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

Signaling: An overview Receptor Cell Membrane This slide can be used to review the basic mechanisms of signal transduction, including common mechanisms of activation and inhibition such as binding, recruitment to substrate, and phosphorylation. Relay Molecules = signaling proteins Output : effectors Slide adapted from NESI 2012

The Receptor Tyrosine Kinase (RTK) – MAP Kinase pathway Signal RTK Plasma membrane Ras Ras Sos (Ras GEF) (G protein) Grb2 (adapter) GTP GDP MAPKKK This is the core of the RTK-MAPK signaling pathway. All signaling molecules in the core MAPK signaling pathway are positive regulators of their downstream targets. Each component of the pathway sequentially activates the next, as follows: 1. Signaling is typically initiated when ligand binds to the extracellular ligand-binding domain of two RTK sub-units, bringing them together so that the RTKs are close enough to phosphorylate each other on tyrosine residues. 2. Grb2 is an adaptor protein that binds both to phosphotyrosines on the RTK and to Sos, a guanine nucleotide exchange factor (GEF). 3. Once Sos is brought to the membrane, it causes the small G protein Ras to dissociate from GDP and bind to GTP, thereby activating Ras. 4. G proteins, such as Ras, are described as a molecular switches because they are self-inactivating. G proteins contain a GTPase domain that will eventually hydrolyze the bound GTP to GDP, deactivating themselves. They are therefore prone to gain-of-function mutations, because any loss-of-function mutation in the GTPase domain will cause the G protein to be constitutively active once it is turned on.. 5. GTP-bound Ras will bind to MAPKKK, activating it. Activated MAPKKK phosphorylates MAPKK. 6. Phosphorylation of MAPKK activates it, causing it to phosphorylate MAPK. 7. A significant proportion of phosphorylated, activated MAPK enters the nucleus, where it phosphorylates transcription factors, modulating gene expression. The activation of the three kinases, MAPKKK, MAPKK and MAPK is an example of a phosphorylation cascade. P MAPKK P MAPK

Optional Clicker Questions: 1. In the MAP kinase pathway, phosphorylation serves to: Turn off the pathway Inhibit the next component in the pathway Activate the next component in the pathway Prevent further phosphorylation of downstream components. Correct answer: C This question checks that the students understand that, in the MAPK cascade, phosphorylation is activating. Since this point is central to understanding the case study, it is an opportunity to go back over the logic of the pathway if a significant proportion of the students miss the question.

Optional Clicker Questions: 2. G proteins are described as molecular switches because: G proteins can be turned on and off by Sos. G proteins can activate or inhibit downstream components. G proteins can activate different downstream components depending on whether they are bound to GDP or GTP. G proteins have inherent GTPase activity and will turn themselves off by hydrolyzing GTP to GDP. Correct answer: D This question checks that the students understand that G proteins deactivation is like a timer switch and will turn off on its own after a period of time. This is an opportunity to go back over the logic of the pathway if a significant proportion of the students miss the question.

Optional Clicker Questions: 3. If a MAPKKK is lost due to homozygous mutation in the MAPKKK gene: The pathway will function normally. MAPK can still be activated if enough ligand is present. MAPK cannot be activated regardless of the presence of ligand. The pathway will be always active. Correct answer: C This question checks that the students have an abstract understanding of the logic of the pathway; each activation is dependent on the previous.

The Receptor Tyrosine Kinase (RTK) – cell survival pathway BAD PI3 Kinase RTK Signal Plasma membrane PIP3 P PDK1 Bcl2 Apoptosis Akt PIP3 This is the core of the RTK-cell survival signaling pathway. Activation of the RTK-survival pathway is required to promote cell survival (prevent apoptosis), through inhibition of BAD, an inhibitor of Bcl2. The signal is transduced as follows: 1. Signaling is initiated when ligand binds to two RTK sub-units, and the RTKs phosphorylate each other on tyrosine residues. 2. Phosphoinositde 3-kinase (PI3K) binds, directly or indirectly, to phosphorylated RTK, bringing it in proximity to its substrate. 3. PI3K phosphorylates the membrane lipid phosphatidylinositol 4,5-bisphosphate (PIP2), which becomes PIP3. 4. PIP3 recruits PIP3-dependent kinase PDK1 to the membrane. 5. PIP3 also recruits Akt to the membrane, where it is phosphorylated by PDK1. Once activated, Akt diffuses away. 6. Akt is a kinase with many downstream targets, including the protein BAD. Phosphorylation inactivates BAD, preventing it from binding to Bcl2. 7. Unbound Bcl2 is able to inhibit apoptosis (by binding to factors that promote release of cytochrome c from the mitochondria).

Optional Clicker Questions: 4. Akt….. prevents apoptosis (promotes cell survival) by inhibiting an inhibitor of Bcl2. is a kinase that activates Bcl2 by phosphorylation. promotes apoptosis but is inhibited by PDK1 phosphorylation. is a phosphatase, and inhibits apoptosis by dephosphorylating PDK1 targets. Correct answer: A If many students miss this question, this is a good point at which to go over the general principle that inhibition of an inhibitor results in activation. The students will need to be able to understand this logic when they do the take-home assignment on the beta-catenin signaling pathway.

Optional Clicker Questions: 5. If all Akt activity is lost due to homozygous mutation in the AKT gene: The pathway will function normally to inhibit apoptosis. The pathway can still be activated if enough ligand is present. The pathway can never be activated regardless of the presence of ligand, and the cell will undergo apoptosis. The pathway will be always active, and the cell will not undergo apoptosis even under appropriate conditions (the absence of the ligand). Correct answer: C This question asks the students to apply the logic assessed in the previous question to a manipulation of the pathway. If Akt is no longer present to phosphorylate Bad, Bad will sequester Bcl2, resulting in apoptosis due to loss of Bcl2’s pro-survival activity.

Example: SCF and Kit activate MITF in melanoblasts RAF (MAPKKK) MEK (MAPKK) ERK (MAPK) Sos (Ras GEF) Grb2 (adapter) Kit SCF Plasma membrane (G protein) Ras GTP GDP MITF CBP Example: SCF and Kit activate MITF in melanoblasts This slide accompanies the interleaving exercise on the importance of differential gene expression.

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

Case Study: Tiger Syndrome P Raf (MAPKKK) MEK (MAPKK) ERK (MAPK) Sos (Ras GEF) Grb2 (adapter) RTK Signal Plasma membrane (G protein) Ras GTP GDP Case Study: Tiger Syndrome

Case Study: Tiger Syndrome How do you think the T266K mutation in Sos might alter its activity? b) How does this affect the upstream components of the MAP kinase pathway? And the downstream components?

Case Study: Tiger Syndrome P Raf (MAPKKK) MEK (MAPKK) ERK (MAPK) Sos (Ras GEF) Grb2 (adapter) RTK Signal Plasma membrane (G protein) Ras GTP GDP Case Study: Tiger Syndrome

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?

✕ Drug Case Study: Tiger Syndrome Signal Plasma membrane RTK Ras Raf (MAPKKK) MEK (MAPKK) ERK (MAPK) Sos (Ras GEF) Grb2 (adapter) RTK Signal Plasma membrane (G protein) Ras GTP GDP Case Study: Tiger Syndrome ✕ Drug