BIOLOGY 2B03 Chapter Notes - Chapter 6: Receptor Tyrosine Kinase, Hematopoietic Stem Cell, Sh2 Domain

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BIOLOGY 2B03 - Module 6 Lecture II
Cytokine Receptors and the JAK-STAT Pathway
About 2 million new erythrocytes (red blood cells) are produced per second in human adults
● Develop in the bone marrow → circulate about 4 months → digested by macrophage
â—Ź Then the RBC are replaced when mitotically-proliferating stem cells/progenitor cells stop
dividing and instead differentiate
â—Ź Erythropoietin (Epo) : cytokine; the signal for maturation of the erythrocytes
â—‹ Its expression is regulated by an oxygen-binding transcription factor in kidney cells
â—‹ Then Epo is released into the circulatory system
â—‹ Only progenitor cells that carry an Epo Receptor (EpoR)
â—Ź EpoR: cytokine receptor that is linked to the JAK-STT signal transduction pathway
â—Ź Cellular responses: inhibition of cell death, changes in pattern of gene expression, and
differentiation
Components of the Pathway
Following the principles of the cell signalling…
â—Ź Signal: Epo
â—‹ Inactive as a monomeric, single-pass transmembrane protein
â—Ź Receptor: EpoR
â—‹ Initiates dimerization
○ The erythropoietin signal interacts with two EpoR’s
â—Ź Intracellular signal transduction pathway: JAK-STAT
â—Ź Responding change in target cell behaviour
Receptor Dimerization and Autophosphorylation
EpoR: has three functional domains
â—Ź The cytosolic domain
â—‹ Each EpoR is associated with JAP kinase
â—Ź The transmembrane domain
â—Ź The extracellular domain
A JAK kinase in the unphosphorylated state: inactive, weak kinase activity
● Dimerization of the receptors by Epo → two JAK kinases are closer together
● Sufficient to phosphorylate a neighbouring JAK kinase → autophosphorylation
â—‹ Phosphorylation of the activation lip on JAK kinases activates kinase activity
â—‹ Targets: tyrosine residues on the intracellular domain of the receptor
○ JAK kinase is specifically a tyrosine kinase → only tyrosine residues are
phosphorylated
Phosphorylation of Protein Docking Sites
Activation of the receptor → a cascade of intracellular events
The phosphorylated docking sites: available for protein-protein interactions
â—Ź Including binding of the STAT transcription factor
● SH2: STAT’s protein-protein interaction domain; specifically recognized phosphorylated
tyrosine residues
STAT Monomers: inactive but dimerization and activation depend upon phosphorylation
â—Ź STAT protein is proximal to the JAK kinase by accumulating on the receptor docking sites
â—Ź STAT itself is a target of phosphorylation by JAK kinase
● Phosphorylation of STAT → dimerization an changes conformation such that nuclear
localization sequence is unmasked
â—Ź Then the STAT dimer can be transported through the nuclear pore complex into the nucleus
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SH2 Protein Binding Domain - P-Tyr
SH2 domain: protein-protein interaction domain; essential to the function of the cytokine signalling
pathway
â—Ź No enzymatic function
â—Ź Allows a protein to bind to specific target substrates
â—Ź May re-localize a protein within the cytoplasm / link together protein in a pathway
The SH2 domain did not change but its target did?
â—Ź Either the docking site tyrosine was phosphorylated or unphosphorylated
Target Peptide Sequence: Pro-Asn-pTyr-Glu-Glu-Ile-Pro
â—Ź SH2 has high affinity to this sequence when tyrosine is phosphorylated (vice versa)
● Precise fit of P-tyrosine and isoleucine → allows for reversible binding of the SH2 domain
to the target peptide
Diverse Protein-Protein interaction Domains
All are responsible for linking together two proteins
â—Ź In some cases, binding is dependent upon reversible modifications to the target peptide
â—Ź Example) the SH2, PTB, and 14-3-3 domains bind to peptides containing phosphorylated
tyrosine with high affinity
â—‹ Allows protein-protein binding to be reversible
â—Ź Other domains bind to sequences that are not modified
â—‹ example)PDZ domains bind to hydrophobic residues at the C-terminus and SH3 and
WW domains bind proline-rich domains
â—‹ These binding would not be reversible
Target Genes of STAT Transcription Factor
STAT5: transcription factor; regulates many genes that are necessary for differentiation of mature red
blood cells
â—Ź Ex) Bcl-xL gene: codes for the Bcl-XL protein (inhibitor of apoptosis)
○ Inhibition of apoptosis → the erythroid progenitor cells persist and differentiate
White Bone Marrow: primary source of erythrogenesis
â—Ź Particularly evident during development when all red blood cells are formed in the fetal liver
â—Ź Provides visual assy for activation of the cytokine/JAK-STAT pathway
â—Ź Mouse lacks the EpoR: mouse is homozygous for a loss of function allele of the epoR gene
○ Liver is still intact but no bright red colouration → no red blood cells are being
made
Turning off the sinal: Short-Term Regulation
Continual activation of the cytokine pathway → overproduction of red blood cells
● Increase the viscosity of the blood → blockage of the narrow capillaries of the circulatory
system → stroke or heart attack
● “Epo Doping”: exogenous erythropoietin to increase haematocrit → carry more oxygen
→ increases endurance
Two mechanism for disabling the pathway
â—Ź Reversing phosphorylation
â—‹ A phosphatase dephosphorylates modified amino acid residues
○ SHP1 phosphatase: has two SH2 domains → dock at the same docking sites as
STAT transcription factor
○ Localization of SHP1 → dephosphorylation of JAK kinase → signalling pathway
turned off
â—‹ Short term because removal of SHP1 allows fast re-activation of JAK kinase
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Turning off the Singal: Long-Term Regulation
SOCS protein: able to bind to the phosphorylated docking sites via an SH2 domain
â—Ź Expressed in response to high oxygen levels in the body
● Multiple SOCS bound to docking site → blocking access to the sites by STAT
â—Ź Is an E3 ubiquitin ligase: targets JAK kinase for ubiquitination and degradation through the
proteasome
● Removal of JAK → inactivation of the signalling pathway
â—‹ Reactivation is very slow b/c of the expression of new JAK kinase proteins
Mutation in EpoR gene: created truncated versions of the EpoR → shorter docking sites
â—Ź decreased sensitivity to the negative regulators of the JAK-STAT pathway, SHP-1 and SOCS
â—Ź Result: higher haematocrit
â—Ź Long-term inactivation of the pathway: receptor recycling and signal release
â—‹ Many signalling pathways can be turned off when the receptor is internalized through
endocytosis and the ligand dissociates
○ Receptor is recycled back to the surface → Epo levels have gone down → receptor
won’t be reactivated
RTK and Ras
RTK is associated with Ras G-protein activation
â—Ź RTK: involved in cell differentiation cell survival or apoptosis, cell division and
proliferation, or changes in cell metabolism signalling pathway
â—‹ Example) Neural growth factor (NGF): able to rapidly induce differentiation of
neural cells including the formation of extensive axon growth
â—‹ Other hormones: platelet derived growth factor (PDGF), epidermal growth factor
(EGF) and insulin
● In all cases the hormone will interact with a transmembrane receptor tyrosine kinases →
activate the intrinsic kinase activity of that receptor
Components of Pathway
It differs from the cytokine receptors in structure
â—Ź Extracellular signal binding domain
â—Ź single -pass transmembrane domain
â—Ź Intrinsic kinase activity on the cytoplasmic domain of the protein
● Ligand binding→ dimerization of the receptor and phosphorylation of the kinase domain
â—Ź The intracellular pathway is much longer than in the cytokine
â—Ź Involves in activation of an intracellular, membrane-anchored protein (Ras)
Regulation of Ras: requires proteins that link it to the activated RTK
â—Ź Adaptor proteins: GRB2
â—Ź Ras effectors: GEF and GAP
Ras G-protein activation → kinase cascade that culminates in activation of MAP kinase
â—Ź MAP kinase: modulates cell behaviour by phosphorylating transcription factor and changing
patterns of gene expression
RTK Activation: Signal Binding and Receptor Dimerization
RTK activation is similar to activation of cytokine receptors
● Ligand binding → dimerization of the transmembrane receptors
● Example) EGF: binds to each of the receptors → change the conformation of the
extracellular domain → inducing dimerization
RTK Activation: Phosphorylation of Docking Sites
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Document Summary

About 2 million new erythrocytes (red blood cells) are produced per second in human adults. Develop in the bone marrow circulate about 4 months digested by macrophage. Then the rbc are replaced when mitotically-proliferating stem cells/progenitor cells stop dividing and instead differentiate. Erythropoietin (epo) : cytokine; the signal for maturation of the erythrocytes. Its expression is regulated by an oxygen-binding transcription factor in kidney cells. Then epo is released into the circulatory system. Only progenitor cells that carry an epo receptor (epor) Epor: cytokine receptor that is linked to the jak-stt signal transduction pathway. Cellular responses: inhibition of cell death, changes in pattern of gene expression, and differentiation. The erythropoietin signal interacts with two epor"s. Each epor is associated with jap kinase. A jak kinase in the unphosphorylated state: inactive, weak kinase activity. Dimerization of the receptors by epo two jak kinases are closer together. Sufficient to phosphorylate a neighbouring jak kinase autophosphorylation.

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