SIGNAL TRANSDUCTION

A. OVERVIEW OF RECEPTORS AND SIGNALLING

RECEPTOR FAMILIES

MECHANISMS OF SIGNALLING BY RECEPTORS

AMPLIFICATION OF SIGNALS

KEY FUNCTION OF PHOSPHORYLATION

PROTEIN INTERACTION DOMAINS

Objectives of this lecture

• Recognize the different types of receptors and the mechanisms they use to signal into cells

• Understand the importance of signal amplification

• Understand the basic mechanisms of protein phosphorylation and the type of kinases

• Identify some of the key protein interaction domains that function in signalling pathways

• Be aware of applicability of these studies to virtually all disease processes (cancer is highlighted)

A Cascade of Signals from Membrane to Nucleus

Downward, Nature 2001

CYTOKINE PRODUCTION / HORMONE ACTION

Inducing Stimulus

Cytokine-producing cell

TargetGene Activation

Biological Effect

Target cell

Receptor PARACRINE

Distant CellCirculation

Nearby Cell

ENDOCRINE

AUTOCRINECytokinegene

CLASSIFICATION OF RECEPTORS IN FAMILIES

A. Receptors for Growth and Differentiation Factors- have associated enzyme activity

B. Serpentine Receptors- coupled to G proteins

C. Intracellular Receptors - bind hormone and act as transcription factors

D. Channel Forming Receptors- receptors for neurotransmitters

E. Immune System Receptors - T cell, B cell, Ig receptors

A. Receptors for Growth and Differentiation Factors

In general, tyrosine kinase activity is involved in receptor signalling (some serine/threonine kinase receptors, some guanylate cyclase-encoding)

Receptors with intrinsic tyrosine kinase domain:

- EGF, PDGF, FGF, SLF/c-kit have single subunits

- insulin, IGF-I have multiple subunits, α2β2

- hepatocyte growth factor (c-met receptor), αβ

Growth FactorReceptors with Tyrosine Kinase Domains Share

Common StructuralFeatures

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Figure 1 Receptor Tyrosine Kinase Families Human receptor tyrosine kinases (RTKs) contain 20 subfamilies, shown here schematically with the family members listed beneath each receptor. Structural domains in the extracellular regions, identified ...

Lemmon & Schlessinger, Cell 2010

DIMERIZATION

is a Key Concept

In Understanding The Signalling

Events TransmittedBy Growth Factor

ReceptorsSchlessinger, Cell 2000

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Lemmon & Schlessinger, Cell 2010

Models depicting various means by which extracellular domains allow for dimerization

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Models of intracellular domain kinase activation

Lemmon & Schlessinger, Cell 2010

Receptors having associated tyrosine kinase

Hemopoietin receptor family - includes receptors for interleukins and colony stimulating factors

-primarily found in hematopoietic cells

- intracellular associated tyrosine kinases (JAK’s) activated by ligand binding to receptor

IL-3, IL-5 and GM-CSF are Examples of Hemopoietin

Receptors That Share a Common Subunit

B. Serpentine receptors, or G-protein coupled receptors (GPCR’s)

7 transmembrane domains; extracellular domains are responsible for creating a ligand binding site

eg. epinephrine, muscarinic acetylcholine receptor, rhodopsin

coupled to G proteins via intracellular portion of receptor

Signalling: via G protein transducers

bind GTP in active state; hydrolyzed to GDP when inactive

amplification of signals

STRUCTURE OF A TYPICAL SERPENTINE RECEPTOR

C. Intracellular Receptors

translocated from cytosol to nucleus when bound with ligand; DNA binding and transcription activation domains

Signalling:

direct binding to DNA, in the presence of ligand, to activate transcription

Intracellular Receptors for Various Hormones have Conserved Structural Features

Inhibitoryprotein complex

Hormone binding site

DNA binding domain

Hormone

DNA binding site exposed

N C

N

CN

CN

C

N C

N C

DNA binding domain

Cortisol R

Estrogen R

Progesterone R

Vitamin D R

Thyroid hormone R

Retinoic Acid R

Transcription activation domain

D. Channel Forming Receptors

in neural and muscle tissue

eg.acetylcholine, dopamine, glycine, γ-aminobutyrate (GABA)

structures with 4 or 5 subunits that each have several transmembrane domains; subunits cluster to form a gated channel

Signalling:

function at nerve and muscle synapses to propagate an electrical signal by transport of ions

E. Immune system receptors

B and T cell receptors; consist of multiple subunits

associated tyrosine kinases activated to phosphorylate ITAM’s – Immune receptor Tyrosine-based Activation Motifs

ITAM's serve as docking sites for other tyrosine kinases that are activated and subsequently activate signalling pathways that involve a series of intermediate tyrosine phosphorylated adaptor proteins.

Signalling

pathways utilized are similar to those of growth factor receptor tyrosine kinases.

The T-Cell Receptor Recognizes Antigens Bound to MHC on Antigen Presenting Cells

Signal transduction is the means by which molecular responses are propagated within a cell

A cell senses its environment by way of signalling molecules (starting with receptors) and the resulting changes in molecular shapes or activities cause corresponding changes in cell behaviour

Signal transduction studies aim to explain (molecularly) all aspects of the behaviour of an individual cell, from its growth and division, to its differentiation into a more specialized cell type, and its death by apoptosis.

B. MECHANISMS OF SIGNAL TRANSDUCTION

G Protein Coupling to Serpentine

Receptor Results in GTP/GDP Exchange and Dissociation of the ‘Active’ G protein

Subunit

Signalling Events

Result in Enormous

Amplification of

Downstream

‘Messengers’

to Affect Many Targets

PHOSPHORYLATION

Protein kinases catalyze the transfer of the g-phosphate from nucleotide triphosphate (usually ATP) to a hydroxyl acceptor site on a protein

Kinase

Protein + NTP Protein-P + NDP

Phosphatase

Serine/Threonine Kinases; eg. cAMP-dependent protein kinase, Protein kinase C; MAP kinases

Tyrosine Kinases; eg. EGF, PDGF, Insulin receptors, src family of oncogenes, JAK family

Dual specificity kinases; eg. MEK’s

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Protein Phosphorylation Network

N-H

H-C-CH2-OH

O=C

N-H

H-C-CH2-O-P-O-

O=C O

O-

Serine Phospho-Serine

Phosphorylation causes a dramatic change in charge on

a protein

Function of Protein Phosphorylation

Addition of a highly charged phosphate group alters a protein's surface charge and its structure

Numerous mechanisms by which phosphorylation can alter the function of a protein (switches)

Tyrosine Phosphorylation - Results in formation of new sites of protein-protein interaction, mediated by SH2 or PTB domains

Ser/Thr Phosphorylation, acts primarily to modulate activity of protein/enzyme that gets phosphorylated, but may also result in altered protein binding

Phosphorylation in

Signal Transduction

Pawson & Scott,TIBS, 2005

Other Kinases

Lipid Kinases; e.g. PI Kinases can phosphorylate various positions on the inositol ring of the lipid Phosphatidylinositol

Phosphorylation of sugars, nucleotides and many other small molecules, all mediated by kinases (these are usually involved in metabolic pathways as opposed to signalling pathways)

“TURN-OFF SIGNALS”

For every signal transmitted into cells there must be a means of regulating, or turning off, the signal

In the case of phosphorylation, phosphatases play a key role in reversing the reaction.

Phosphatases include tyrosine, serine/threonine and lipid phosphatases.

In the case of G proteins, reversal is by breaking down the guanine nucleotide by GTPase activity

Degradation of ligand or its dissociation from receptor stops signalling at the receptor, although ‘downstream’ events may still proceed

Protein Interaction Domains

Many signalling pathways proceed via protein-protein interaction events

Several domains identified that serve as 'cassettes' of protein 3D structure.

In some cases, sequence homology is very weak, yet similar 3D structures have been demonstrated.

The primary functions are to alter activity of an enzyme, or to change the location of an enzyme so it is placed close to its substrate (e.g. enzymes acting on lipids translocated to the plasma membrane)

Protein Interaction Domains

Function of some domains depends on phosphorylation state; e.g. SH2 binding to phosphotyrosine

Some show constitutive binding; e.g. SH3 to poly-proline motifs

Others may bind specific second messengers to alter function of a protein, or its location in the cell; PH domains binding to lipids

Protein Modules and Docking Proteins

Schlessinger, Cell 2000

SH Domains

Src Homology - first noticed by comparison of src (the first oncogene) sequence with other proteins SH1 - tyrosine kinase domain SH2 - binds specific phosphotyrosines, with hydrophobic a.a.'s on C-term side of PY SH3 - binds polyproline motifs; binding constitutive PTB Domains

Phosphotyrosine Binding Domains Bind phosphotyrosine in a binding pocket, like SH2, but specificity is determined by residues on N-term. side of PY

Pleckstrin Homology (PH) Domains

First identified in Pleckstrin, a major protein kinase substrate first identified in platelets

Shown to mediate protein interactions in a few cases, but primarily protein binding to lipids (mainly various forms of phosphorylated phosphatidylinositols)

Ras(GDP)

P

GRB2(adaptor with both

SH2 and SH3)SH2 binds to P-Y

SOS

Guanine nucleotideexchange factor;Bound to SH3 of GRB2Via poly-proline

Ras(GTP)

raf

MEK

erk1/2

TranscriptionFactors

Phosphorylation

Phosphorylation(threonine/tyrosine)

Phosphorylation

GTP exhanges with GDPAssociationvia ras bindingdomain

p21ras to erk - a key signalling pathway

OUT

IN

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A Genetic vs Molecular Description of the Ras Pathway

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Extracellular Signal

TKR

P85/p110PI3K

4,5 3,4,5

3,45P’ase

PDK1 PKB308 473

?PDK2

NUCLEUSCYTOPLASM

The Basics of PI 3-kinase Signalling

PI 3-kinase phosphorylates PI(4,5)P2 in the plasma membrane

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Control of Cell survival by PI3K/PKB

Duronio, Biochem J, 2008

Many Signalling Proteins May Act as OncogenesWhen Mutated or Overexpressed

Nuclearproteins

Mycfos jun

PDGFEGF Growth FactorsM-CSF

Membrane-associated Tyrosine kinases

srcras proteins

GTP-bindingproteins

PDGF ReceptorEGF Receptor (erb B)M-CSF Receptor (fms)

Tyrosine kinaseReceptors

Cytoplasmic Tyrosine kinases

(fps/fes)(raf)

Cytoplasmic Ser/Thr kinases

Thyroid Hormone Receptor (erb B)

Steroid-typeGrowth factorReceptors

REFERENCES – Signal transduction (Duronio, lecture 1)

The Ins and Outs of Signalling. J. Downward. Nature 411: 759 - 762 (2001).

Kinome signaling through regulated protein-protein interactions in normal and cancer cells. T. Pawson and M. Kofler. Curr Opinion Cell Biol 21: 147-53 (2009).

Protein phosphorylation in signaling - 50 years and counting. T. Pawson and J. Scott. TIBS 30: 286-290 (2005).

Cell signaling by receptor tyrosine kinases. J. Schlessinger. Cell 103: 211 - 225 (2000).

Cell signaling by receptor tyrosine kinases. M.A. Lemmon and J. Schlessinger. Cell 141: 1117- 1134 (2010).

V. Duronio, The Life of a Cell – Apoptosis Regulation by the PI3K/PKB Pathway. Biochem J. 415: 333-344 (2008).

For further in depth study: STKE, Science website http://stke.sciencemag.org/

Mechanisms of Signalling by RTK’s

A. PKB Activation by Phosphorylation

B. PI3K activation by pY binding and localization to plasma membrane

C. PLCg activation by pY binding, phosphorylation and localization to membrane

Multiple Effectors Regulated by RTK’s