11 RTK/RAS/MAP kinase pathway|Tulane

overview

• Receptor Tyrosine Kinase:
  • structure
  • mechanism of activation
• Adaptor proteins/RAS:
  • mechanism of activation
• MAP Kinase pathway:
  • Signaling cascade
  • mechanism of activation

Kinase

• Kinase: Phosphorylate protein to active/inactive cell signal pathways.

• Phosphorylate: Ser, Thr and/or Tyr residues

• Kinase: conserved and has 11 subdomains.
  Conserved K to γ phosphate from ATP, can not be substitute by Arg.

Conserved kinase catalytic domain: 11 subdomains

Piedaldism

Piebaldism is a rare autosomal dominant disorder of melanocyte development characterized by a congenital white forelock and multiple symmetrical hypopigmented or depigmented skin.
KIT heterozygous mutation

An autosomal dominant disorder of melanolcyte development characterized by a congenital white foreloc k and multiple symmetrical hypopigmented or depigmented skin.

KIT heterozygous mutation.

Similar mutation induced into mouse could cause similar symptom.

• Several common cell-surface receptors and signal transduction pathways
• Overview of signal transduction pathways triggered by receptors that activate protein tyrosine kinases.

Structure and dimerization of RTK

Dimerization could either happend in cis and trans.

• Different modes of ligand-mediated dimerization
• Dimerization of FGF family receptors is aided by heparan sulfate (polysaccharide)
• Dimerization of Receptor Tyrosine Kinases
  1. When not activated, RTKs are monomeric (almost all).
  2. Binding of ligand to the extracellular domain causes dimerization (also called cross-linking) of two RTK molecules. This can be accomplished by:
  • The ligand is a monomer and causes a conformational change in the receptor that exposes a binding domain in the RTK that promotes its dimerization (EGF).
  • The ligand is a homo- or heterodimer that thus automatically dimerizes the receptor (PDGF, NGF).
  • The receptor already exists as a dimer (insulin).
  • The ligand is clustered together by binding to extracellular sulfate proteoglycans (FGFs).
  3. The RTK undergoes cis or trans autophosphorylation on tyrosine
     residues. This further stimulates the kinase activity of the RTK.
• Dimerization of EGF family receptors (HER)
• Different mode of dimerization of EGF family receptors
  • In 25% of breast cancers, HER2 gene is amplified and cancer cells overexpress HER2. What might be the effects of HER2 overexpression?
  • Increase in HER2 expression on the cell surface will make the cell more sensitive to signaling by EGF family ligands. Why?

Do the ligands activate same cellular response?

• Different ligands induce different biological responses
  via inducing phosphorylations on different Tyr residues

Membrane-bound ligand and bidirectional signaling

1. Ephrins are “repulsive” signals that stop neurons from growing into the incorrect areas of the brain.
2. Ephrins are unusual ligands in that many of them are transmembrane proteins.
3. Even though ephrins are monomeric, they are clustered in the plasma membrane of the cell that presents them. This allows for the cross-linking of their receptors, the Eph receptors.
4. Ephrins and Eph receptors carry out bidirectional signaling: when ephrin binds to the Eph receptor, the ephrin undergoes conformational changes that promotes a signal into the cell that presented the ephrin.

Monomeric RTK kinase domain is in an inactive state

• Activation of EGF receptor by EGF results in the formation of an asymmetric kinase domain dimer.
• Different mode of activation of EGF family receptors
• Different ways to inhibit RTK
• Dominant negative Receptor Tyrosine Kinase
• RTK signaling is downregulated by endocytosis
• The PDGF Receptor Signaling Complex
  1. The PDGF Receptor Signaling Complex The PDGF receptor is phosphorylated on several tyrosines (5 are shown here)
  2. Different phosphorylated tyrosine residues activate different signaling pathways

Adaptor proteins and RAS Switch

• The compound eye of Drosophila melanogaster.
  Sevenless mutants fail to produce R7 receptors
  Sevenless encodes a receptor tyrosine kinase
• Genetic studies reveal that activation of Ras induces development of R7 photoreceptors in the Drosophila eye.
• Receptor tyrosine kinases (RTKs)
  • Two different classes of proteins associate with phosphotyrosine residues in RTKs.
  1. Adapter or Docking proteins: These proteins couple the activated receptor to other signaling molecules but have no intrinsic signaling properties themselves.
  2. Enzymes involved in signaling pathways, GEFs and GAPs

Adaptor proteins provide additional docking sites for downstream signaling molecules

Surface model of an SH2 domain bound to a phosphotyrosine-containing peptide.

• Activation of Ras following ligand binding to receptor tyrosine kinases (RTKs) or cytokine receptors.
• RAS is a GTPase that can be turned on and off
  • Ras promotes proliferation and differentiation pathways activated by RTKs.
  • Dominant-negative forms of Ras block these two pathways. Conversely, hyperactive forms of Ras promote proliferation without any extracellular signal.
  • Ras is a major mediator of cancer. ~30% of human cancers have hyperactive forms of Ras.
• Structures of Ras bound to GDP, Sos protein, and GTP.
  • There are 3 different RAS proteins H-RAS, K-RAS and N-RAS They are all oncogenes

MAP kinase pathway

RTK (inactive) → bind → RTK dimer → GRB2 SOS → RAS(Inactivate) → RAS(activate) → RAF(MAPKK) → MEK(MAPKK) → MARK (ERK) → Activation of Transcription

Active RAS activates the MAP kinase pathway

• Induction of gene transcription by MAP kinase
• Outline of the yeast MAP kinase pathways, illustrating similarities and difference in architecture
• Different scaffold proteins activate different MAP kinases
• RTK activates other signaling pathways
• Recruitment and activation of protein kinase B (PKB) in PI-3 kinase pathways

Signaling Networks

1. Signal diversity: most receptors activate multiple intracellular signaling pathways. This is one mechanism that allows a single receptor to have multiple effects on a cell.
2. Cross-talk: most signaling pathways contain points where they can be regulated by other signaling pathways. This enables one signaling pathway to “branch out” and affect other target proteins.
3. Redundancy: many pathways are activated by more than one receptor. Thus, different signal molecules can have similar effects on a cell.
4. Signal amplification: multiple steps in an intracellular signaling pathway allows for the signal to be amplified along the way. Thus, very small amounts of a ligand can have dramatic effects.

Kinases identified or implicated as the driver of cancer

Pancreas: Insulin and regulation of glucose

• Pancreas is made up of exocrine and endocrine cells
• Insulin and regulation of glucose

Insulin stimulates glucose uptake via small GTPases, and PI3K and AKT signaling pathway

• Multiple signal transduction pathways interact to regulate adipocyte differentiation