1. Mitogen-Activated Protein Kinase Pathway

2. Mitogen- a compound that encourages a cell to commence division, triggering mitosis Cell division requires the regulation of specific genes (including migration of cell-division specific proteins to key areas of the cell, manipulation of the cell cycle, and at various stages, growth of cell; ie: DNA synthesis, accumulation of energy for division) The Mitogen-Activated Protein Kinase Pathway has evolved to regulate numerous transcriptional events in practically every eukaryotic organism. THE MAPK Pathway controls everything from cell growth, to cell division, to inducing mating in haploid yeast.

3. Basic MAPK Pathway has the following Mitogen Receptor G-protein (GTPase) MAPKKKMAPKK/MEKMAPK/ERK Transcription factor DNA sequence Cell Membrane Nucleus

4. Some MAPK Pathways are activated by Receptor Tyrosine Kinases (RTK), like the ones in growth factor pathways in humans. RTK G-protein MAPKKK MAPKK MAPK In between kinases Txn factors Growth factor

5. However, yeast lack receptor tyrosine kinases (but not tyrosine kinases), and so the yeast mating pathway uses a G- protein Coupled Receptor (GPCR)

6. Mitogen GPCR Heterotrimeric G-protein Rho-like G-protein MAPKKK MAPKK MAPKs Txn Factors Scaffold Protein Mitogen: a or alpha factor G-protein Coupled Receptor (GPCR): 7- transmembrane domain receptor that, upon binding of ligand (mitogen), develops an intra-cellular conformational change, activating the hydrolytic activity of the coupled G- protein (in this case G- alpha) G-alpha: one of three G-proteins in the heterotrimeric g-protein that is coupled to the GPCR. G-alpha is a GTPase, meaning it hydrolizes GTP into GDP + P. In its active form, G-alpha is bound to GTP, and in this active form it sequesters and inactivates G- beta/gamma. When the mitogen binds the GPCR, the conformational change in the GPCR allows G-alpha to utilize its GTPase ability. It hydrolizes its GTP into GDP. G-alpha + GDP can no longer bind G-beta/gamma, which is now free to act on the other components of the pathway.

7. Explanation of the MAPKKK, MAPKK, MAPK portion of the pathway. MAPKKK MAPKK MAPKs THE MAPK pathway is unusual in that there are multiple members between the receptor and transcription factors. If the purpose of the mitogen is to activate certain transcription factors, why use so many sequential members? Why not just have one kinase that can activate all the necessary transcription factors? Answers: 1.More members means more points for regulation. 2.Combinations of feedback and cooperative binding allow for ultra-sensitivity and switch like behavior in the pathway. 3.Allows for more elaborate, well-regulated cross-talk between different MAPK pathways in the cell.

8. What’s ultra-sensitivity and switch-like behavior? A quick explanation of Michaelis-Menton behavior, cooperativity, and Hill’s Co-efficient. Simple Michaelis-Menton dynamics predicts the manner in which a protein-ligand interaction will behavior under varying concentrations. In general, a simple Michaelian behavior occurs as the image to the right, where only one protein-substrate collision is necessary: However, if many collisions are necessary, each step limits the reaction rate just a little, so the behavior looks more like this (blue lines, the lower the blue line, the more steps in the pathway): Note: Negative feedback has a somewhat similar behavior, except by a different mechanism, and pathway activity tends to not only rise more slowly, but eventually fall.

9. What’s ultra-sensitivity and switch-like behavior? A quick explanation of Michaelis-Menton behavior, cooperativity, and Hill’s Co-efficient. There are many collisions necessary for signal transduction in the MAPK Pathway. For example, MAPKKK must be phosphorylated on two residues, in turn phosphorylating two serine residues on MAPKK, which in turn must phosphorylate a tyrosine and a threonine residue on MAPK. Each phosphorylation is a separate event, and the next step in the pathway cannot occur without both residues in each kinase being phosphorylated. That makes for six sequential collisions, not including the steps before MAPKKK and after MAPK. Therefore, at low to moderate mitogen concentrations, the MAPK pathway tends to be stable at low rates, and is therefore desensitized to the mitogen.

10. Cooperativity/Cooperative binding: a special case of allosteric binding in which binding of a ligand at one site increases the proteins affinity for a ligand at another site. An example of this is binding of the first oxygen molecule to hemoglobin, which increases hemoglobins affinity for the next oxygen molecule. What’s ultra-sensitivity and switch-like behavior? A quick explanation of Michaelis-Menton behavior, cooperativity, and Hill’s Co-efficient. Cooperative binding exists in the MAPK Pathway in the form of a scaffold protein. The scaffold recruits the three MAP Kinases in the pathway, bring them closely into juxtaposition with each other. This increases the likelihood of their interacting with each other. The effect is positive cooperativity. The effect of this scaffold is that it increases the affinity of these three kinases for each other, despite the fact that six collisions (reactions) are necessary. So if MAPKKK is partially active (one phosphate), the reaction won’t continue. But if it is fully activated (two phosphates) it can activate MAPKK a little easier, because MAPKK is closer (two activations are needed again, but these activations are a little easier). The effect of this cooperative binding is a sudden jump in the reaction rate when a threshold level is hit. Hill’s coefficient provides a quantitative method of measuring binding cooperativity. A coefficient greater than 1 means positive cooperativity, while less than 1 means negative cooperativity. The larger the coefficient the steeper the jump in the reactions (a taller switch behavior). Switch behavior

11. Summary: A combination of negative feedback at the beginning, multiple requisite sequential reaction steps, cooperative binding, and positive control at the end, gives the MAPK pathway its distinctive, non- Michaelian, but switch-like behavior. A threshold level of mitogen is needed in order for the pathway to be able to induce a specific transcriptional response.