M6L2 - Phosphorylation in Signaling Pathways

RBC Production

• 2 million made per sec

• Cells develop in bone marrow and circulate for 4 months

• They’re digested and recycled by macrophages (WBC)

How are RBC Replaced

• When progenitor cells stop dividing 

• They start to differentiate instead 

• The signal for maturation is Epo

  • Signaling cytokine protein (erythropoietin)

How is erythropoietin expression regulated

• by an O2 binding transcription factor in our kidney

• It goes into circulatory systems where only the erythrocyte progenitor cells carry the right receptor

What’s the receptor for Epo

• Erythropoietin receptor (EpoR)

• It’s a cytokine receptor bc erythropoietin is a cytokine 

• It’s linked to the JAK-STAT STP

• It’s activation inhibits cell death

Components of JAK-STAT Pathway

• Signal: Epo

• Receptor: EpoR

• Intracellular STP: JAK-STAT

EpoR activation Mechanism

• It’s inactive as a monomeric single-pass transmembrane protein

• When Epo is available, its signal interacts with 2 EpoR receptors 

• This initiates dimerization

What are the 3 functional domains of the EpoR

1. Cytosolic domain

2. Transmembrane alpha-helix domain

3. extracellular domain

Autophosphorylation of JAK Kinases

• Each EpoR has a JAK kinase in its cytosolic domain

• It’s unphosphorylated state is inactive with weak kinase activity

• When Epo binds, it leads to dimerization of EpoR

• The 2 JAK kinases becoming closer allow the neighbour to be phosphorylated to activate it 

Phosphorylation Targets of JAK kinases

• Many

• Includes tyrosine residues on intracellular domain of EpoR

• JAK kinase is a tyrosine kinase 

  • Only tyrosine residues are phosphorylated 

Intracellular Events Following EpoR activation

• The phosphorylated docking sites can now bind with STAT transcription factors

• They then dimerize to become activated 

How does STAT become phosphorylized

• STAT has a domain (SH2) that specifically recognizes phosphorylated tyrosine residues 

• They accumulate on the EpoR docking sites to be proximal to JAK kinase 

• STAT becomes the target of phosphorylation by JAK kinase 

• It’s phosphorylization allows STAT dimerization 

• This changes the conformation to unmask a nuclear localization sequence 

• It goes through nuclear pores to activate transcription of target genes

How does STAT recognize phosphorylated tyrosine residues on EpoR

• SH2 domain can re-localize proteins (like sending it to the nuclear pore when active) 

  • It’s a domain ONLY for protein-protein interaction

• SH2 can link together proteins in a pathway 

• SH2 binding pocket fits perfectly with target peptide 

• Target Sequence: Pro-Asn-pTyr- Glu-Glu-Ile

  Pro

• It binds with high affinity to this

• Binds with low affinity when tyrosine is unphosphorylated 

• The binding is reversible

Different Domains with Different protein-protein interactions

• They all link together 2 proteins

• SH2 / PTB / 14-3-3: Bind with phosphorylated tyrosine but not unphosphorylated 

  • Allows reversibility

• PDZ: Bind hydrophobic residues at C-terminus 

  • Not reversible

• SH3 / WW: Bind proline-rich domains 

  • Not reversible 

What STAT protein is erythrogenesis (RBC Production) associated with and what gene is coded for by its activation

• The activation of the STAT5 transcription factor

• Many genes are regulated by this activated STAT5 needed for differentiation of progenitor cells 

• Ex. of a gene is Bcl-XL protein

  • It inhibits apoptosis 

  • Allows progenitor cells to persist and differentiate 

What happens when you produce too many RBC (when signal wont turn off bc mutation or smth)

• Results in elevated hematocrit

• Increases blood viscosity which can block narrow capillaries 

• This can result in stroke / heart-attack

RTK extracellular domain

Ligand-binding domain of RTKs; binds growth factors like EGF

RTK transmembrane domain

Single-pass α-helix that spans membrane

RTK cytoplasmic domain

Contains intrinsic tyrosine kinase + activation loop + docking tyrosines

Ligand-induced dimerization

Ligand binds two RTK monomers → stabilizes dimer formation

RTK autophosphorylation

Kinase domains phosphorylate activation loop + docking tyrosines after dimerization

Activation loop phosphorylation effect

Increases kinase activity + creates phosphotyrosine docking sites

RTK docking sites

Phosphotyrosines that recruit SH2/PTB-containing proteins

Adaptor protein

Protein with multiple interaction domains that links RTKs to downstream effectors

Scaffold protein

Larger adaptor organizing several pathway proteins for efficiency + specificity

GRB2 structure

One SH2 domain + two SH3 domains

GRB2 SH2 function

Binds phosphotyrosines on activated RTKs; phosphorylation-dependent

GRB2 SH3 function

Binds proline-rich motifs (e.g., SOS); phosphorylation-independent

SH3–proline interaction

SH3 cleft recognizes proline-rich sequences via structural complementarity

Ras definition

Small monomeric GTPase molecular switch

Ras-GTP state

ON; switch regions pulled inward; can bind effectors

Ras-GDP state

OFF; gamma phosphate gone → switches relax; cannot bind effectors

Intrinsic GTPase activity

Ras slowly hydrolyzes GTP → GDP; required for OFF state

GDP–GTP exchange

GDP dissociates (low affinity); high intracellular GTP binds → Ras turns ON

GEF function

Promotes GDP release; activates Ras (SOS is Ras-GEF)

GAP function

Accelerates Ras GTP hydrolysis ~100×; inactivates Ras (NF1 is Ras-GAP)

GDI function

Prevents GDP dissociation; keeps Ras OFF

Ras cellular clock

Duration of Ras-GTP determined by GAP activity; controls signal length

SOS recruitment mechanism

GRB2 binds RTK via SH2; GRB2 SH3 binds SOS → brings SOS to membrane to activate Ras

NF1 role

Ras-GAP that shortens Ras-ON time; loss causes excessive Ras signaling

Three Ras conformations

Ras-GDP (inactive)

Full cascade order

RTK → GRB2 → SOS → Ras → Raf → MEK → MAPK

Raf definition

MAPKKK; serine/threonine kinase activated by Ras

14-3-3 binding to Raf

Keeps Raf inhibited; Ras binding releases 14-3-3

MEK definition

MAPKK; dual-specificity kinase (phosphorylates Thr and Tyr on MAPK)

MAPK/ERK activation mechanism

MEK dual-phosphorylates MAPK → MAPK dimerizes and becomes active

MAPK dimer effect

Dimerization reveals ATP + substrate binding pockets; enables nuclear entry

MAPK cytoplasmic target

p90RSK kinase

p90RSK nuclear effect

Phosphorylates SRF transcription factor

MAPK nuclear target

TCF transcription factor

TCF + SRF role

Bind SRE enhancer → recruit RNA polymerase → increase transcription

SRE

Serum Response Element; upstream enhancer of immediate early genes

c-fos

Immediate early gene encoding transcription factor promoting cell cycle entry

MAPK pathway outcomes

Cell division, differentiation, survival/apoptosis, metabolic changes depending on context

Signal amplification

Each kinase phosphorylates many substrates → millions of MAPK molecules from one ligand

Hormone sensitivity

RTK pathways respond to nanomolar (10⁻⁹ M) ligand concentrations

Experiment: anti-Ras antibody

Blocking Ras prevents EGF-induced division → Ras is required downstream of RTK

Experiment: Ras-D mutant

Constitutively active Ras drives division without EGF → Ras is downstream and sufficient

Experimental conclusion

Ras functions downstream of RTK and is essential for the pathway

Constitutively active Ras mutation

GTPase-impaired mutant locked in ON state; common in cancers

Her2 (ERBB2) mutation

RTK amplified/mutant → ligand-independent dimerization → persistent signaling → breast cancer

NF1 loss-of-function

Loss of Ras-GAP → prolonged Ras-GTP → neurofibromatosis type 1

Autophosphorylation purpose

Activates kinase activity + creates docking sites for SH2/PTB proteins

SH2 domain

Binds phosphotyrosine motifs; phosphorylation-dependent

SH3 domain

Binds proline-rich motifs; phosphorylation-independent

Activation loop function

Regulates access to ATP/substrate pockets; phosphorylation opens kinase

MEK dual-specificity

Phosphorylates MAPK on Thr + Tyr

RTK pathway full flowchart

Ligand → RTK dimer → autophosphorylation → GRB2 → SOS → Ras-GTP → Raf → MEK → MAPK → nuclear transcription

Immediate early genes

Genes rapidly induced (e.g., c-fos) driving later transcription for cell cycle