioesterase 1 (PPT1) was first isolated from bovine brain extracts

based on its ability to depalmitoylate [3H]-palmitate-labeled H-Ras [73] and was also

found to depalmitoylate Gα subunits and acyl-CoAs in vitro, with a preference for a

chain length between 14 and 18 carbons [74]. Further expression studies revealed that

PPT1 is a lysosomal enzyme [75] and it is therefore unlikely to play a role in regulated

deacylation of cytoplasmic proteins. The gene encoding PPT1 was found to be located

on chromosome 1p32, the region linked to the neurodegenerative disease infantile

neuronal ceroid lipofuscinosis (INCL), and as mutations in the PPT gene were found in

INCL patients, mutated PPT1 was identified as the underlying cause of INCL [76]. One

of the features of INCL is the accumulation of granular deposits inside the cells.

[35S]cysteine-labeled lipid thioesters accumulate in immortalized lymphoblasts from

patients with INCL, and this accumulation can be reversed by adding recombinant

PPT1 to the cells [77], which is taken up and trafficked to lysosomes [78]. The normal

function of PPT1 is thus to remove the acyl chains from proteins being degraded in the

lysosome.

12

A homologue of PPT1, called PPT2, has been cloned. It is also a lysosomal

thioesterase, but has a substrate specificity for palmitoyl-CoA and not palmitoylated

proteins [79] and therefore likely does not act as a thioesterase for acylated proteins

during the degradation process.

Deregulation of S-acylation implicated in diseases

Aberrant regulation of S-acylation and deacylation has been implicated in a number of

human diseases. The dysregulation of S-acylation can be caused by mutations in the

acylated proteins, or by abnormal expression of either the PAT or thioesterase involved.

Huntington’s disease, a neurodegenerative disease, is caused by a dominant mutation in

the protein huntingtin that expands the number of glutamines within an existing

polyglutamine stretch [80]. Within this region, there is a cysteine that is transiently

acylated, both in the wild-type and mutated huntingtin, but the level of acylation is

lower in the glutamine-expanded mutant than in the wild-type protein. Less acylated

forms of huntingtin display increased aggregation and formation of insoluble inclusions

in neuronal cells. Overexpression of the PAT responsible for acylation of huntingtin,

DHHC17/HIP14, reduces the number of inclusions in cells expressing mutant

huntingtin [42]. Finding a way to decrease the activity of the thioesterase responsible

for deacylation of huntingtin, which still needs to be identified, or increasing the

activity of DHHC17/HIP14 in the affected neuronal cells in patients would be an

attractive model for Hungtington’s disease treatment or prevention.

Targeting the S-acylation regulation machinery could also be a potential taget for anti-

cancer therapies. Ras proteins, which can be both farnesylated and S-acylated, play an

13

important role in tumour progression and Ras mutations are common in human cancers

[81]. Oncogenic H-Ras mutants undergo increased cycles of acylation and deacylation

compared to wild-type H-Ras and are GTP-bound to a higher extent [8]. This opens up

the possibility to modulate Ras activity by preventing dynamic acylation of Ras and also

suggests that either inhibiting the acylation or stimulating the deacylation could have

similar effects on reducing the oncogenicity of Ras. Ras lipidation is already an anti-

oncogenic drug target; farnesyl transferase inhibitors (FTIs) have been tested for their

efficacy as cancer drugs. Despite initial promising results, the FTIs have not been

efficient anti-tumour drugs in clinical trials, presumably because Ras is instead

geranylgeranylated following FTI treatment [82]. It is possible that S-

acylation inhibitors could prove a more efficient route in the development of treatments

for Ras-mediated tumours.

In fact, the deacylation of another protein, tubulin, might be a mechanism already in use

in anti-cancer therapy. Treatment of leukemic lymphoblasts with clinically relevant

concentrations of vinblastine was reported to lower the levels of [3H]-palmitate

incorporated into tubulin, as well as resulting in the known effects of vinblastine,

microtubule disassembly and apoptosis [83]. As tubulin can autopalmitoylate [84], the

regulating step in the tubulin acylation/deacylation control could be at the level of

thioesterases. Modulating thioesterase activity in cancer cells sensitive to vinblastine

might be a useful therapeutic strategy.

Future perspectives

In summary, only a single thioesterase, APT1, is known to act on cytoplasmic S-

acylated proteins, although the protein acylthioesterase activity of other candidates has

14

not been thoroughly tested. Whether the substrate specificity and/or activity of this

enzyme are regulated by post-translational modification or subcellular localisation is an

open question. In this context, the association of a substantial fraction of APT1 with

cellular membranes is intriguing. However, comparison to the large family of DHHC-

containing PATs for cytoplasmic proteins (23 in man) might suggest that APT1 cannot

confer enough specificity in substrate deacylation to be a likely regulatory point for all

of them. Nevertheless, in order fully to understand the role of S-acylation in

contributing to the regulation of the function of cytoplasmic proteins it is essential to

study thioesterases as well as PATs and to elucidate the control of their opposing

effects in vivo.

Current evidence suggests that PPT1 is mainly involved in the lysosomal degradation of

acylated proteins. However, with respect to extracellular acylated proteins it will be

interesting to see whether PPT1, which can be released from cells before being taken up

into lysosomes, could deacylate some signalling molecules (e.g. Wnts) extracellularly

and thus modulate their spread within tissues and function.

References

1. Smotrys JE, Linder ME. 2004. Palmitoylation of intracellular signaling proteins:

Regulation and Function. Annu Rev Biochem. 73: 559-587.

2. Resh MD. 2006. Palmitoylation of ligands, receptors, and intracellular signaling

molecules. Sci STKE. 2006: 1-12.

3. Resh MD. 2006. Trafficking and signaling by fatty-acylated and prenylated proteins.

Nat Chem Biol. 2: 584-590.

15

4. Pepinsky RB, Zeng C, Wen D, Rayhorn P, Baker DP, Williams KP, Bixler SA,

Ambrose CM, Garber EA, Miatkowski K, Taylor FR, Wang EA, Galdes A. 1998.

Identification of a palmitic acid-modified form of human Sonic hedgehog. J Biol Chem.

273: 14037-14045.

5. Kleuss C, Krause E. 2003. Galpha(s) is palmitoylated at the N-terminal glycine.

EMBO J. 22: 826-832.

6. Wolven A, Okamura H, Rosenblatt Y, Resh MD. 1997. Palmitoylation of p59fyn is

reversible and sufficient for plasma membrane association. Mol Biol Cell. 8: 1159-

1173.

7. Magee AI, Gutierrez L, McKay IA, Marshall CJ, Hall A. 1987. Dynamic fatty

acylation of p21N-ras. EMBO J. 6: 3353-3357.

8. Baker TL, Zheng H, Walker J, Coloff JL, Buss JE. 2003. Distinct rates of palmitate

turnover on membrane-bound cellular and oncogenic H-Ras. J Biol Chem. 278: 19292-

19300.

9. El-Husseini AE-D, Schnell E, Dakoji S, Sweeney N, Zhou Q, Prange O, Gauthier-

Campbell C, Aguilera-Moreno A, Nicoll RA, Bredt DS. 2002. Synaptic strength

regulated by palmitate cycling on PSD-95. Cell. 108: 849-863.

10. Yeh DC, Duncan JA, Yamashita S, Michel T. 1999. Depalmitoylation of

endothelial nitric-oxide synthase by acyl-protein thioesterase 1 is potentiated by Ca(2+)-

calmodulin. J Biol Chem. 274: 33148-33154.

11. Hawtin SR, Tobin AB, Patel S, Wheatley M. 2001. Palmitoylation of the

vasopressin V1a receptor reveals different conformational requirements for signaling,

agonist-induced receptor phosphorylation, and sequestration. J Biol Chem. 276: 38139-

38146.

16

12. Ponimaskin EG, Schmidt MF, Heine M, Bickmeyer U, Richter DW. 2001. 5-

Hydroxytryptamine 4(a) receptor expressed in Sf9 cells is palmitoylated in an agonist-

dependent manner. Biochem J. 353: 627-634.

13. Hayashi MK, Haga T. 1997. Palmitoylation of muscarinic acetylcholine receptor

m2 subtypes: reduction in their ability to activate G proteins by mutation of a putative

palmitoylation site, cysteine 457, in the carboxyl-terminal tail. Arch Biochem Biophys.

340: 376-382.

14. Kennedy ME, Limbird LE. 1994. Palmitoylation of the alpha 2A-adrenergic

receptor. Analysis of the sequence requirements for and the dynamic properties of alpha

2A-adrenergic receptor palmitoylation. J Biol Chem. 269: 31915-31922.

15. Loisel TP, Adam L, Hebert TE, Bouvier M. 1996. Agonist stimulation increases the

turnover rate of beta 2AR-bound palmitate and promotes receptor depalmitoylation.

Biochemistry. 35: 15923-15932.

16. Petaja-Repo UE, Hogue M, Leskela TT, Markkanen PMH, Tuusa JT, Bouvier M.

2006. Distinct Subcellular Localization for Constitutive and Agonist-modulated

Palmitoylation of the Human d Opioid Receptor. J Biol Chem. 281: 15780-15789.

17. Moffett S, Rousseau G, Lagacé M, Michel Bouvier. 2001. The palmitoylation state

of the b2-adrenergic receptor regulates the synergistic action of cyclic AMP-dependent

protein kinase and b-adrenergic receptor kinase involved in its phosphorylation and

desensitization. Journal of Neurochemistry. 76: 269-279.

18. Soskic V, Nyakatura E, Roos M, Muller-Esterl W, Godovac-Zimmermann J. 1999.

Correlations in palmitoylation and multiple phosphorylation of rat bradykinin B2

receptor in Chinese hamster ovary cells. J Biol Chem. 274: 8539-8545.

19. Ponimaskin E, Dumuis A, Gaven F, Barthet G, Heine M, Glebov K, Richter DW,

Oppermann M. 2005. Palmitoylation of the 5-Hydroxytryptamine4a receptor regulates

17

receptor phosphorylation, desensitization, and b-arrestin-mediated endocytosis. Mol

Pharmacol. 67: 1434-1443.

20. Mumby SM, Kleuss C, Gilman AG. 1994. Receptor regulation of G-protein

palmitoylation. Proc Natl Acad Sci U S A. 91: 2800-2804.

21. Degtyarev MY, Spiegel AM, Jones TL. 1993. Increased palmitoylation of the Gs

protein alpha subunit after activation by the beta-adrenergic receptor or cholera toxin. J

Biol Chem. 268: 23769-23772.

22. Gurdal H, Seasholtz TM, Wang HY, Brown RD, Johnson MD, Friedman E. 1997.

Role of G alpha q or G alpha o proteins in alpha 1-adrenoceptor subtype-mediated

responses in Fischer 344 rat aorta. Mol Pharmacol. 52: 1064-1070.

23. Bhamre S, Wang HY, Friedman E. 1998. Serotonin-mediated palmitoylation and

depalmitoylation of G alpha proteins in rat brain cortical membranes. J Pharmacol Exp

Ther. 286: 1482-1489.

24. Chen CA, Manning DR. 2000. Regulation of galpha i palmitoylation by activation

of the 5-hydroxytryptamine-1A receptor. J Biol Chem. 275: 23516-23522.

25. Duncan JA, Gilman AG. 1998. A cytoplasmic acyl-protein thioesterase that

removes palmitate from G protein a subunits and p21RAS. J Biol Chem. 273: 15830-

15837.

26. Duncan JA, Gilman AG. 1996. Autoacylation of G protein a subunits. J Biol Chem.

271: 23594-23600.

27. Veit M, Sachs K, Heckelmann M, Maretzki D, Hofmann KP, Schmidt MFG. 1998.

Palmitoylation of rhodopsin with S-protein acyltransferase: enzyme catalyzed reaction

versus autocatalytic acylation. Biochim Biophys Acta 1394: 90-98.

18

28. Veit M. 2000. Palmitoylation of the 25-kDa synaptosomal protein (SNAP-25) in

vitro occurs in the absence of an enzyme, but is stimulated by binding to syntaxin.

Biochem J. 345 145-151.

29. Corvi MM, Soltys C-LM, Berthiaume LG. 2001. Regulation of Mitochondrial

Carbamoyl-phosphate Synthetase 1 Activity by Active Site Fatty Acylation. J Biol

Chem. 276: 45704-45712.

30. Kümmel D, Heinemann U, Veit M. 2006. Unique self-palmitoylation activity of the

transport protein particle component Bet3: A mechanism required for protein stability.

Proc Natl Acad Sci U S A. 103: 12701-12706.

31. Bélanger C, Ansanay H, Qanbar R, Bouvier M. 2001. Primary sequence

requirements for S-acylation of b2-adrenergic receptor peptides. FEBS Letters. 499: 59-

64.

32. Bañó MC, Jackson CS, Magee AI. 1998. Pseudo-enzymatic S-acylation of a

myristoylated yes protein tyrosine kinase peptide in vitro may reflect non-enzymatic S-

acylation in vivo. Biochem J. 330 723-731.

33. Lobo S, Greentree WK, Linder ME, Deschenes RJ. 2002. Identification of a Ras

palmitoyltransferase in Saccharomyces cerevisiae. J Biol Chem. 277: 41268-41273.

34. Roth AF, Feng Y, Chen L, Davis NG. 2002. The yeast DHHC cysteine-rich domain

protein Akr1p is a palmitoyl transferase. J Cell Biol. 159: 23-28.

35. Roth AF, Wan J, Bailey AO, Sun B, Kuchar JA, Green WN, Phinney BS, Yates III

JR, Davis NG. 2006. Global analysis of protein palmitoylation in yeast. Cell. 125: 1003-

1013.

36. Fukata M, Fukata Y, Adesnik H, Nicoll RA, Bredt DS. 2004. Identification of PSD-

95 palmitoylating enzymes. Neuron. 44: 987-996.

19

37. Mitchell DA, Vasudevan A, Linder ME, Deschenes RJ. 2006. Protein

palmitoylation by a family of DHHC protein S-acyltransferases. J Lipid Res. 47: 1118-

1127.

38. Tsutsumi R, Fukata Y, Fukata M. 2008. Discovery of protein-palmitoylating

enzymes. Pflugers Arch.

39. Swarthout JT, Lobo S, Farh L, Croke MR, Greentree WK, Deschenes RJ, Linder

ME. 2005. DHHC9 and GCP16 constitute a human protein fatty acyltransferase with

specificity for H- and N-Ras. J Biol Chem. 280: 31141-31148.

40. Fernandez-Hernando C, Fukata M, Bernatchez PN, Fukata Y, Lin MI, Bredt DS,

Sessa WC. 2006. Identification of Golgi-localized acyl transferases that palmitoylate

and regulate endothelial nitric oxide synthase. J Cell Biol. 174: 369-377.

41. Huang K, Yanai A, Kang R, Arstikaitis P, Singaraja RR, Metzler M, Mullard A,

Haigh B, Gauthier-Campbell C, Gutekunst CA, Hayden MR, El-Husseini A. 2004.

Huntingtin-interacting protein HIP14 is a palmitoyl transferase involved in

palmitoylation and trafficking of multiple neuronal proteins. Neuron. 44: 977-986.

42. Yanai A, Huang K, Kang R, Singaraja RR, Arstikaitis P, Gan L, Orban PC, Mullard

A, Cowan CM, Raymond LA, Drisdel RC, Green WN, Ravikumar B, Rubinsztein DC,

El-Husseini A, Hayden MR. 2006. Palmitoylation of huntingtin by HIP14 is essential

for its trafficking and function. Nat Neurosci. 9: 824-831.

43. Keller CA, Yuan X, Panzanelli P, Martin ML, Alldred M, Sassoe-Pognetto M,

Luscher B. 2004. The gamma2 subunit of GABA(A) receptors is a substrate for

palmitoylation by GODZ. J Neurosci. 24: 5881-5891.

44. Miura GI, Treisman JE. 2006. Lipid Modification of Secreted Signaling Proteins.

Cell Cycle. 5: 1184-1188.

20

45. Hofmann K. 2000. A superfamily of membrane-bound O-acyltransferases with

implications for Wnt signaling. Trends Biochem Sci. 25: 111-112.

46. Buglino JA, Resh MD. 2008. Hhat Is a palmitoylacyltransferase with specificity for

N-palmitoylation of Sonic Hedgehog. J Biol Chem. 283: 22076-22088.

47. Miura GI, Buglino J, Alvarado D, Lemmon MA, Resh MD, Treisman JE. 2006.

Palmitoylation of the EGFR ligand Spitz by Rasp increases Spitz activity by restricting

its diffusion. Dev Cell. 10: 167-176.

48. Zhai L, Chaturvedi D, Cumberledge S. 2004. Drosophila Wnt-1 undergoes a

hydrophobic modification and is targeted to lipid rafts, a process that requires

porcupine. J Biol Chem. 279: 33220-33227.

49. Yang J, Brown MS, Liang G, Grishin NV, Goldstein JL. 2008. Identification of the

acyltransferase that octanoylates Ghrelin, an appetite-stimulating peptide hormone. Cell.

132: 387-396.

50. Veit M. 2004. The human SNARE protein Ykt6 mediates its own palmitoylation at

C-terminal cysteine residues. Biochem J. 384: 233-237.

51. Dietrich LE, Gurezka R, Veit M, Ungermann C. 2004. The SNARE Ykt6 mediates

protein palmitoylation during an early stage of homotypic vacuole fusion. EMBO J. 23:

45-53.

52. Smotrys JE, Schoenfish MJ, Stutz MA, Linder ME. 2005. The vacuolar DHHC-

CRD protein Pfa3p is a protein acyltransferase for Vac8p. J Cell Biol. 170: 1091-1099.

53. Meiringer CTA, Auffarth K, Hou H, Ungermann C. 2008. Depalmitoylation of

Ykt6 prevents its entry into the multivesicular body pathway. Traffic. 9: 1510 - 1521.

54. Sugimoto H, Hayashi H, Yamashita S. 1996. Purification, cDNA Cloning, and

Regulation of Lysophospholipase from Rat Liver. J Biol Chem. 271: 7705-7711.

21

55. Toyoda T, Sugimoto H, Yamashita S. 1999. Sequence, expression in Escherichia

coli, and characterization of lysophospholipase II. Biochim Biophys Acta. 1437: 182-

193.

56. Duncan JA, Gilman AG. 2002. Characterization of Saccharomyces cerevisiae acyl-

protein thioesterase 1, the enzyme responsible for G protein a subunit deacylation in

vivo. J Biol Chem. 277: 31740-31752.

57. Flaumenhaft R, Rozenvayn N, Feng D, Dvorak AM. 2007. SNAP-23 and syntaxin-

2 localize to the extracellular surface of the platelet plasma membrane. Blood. 110:

1492-1501.

58. Veit M, Schmidt MFG. 2001. Enzymatic depalmitoylation of viral glycoproteins

with acyl-protein thioesterase 1 in vitro. Virology. 288: 89-95.

59. Sim DS, Dilks JR, Flaumenhaft R. 2007. Platelets possess and require an active

protein palmitoylation pathway for agonist-mediated activation and in vivo thrombus

formation. Arterioscler Thromb Vasc Biol. 27: 1478-1485.

60. Makita N, Sato J, Rondard P, Fukamachi H, Yuasa Y, Aldred MA, Hashimoto M,

Fujita T, Iiri T. 2007. Human Gsa mutant causes pseudohypoparathyroidism type

Ia/neonatal diarrhea, a potential cell-specific role of the palmitoylation cycle.

Proceedings of the National Academy of Sciences. 104: 17424-17429.

61. Robinson LJ, Michel T. 1995. Mutagenesis of palmitoylation sites in endothelial

nitric oxide synthase identifies a novel motif for dual acylation and subcellular

targeting. Proc Natl Acad Sci U S A. 92: 11776-11780.

62. Degtyarev M, Spiegel A, Jones T. 1994. Palmitoylation of a G protein a i subunit

requires membrane localization not myristoylation. J Biol Chem. 269: 30898-30903.

63. Hancock JF, Magee AI, Childs JE, Marshall CJ. 1989. All ras proteins are

polyisoprenylated but only some are palmitoylated. Cell. 57: 1167-1177.

22

64. Pallavi B, Nagaraj R. 2003. Palmitoylated peptides from the cysteine-rich domain

of SNAP-23 cause membrane fusion depending on peptide length, position of cysteines,

and extent of palmitoylation. J Biol Chem. 278: 12737-12744.

65. Vogel K, Roche PA. 1999. SNAP-23 and SNAP-25 are palmitoylated in vivo.

Biochem Biophys Res Commun. 258: 407-410.

66. Rose JK, Adams GA, Gallione CJ. 1984. The presence of cysteine in the

cytoplasmic domain of the vesicular stomatitis virus glycoprotein is required for

palmitate addition. Proc Natl Acad Sci U S A. 81: 2050-2054.

67. Veit M, Kretzschmar E, Kuroda K, Garten W, Schmidt MF, Klenk HD, Rott R.

1991. Site-specific mutagenesis identifies three cysteine residues in the cytoplasmic tail

as acylation sites of influenza virus hemagglutinin. J Virol. 65: 2491-2500.

68. Dietzen DJ, Hastings WR, Lublin DM. 1995. Caveolin is palmitoylated on multiple

cysteine residues. J Biol Chem. 270: 6838-6842.

69. Veit M, Reverey H, Schmidt MF. 1996. Cytoplasmic tail length influences fatty

acid selection for acylation of viral glycoproteins. Biochem J. 318 163-172.

70. Devedjiev Y, Dauter Z, Kuznetsov SR, Jones TL, Derewenda ZS. 2000. Crystal

structure of the human acyl protein thioesterase I from a single X-ray data set to 1.5 A.

Structure. 8: 1137-1146.

71. Biel M, Deck P, Giannis A, Waldmann H. 2006. Synthesis and evaluation of acyl

protein thioesterase 1 (APT1) inhibitors. Chemistry. 12: 4121-4143.

72. Gregory SG, Barlow KF, McLay KE, Kaul R, Swarbreck D, Dunham A, Scott CE,

Howe KL, Woodfine K, Spencer CCA, Jones MC, Gillson C, Searle S, Zhou Y,

Kokocinski F, McDonald L, Evans R, Phillips K, Atkinson A, Cooper R, Jones C, Hall

RE, Andrews TD, Lloyd C, Ainscough R, Almeida JP, Ambrose KD, Anderson F,

Andrew RW, Ashwell RIS, Aubin K, Babbage AK, Bagguley CL, Bailey J, Beasley H,

23

Bethel G, Bird CP, Bray-Allen S, Brown JY, Brown AJ, Buckley D, Burton J, Bye J,

Carder C, Chapman JC, Clark SY, Clarke G, Clee C, Cobley V, Collier RE, Corby N,

Coville GJ, Davies J, Deadman R, Dunn M, Earthrowl M, Ellington AG, Errington H,

Frankish A, Frankland J, French L, Garner P, Garnett J, Gay L, Ghori MRJ, Gibson R,

Gilby LM, Gillett W, Glithero RJ, Grafham DV, Griffiths C, Griffiths-Jones S, Grocock

R, Hammond S, Harrison ESI, Hart E, Haugen E, Heath PD, Holmes S, Holt K,

Howden PJ, Hunt AR, Hunt SE, Hunter G, Isherwood J, James R, Johnson C, Johnson

D, Joy A, Kay M, Kershaw JK, Kibukawa M, Kimberley AM, King A, Knights AJ, Lad

H, Laird G, Lawlor S, Leongamornlert DA, Lloyd DM, Loveland J, Lovell J, Lush MJ,

Lyne R, Martin S, Mashreghi-Mohammadi M, Matthews L, Matthews NSW, McLaren

S, Milne S, Mistry S, Moore MJF, Nickerson T, O'Dell CN, Oliver K, Palmeiri A,

Palmer SA, Parker A, Patel D, Pearce AV, Peck AI, Pelan S, Phelps K, Phillimore BJ,

Plumb R, Rajan J, Raymond C, Rouse G, Saenphimmachak C, Sehra HK, Sheridan E,

Shownkeen R, Sims S, Skuce CD, Smith M, Steward C, Subramanian S, Sycamore N,

Tracey A, Tromans A, Van Helmond Z, Wall M, Wallis JM, White S, Whitehead SL,

Wilkinson JE, Willey DL, Williams H, Wilming L, Wray PW, Wu Z, Coulson A,

Vaudin M, Sulston JE, Durbin R, Hubbard T, Wooster R, Dunham I, Carter NP,

McVean G, Ross MT, Harrow J, Olson MV, Beck S, Rogers J, Bentley DR. 2006. The

DNA sequence and biological annotation of human chromosome 1. Nature. 441: 315-

321.

73. Camp L, Hofmann S. 1993. Purification and properties of a palmitoyl-protein

thioesterase that cleaves palmitate from H-Ras. J Biol Chem. 268: 22566-22574.

74. Camp L, Verkruyse L, Afendis S, Slaughter C, Hofmann S. 1994. Molecular

cloning and expression of palmitoyl-protein thioesterase. J Biol Chem. 269: 23212-

23219.

24

75. Verkruyse LA, Hofmann SL. 1996. Lysosomal targeting of palmitoyl-protein

thioesterase. J Biol Chem. 271: 15831-15836.

76. Vesa J, Hellsten E, Verkruyse LA, Camp LA, Rapola J, Santavuori P, Hofmann SL,

Peltonen L. 1995. Mutations in the palmitoyl protein thioesterase gene causing infantile

neuronal ceroid lipofuscinosis. Nature. 376: 584-587.

77. Lu JY, Verkruyse LA, Hofmann SL. 1996. Lipid thioesters derived from acylated

proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect

in lymphoblasts by recombinant palmitoyl-protein thioesterase. Proc Natl Acad Sci U S

A. 93: 10046-10050.

78. Hellsten E, Vesa J, Olkkonen VM, Jalanko A, Peltonen L. 1996. Human palmitoyl

protein thioesterase: evidence for lysosomal targeting of the enzyme and disturbed

cellular routing in infantile neuronal ceroid lipofuscinosis. Embo J. 15: 5240-5245.

79. Soyombo AA, Hofmann SL. 1997. Molecular cloning and expression of palmitoyl-

protein thioesterase 2 (PPT2), a homolog of lysosomal palmitoyl-protein thioesterase

with a distinct substrate specificity. J Biol Chem. 272: 27456-27463.

80. MacDonald ME, Ambrose CM, Duyao MP, Myers RH, Lin C, Srinidhi L, Barnes

G, Taylor SA, James M, Groot N, MacFarlane H, Jenkins B, Anderson MA, Wexler

NS, Gusella JF, Bates GP, Baxendale S, Hummerich H, Kirby S, North M, Youngman

S, Mott R, Zehetner G, Sedlacek Z, Poustka A, Frischauf A-M, Lehrach H, Buckler AJ,

Church D, Doucette-Stamm L, O'Donovan MC, Riba-Ramirez L, Shah M, Stanton VP,

Strobel SA, Draths KM, Wales JL, Dervan P, Housman DE, Altherr M, Shiang R,

Thompson L, Fielder T, Wasmuth JJ, Tagle D, Valdes J, Elmer L, Allard M, Castilla L,

Swaroop M, Blanchard K, Collins FS, Snell R, Holloway T, Gillespie K, Datson N,

Shaw D, Harper PS. 1993. A novel gene containing a trinucleotide repeat that is

expanded and unstable on Huntington's disease chromosomes. Cell. 72: 971-983.

25

81. Karnoub AE, Weinberg RA. 2008. Ras oncogenes: split personalities. Nat Rev Mol

Cell Biol. 9: 517-531.

82. Sousa SF, Fernandes PA, Ramos MJ. 2008. Farnesyltransferase inhibitors: a

detailed chemical view on an elusive biological problem. Curr Med Chem. 15: 1478-

1492.

83. Caron JM, Herwood M. 2007. Vinblastine, a chemotherapeutic drug, inhibits

palmitoylation of tubulin in human leukemic lymphocytes. Chemotherapy. 53: 51-58.

84. Wolff J, Zambito A, Britto P, Knipling L. 2000. Autopalmitoylation of tubulin

Protein Sci. 9: 1357-1364.

85. Cantrell DA, Smith KA. 1984. The interleukin-2 T-cell system: a new cell growth

model. Science. 224: 1312-1316.

Figure legend

Figure 1: Dynamic S-acylation of Lck.

A: The S-acylation (represented by thick grey lines) of Lck at two cysteines near an N-

terminal myristoylation (represented by thin black line). A protein acyl transferase

(PAT) mediates the addition of palmitate from palmitoyl coenzyme A (PalCoA)

whereas a thioesterase (TE) catalyzes the hydrolysis of palmitic acid (PalCOOH) from

Lck. B: Accumulation of proteins with phosphorylated tyrosines in lysates from human

blast T cells exposed to 1 mM pervanadate (PV) for the times indicated, analyzed by

phosphotyrosine (PY) western blotting. C: Lck immunoprecipitated from human T

lymphoblast cells pulsed with 3H-palmitate, and chased for the indicated times.

Pervanadate (PV) treatment causes increased activation of Lck, seen as increasing

26

amounts of active, serine-phosphorylated Lck (p60 SP) and also increases the palmitate

turnover on Lck compared to control cells not treated with pervanadate (ctrl).

Method: T lymphoblasts were a kind gift of Dr. Julian Ng and Dr. Doreen Cantrell at

CRUK-LRI. They were prepared from outdated blood bags and maintained for up to 2

weeks in RPMI 1640 medium containing 10% foetal calf serum and Interleukin 2

(20ng/ml) at 50-200 x 104 cells/ml [85]. Cells were quiesced by removal of IL2 for 2-3

days, spun down and resuspended in labelling medium containing 5mM pyruvate, then

labelled with 3H palmitic acid (30-60Ci/mmol, approx. 200 µCi/ml) and incubated at

37C for 45 min, then spun down and resuspended in chase medium (labelling medium

containing 80µM palmitic acid, approx. 20 fold excess over tritiated palmitic acid, with

or without 1 mM PV). This was followed by rapid washing with ice-cold PBS, lysis,

immunoprecipitation , separation by SDS-PAGE and fluorography to detect 3H-

palmitate labelled bands.

Acknowledgements

We thank Julian Ng and Doreen Cantrell at Cancer Research UK-London Research

Institute for the kind gift of human T lymphoblasts. Work in the Magee laboratory is

supported by the UK Medical Research Council and Biotechnology and Biological

Sciences Research Council.

27