Enzyme linked plasma membrane surface receptors

Tyr kinases

(fibroblast, glia cells, keratinocyte, epithelial cells, endothelial cells, chondrocyte)

Growth factors - regulation of proliferation

Endothelial cell growth factor - ECGF

Epidermal growth factor - EGF

its receptor coding gene: c erb B

Fibroblast growth factor - FGH(endothelial cells, fibroblast, smooth muscle cell, granulosa cell, chondrocyte)

InsulinInsulin like growth factor - IGF

Nerve growth factor - NGFInterleukins

(neurons)

Platelet derived growth factor - (PDGF)factor coding gene: c-sis

Four intracellular pathways

cross talk

A Single Signal Can Activate Several PathwaysA Single Signal Can Activate Several Pathways

rasrafras

GTP

PLCDAG

PKC

Multiple effects e.g.Differentiation

Proliferation

MAP kinase cascade

Binding of epidermal growth factor to its receptor activates a MAP

kinase pathway via ras

Binding of epidermal growth factor to its receptor activates phospholipase C (PLC) leading to

production of diacylglycerol (DAG) and activation of protein kinase C (PKC)

protein kinases activated through receptors

1. AGC group: PKA, PKG, PKC, Rac, G-protein kinases

2. CaMK group: kinases regulated by Ca2+/CaM3. CMGC group: cyclin-dependent kinases ERK,

MAP, Casein kinase4. PTK group: conventional protein tyrosine

kinases Src, Abl, Fak, PDGF, IR5. OPK: Other Protein Kinases

appr. 2000 protein kinases in human genome

Protein tyrosine kinases (PTK)

1. Plasma membrane receptors - receptor tyrosine kinases dimer formation, autophosphorylation

2. Cytoplasmic tyrosine kinases

Src, is a non-receptor tyrosine kinase

Src, is the product of the first proto-oncogene to be characterized.

Src kinase -„eldest” Tyr kinase – SH: Src Homology domain

Tyrosine kinases can be cytosolic or integral membrane receptors

.

Single Membrane spanning Hydrophobic domain. No membrane-spanning domain

Substrate

Src homology domains

SH2 binding to Tyr P

SH3 binding to Pro rich regions

Grb2: SH2, SH3 domains – receptor –effector connectionDocking proteins: SH2 domain IRS: insulin receptor substrate

Receptor Tyrosine Kinases

Receptor tyrosine kinasesThe interaction of the external domain of a receptor tyrosine kinase with the ligand, often a growth factor, up-regulates the enzymatic activity of the intracellular catalytic domain, which causes tyrosine phosphorylation of cytoplasmic signaling molecules.

Receptor tyrosine kinases

General Relevance

• Amplification by downstream signaling elements greatly amplifies the effects of low levels of tyrosine phosphorylation that are most directly induced by extracellular triggers. Example: PLC and PI3K

• Activation of multiple kinases (kinase cascades) including ser/thr as

well as tyrosine kinases, is a frequent consequence of these early events. Example: MAP Kinase

Module 1: Figure PDGFR activation Module 1: Figure PDGFR activation

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Tyr-P docking sites on receptor protein or binding of SH2 proteins to plasma membrane

activation of PLC IP3

DAG

activation of Ras MAP kinase

PI3 - kinase

PKB (protein kinase B)

Survivalinhibition of apoptosis

increase inglycogen synthesis

stimulation oftranslation (mTor)

Functions of receptor tyrosine kinases:

•Growth control

•Cell-cell recognition

•Cell cycle control

•Immune responses

•Development

•Differentiation

Tyrosine Kinases, associated genes and proteins are implicated in

developmental defects and cancer• Excessive activation of receptor tyrosine kinases can lead

to uncontrolled growth and malignant transformation.

• Many defective or viral forms of tyrosine kinases and associated proteins are oncogenic:

• v-src

• abl

• erbB

Module 1: Figure tyrosine kinase-linked receptors Module 1: Figure tyrosine kinase-linked receptors

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Response of the insulin receptor kinase (IRK) to ligand binding

• Heterotetramer (2, 2)

• Insulin binding leads to change in structure (different from other RTKs)

• Conformation change activates -subunit TK activity

subunit phosphorylates Tyr residues on cytoplasmic domains as well as downstream substrates (IRS)

Three-dimensional structures of the insulin receptor tyrosine kinase (IRK)

IRK conformational change upon activation loop phosphorylation. The N-terminal lobe of IRK is colored white and the C-terminal lobe is colored dark grey. The activation loop (green) contains autophosphorylation sites Y1158, Y1162 and Y1163, and the catalytic loop (orange) contains the putative catalytic base, D1132. Also shown are the unbound/bound ATP analog and tyrosine-containing substrate peptide (pink). [Hubbard, EMBO J. 16, 5572 (1997)]

Once Tyr-phosphorylated, the IRK activity triggers a number of signaling pathways

• Phosphatidylinositol 3-hydroxy kinase, makes PIP2,PIP3

• Grb2, Sos, activates Ras

• Activation of PI-PLC

Sos: exchange protein

Module 2: Figure insulin receptor Module 2: Figure insulin receptor

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IR (insulin receptor)

IRS (insulin receptor substrate) (IRS1, IRS2)

IRS dependent phosphatidylinositol 3-kinase (PI3K)

PIP3 (PI – 3,4,5 trisphosphate)

aPKC (atypical protein kinase C) PKB/Akt

Sterol regulatory element bindingprotein -1c (SREBP-1c) liver

FAS (fatty acid synthase)Acetyl CoA carboxylase

On-Off SwitchesOn-Off Switches

The majority of signals are transient and the response should be proportionately transient too. If you switch the signal on, you need a way of switching it off again. For example, failure to switch off mitogenic signals is one way to induce a tumour.

So, what we are looking for are biochemical systems that are capable of rapidly switching between two states.

In many signalling systems the ‘on-off’ switch is operated by GTP-binding proteins and/or protein phosphorylation

On-Off Switches – GTP-Binding ProteinsOn-Off Switches – GTP-Binding Proteins

GTP-binding proteins come in two flavours, small monomeric GTP-binding proteins (e.g. p21ras) and heterotrimeric G proteins. The basic GTP/GDP binding cycle is the same in both cases.

GDP

GTP

Pi

GTP

GDPGDP

ACTIVE

INACTIVE

Exchange of bound GDP for GTP

Active subunit can interact with and

activate the next step in the signalling

pathway

subunit dissociates from

subunit GTPase activity GTP > GDP+Pi

subunit reassociates with

On-Off Switches – GTP-Binding ProteinsOn-Off Switches – GTP-Binding Proteins

Ras (p21ras) is a good example of this type of switch. Ras is a small (21 kDa) monomeric protein that binds GTP or GDP and has intrinsic GTPase activity

p21ras GDP p21rasGTP

p21ras

p21ras GDP

On

Off

GTP

Pi

GDP

ACTIVEINACTIVE

Guanine nucleotide exchange factor interacts with ras

This causes exchange of bound GDP for GTP

Intrinsic GTPase activity hydrolyses GTP to GDP and Pi

Ras GTPase stimulated by

association with GTPase-activating

protein (GAP)

Activated ras interacts with and activates the next component in the

signalling pathway

RTKs can activate the Ras pathway of cellular signaling

• Ras is a small G-protein (monomeric 21-kD)

• Mutant Ras proteins are unable to dissociate GTP, so they are stuck in the ON or proliferative state: ras (gene) mutations found in 30% of human cancers.

• Mutations in Ras-GAPs can lead to disease.

Unlike IRK, most RTKs are present as a monomer in the

resting cell membrane

Steps in the activation of Ras by RTKs

Ras-GEF

Raf

Raf is a PK that triggers MAP-K pathway

SH2 binds RTK, SH3 binds SOS

c-fos, c-junCell proliferation

Cascading KinasesCascading Kinases

ras rafraf

Mek1

Erk1

ras

Mek1

ATP

Erk1

GTP

GDP

Nucleus

P P

ATP

ADPErk1

ATP

ADP

ADP

P

P

P

Binding of epidermal growth factor to its

receptor activates ras

Ras activates the serine/threonine

kinase raf

Raf phosphorylates and activates the

dual-specificity kinase Mek-1

Mek-1 phosphorylates the serine/threonine kinase Erk-1 which

migrates to the nucleus

Erk-1 phosphorylates the transcription factor

myc and activates transcription

Signal transduction of cytokines 1.

protein signals (IL - 1 IL - 13)

paracrine, autocrine regulation

heterodimer cytokine receptors

gp130 - common constituent in several receptors

signal - receptor complex

binding of cytoplasmic tyrosine kinases to the complex (JAK etc.)

autophosphorylation (tyr kinases + receptor)

SH2 proteins

JAK: Janus kinase

Signal transduction of cytokines 2.

STAT proteins

transcription factors

phosphorylation at Tyr dimer formation

translocation into nucleus (Ser-P)

binding to specific enhancer elements

activation of specific genes

Module 2: Figure JAK/STAT function Module 2: Figure JAK/STAT function

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A Simple Signalling SystemA Simple Signalling System

Nucleus

P

ATP

ADP

Interferon (IFN)

INF receptor

JAK tyrosine kinaseSTAT transcription factor

Activated IFN receptor

recruits JAK kinase

JAK phosphorylates STAT monomer

Phosphorylation causes STATs to

dimerise and migrate to nucleus…

…where they initiate transcription

Module 2: Figure JAK/STAT heterogeneity Module 2: Figure JAK/STAT heterogeneity

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Module 1: Figure cytokines Module 1: Figure cytokines

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