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