Cell Signaling Pathways A Case Study Approach By

Cell Signaling Pathways – A Case Study Approach By L. Emtage, L. Bradbury, N. Coleman, D. Davenport, A. Dunning and J. Grew

Signaling: An overview Signal Receptor Cell Membrane Relay Molecules = signaling proteins Output : effectors Slide adapted from NESI 2012

Signal The Receptor Tyrosine Kinase (RTK) – MAP Kinase pathway RTK Plasma membrane Grb 2 Sos (Ras GEF) (adapter) Ras GTP MAPKKK P MAPK Ras GDP (G protein)

Optional Clicker Questions: 1. In the MAP kinase pathway, phosphorylation serves to: A. Turn off the pathway B. Inhibit the next component in the pathway C. Activate the next component in the pathway D. Prevent further phosphorylation of downstream components.

Optional Clicker Questions: 2. G proteins are described as molecular switches because: A. G proteins can be turned on and off by Sos. B. G proteins can activate or inhibit downstream components. C. G proteins can activate different downstream components depending on whether they are bound to GDP or GTP. D. G proteins have inherent GTPase activity and will turn themselves off by hydrolyzing GTP to GDP.

Optional Clicker Questions: 3. If a MAPKKK is lost due to homozygous mutation in the MAPKKK gene: A. The pathway will function normally. B. MAPK can still be activated if enough ligand is present. C. MAPK cannot be activated regardless of the presence of ligand. D. The pathway will be always active.

Signal The Receptor Tyrosine Kinase (RTK) – cell survival pathway RTK Plasma membrane PI 3 Kinase PIP 3 PDK 1 P PIP 3 P P P Akt P P BAD Apoptosis Bcl 2

Optional Clicker Questions: 4. Akt…. . A. prevents apoptosis (promotes cell survival) by inhibiting an inhibitor of Bcl 2. B. is a kinase that activates Bcl 2 by phosphorylation. C. promotes apoptosis but is inhibited by PDK 1 phosphorylation. D. is a phosphatase, and inhibits apoptosis by dephosphorylating PDK 1 targets.

Optional Clicker Questions: 5. If all Akt activity is lost due to homozygous mutation in the AKT gene: A. The pathway will function normally to inhibit apoptosis. B. The pathway can still be activated if enough ligand is present. C. The pathway can never be activated regardless of the presence of ligand, and the cell will undergo apoptosis. D. The pathway will be always active, and the cell will not undergo apoptosis even under appropriate conditions (the absence of the ligand).

Example: SCF and Kit activate MITF in melanoblasts SCF Plasma membrane Kit Grb 2 Sos (Ras GEF) (adapter) Ras GTP Ras GDP RAF (MAPKKK) P MEK (MAPKK) P ERK (MAPK) P MI CB TF P MI P TF (G protein)

Case Study: Tiger Syndrome Instructions: • Read the text carefully • Think-Collaborate-Share (work in groups of approx. 4) • Write or draw your group’s answer down (to be collected) • You have 20 minutes to complete the activity

Signal Case Study: Tiger Syndrome RTK Plasma membrane Grb 2 Sos (Ras GEF) (adapter) Ras GTP Ras (G protein) GDP Raf (MAPKKK) P MEK (MAPKK) P ERK (MAPK)

Case Study: Tiger Syndrome a) How do you think the T 266 K mutation in Sos might alter its activity? b) How does this affect the upstream components of the MAP kinase pathway? And the downstream components?

Signal Case Study: Tiger Syndrome RTK Plasma membrane Grb 2 Sos (Ras GEF) (adapter) Ras GTP Ras (G protein) GDP Raf (MAPKKK) P MEK (MAPKK) P ERK (MAPK)

Case Study: Tiger Syndrome c) The other five individuals do not have mutations in RAF or Sos. Which components of the pathway would you sequence next? Why?

Signal Case Study: Tiger Syndrome RTK Plasma membrane Grb 2 Sos (Ras GEF) (adapter) Drug Ras GTP ✕ (G protein) GDP Raf (MAPKKK) P MEK (MAPKK) P ERK (MAPK)