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Enzyme linked plasma membrane surface receptors Tyr kinases. 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.

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slide3

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)

Insulin

Insulin like growth factor -

IGF

Interleukins

Nerve growth factor -

NGF

(neurons)

Platelet derived growth factor -

(PDGF)

factor coding gene: c-sis

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

slide5

A Single Signal Can Activate Several Pathways

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)

Binding of epidermal growth factor to its receptor activates a MAP kinase pathway via ras

DAG

ras

PLC

raf

PKC

ras

GTP

MAP kinase cascade

Multiple effects e.g.

Differentiation

Proliferation

p rotein k inases activated through receptors
protein kinases activated through receptors
  • AGC group: PKA, PKG, PKC, Rac, G-protein kinases
  • CaMK group: kinases regulated by Ca2+/CaM
  • CMGC group: cyclin-dependent kinases ERK, MAP, Casein kinase
  • PTK group: conventional protein tyrosine kinases Src, Abl, Fak, PDGF, IR

5. OPK: Other Protein Kinases

appr. 2000 protein kinases in human genome

slide7

Protein tyrosine kinases (PTK)

  • Plasma membrane receptors - receptor tyrosine kinases
  • dimer formation, autophosphorylation

2. Cytoplasmic tyrosine kinases

src is a non receptor tyrosine kinase
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
Tyrosine kinases can be cytosolic or integral membrane receptors

.

Substrate

Single Membrane spanning

Hydrophobic domain.

No membrane-spanning domain

slide10

Src homology domains

SH2

binding to Tyr P

SH3

binding to Pro rich regions

Grb2: SH2, SH3 domains – receptor –effector connection

Docking proteins: SH2 domain

IRS: insulin receptor substrate

receptor tyrosine kinases1
Receptor tyrosine kinases

The 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 kinases2
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
slide14

Module 1: Figure PDGFR activation

Cell Signalling Biology www.cellsignallingbiology.org 2007

slide15

Tyr-P docking sites on receptor protein or binding of SH2

proteins to plasma membrane

activation of PLC 

IP3

DAG

MAP kinase

activation of Ras

PI3 - kinase

PKB

(protein kinase B)

Survival

inhibition of apoptosis

increase in

glycogen synthesis

stimulation of

translation (mTor)

slide16

Functionsof 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
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
slide18

Module 1: Figure tyrosine kinase-linked receptors

Cell Signalling Biology www.cellsignallingbiology.org 2007

response of the insulin receptor kinase irk to ligand binding
Response of the insulin receptor kinase (IRK) to ligand binding
  • Heterotetramer (2a, 2b)
  • Insulin binding leads to change in structure (different from other RTKs)
  • Conformation change activates b-subunit TK activity
  • b subunit phosphorylates Tyr residues on cytoplasmic domains as well as downstream substrates (IRS)
three dimensional structures of the insulin receptor tyrosine kinase irk
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 p hosphorylated the irk activity trig g ers a number of signaling pathways
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

slide22

Module 2: Figure insulin receptor

Cell Signalling Biology www.cellsignallingbiology.org 2007

slide23

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 binding

protein -1c (SREBP-1c) liver

FAS (fatty acid synthase)

Acetyl CoA carboxylase

slide24

On-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

slide25

On-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.

g

Exchange of bound GDP for GTP

b

GTP

a

INACTIVE

GDP

GDP

a

a subunit dissociates from bg

GTP

ACTIVE

Pi

Active a subunit can interact with and activate the next step in the signalling pathway

a

a subunit GTPase activity GTP > GDP+Pi

GDP

a subunit reassociates with bg

slide26

p21ras

p21ras

p21ras

GDP

GDP

GTP

On-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

This causes exchange of bound GDP for GTP

Guanine nucleotide exchange factor interacts with ras

p21ras

Activated ras interacts with and activates the next component in the signalling pathway

On

GTP

GDP

ACTIVE

INACTIVE

Pi

Ras GTPase stimulated by association with GTPase-activating protein (GAP)

Off

Intrinsic GTPase activity hydrolyses GTP to GDP and Pi

rtks can activate the ras pathway of cellular signaling
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.
steps in the activation of ras by rtks
Steps in the activation of Ras by RTKs

Raf is a PK that triggers MAP-K pathway

Raf

SH2 binds RTK, SH3 binds SOS

c-fos, c-jun

Cell proliferation

Ras-GEF

slide30

Cascading Kinases

Binding of epidermal growth factor to its receptor activates ras

Ras activates the serine/threonine kinase raf

Erk-1 phosphorylates the transcription factor myc and activates transcription

ras

raf

raf

ras

GTP

GDP

ADP

ATP

Raf phosphorylates and activates the dual-specificity kinase Mek-1

P

Mek1

Mek1

Nucleus

ATP

P

Erk1

ATP

ADP

ADP

P

P

P

Erk1

Erk1

Mek-1 phosphorylates the serine/threonine kinase Erk-1 which migrates to the nucleus

slide31

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

slide32

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

slide33

Module 2: Figure JAK/STAT function

Cell Signalling Biology www.cellsignallingbiology.org 2007

slide34

P

Nucleus

A Simple Signalling System

INFg receptor

Interferon (IFN) g

Activated IFNg receptor recruits JAK kinase

Phosphorylation causes STATs to dimerise and migrate to nucleus…

…where they initiate transcription

ADP

ATP

JAK phosphorylates STAT monomer

STAT transcription factor

JAK tyrosine kinase

slide35

Module 2: Figure JAK/STAT heterogeneity

Cell Signalling Biology www.cellsignallingbiology.org 2007

slide36

Module 1: Figure cytokines

Cell Signalling Biology www.cellsignallingbiology.org 2007