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
Cell Signalling Biology www.cellsignallingbiology.org 2007 Cell Signalling Biology www.cellsignallingbiology.org 2007
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
Cell Signalling Biology www.cellsignallingbiology.org 2007 Cell Signalling Biology www.cellsignallingbiology.org 2007
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
Cell Signalling Biology www.cellsignallingbiology.org 2007 Cell Signalling Biology www.cellsignallingbiology.org 2007
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
Cell Signalling Biology www.cellsignallingbiology.org 2007 Cell Signalling Biology www.cellsignallingbiology.org 2007
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
Cell Signalling Biology www.cellsignallingbiology.org 2007 Cell Signalling Biology www.cellsignallingbiology.org 2007
Module 1: Figure cytokines Module 1: Figure cytokines
Cell Signalling Biology www.cellsignallingbiology.org 2007 Cell Signalling Biology www.cellsignallingbiology.org 2007