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Lecture 9

Lecture 9 - tyrosine kinase.pdf

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University of Toronto St. George
Maurice Ringuette

Lecture 9 November 8, 20135:39 PM Non covalent interactions. We saw how small molecules interacted with proteins. Small molecules , they interacted through non covalent interactions. Changed the structure of the protein through downstream signaling. Today we center on two covalent modifications 1. Phosphorylation - forming covalent binds 2. Proteolysis - breaking covalent bonds There are 6 main classes. BIO230 Page 1 Here we have receptor tyrosine kinases. - Transmembrane proteins which are receptors for signals. - With tyrosine kinases on the cytoplasmic face - Two main classes. 60 encoded in human genome - some bind to secreted proteins. Proteins in the extracellular face diffusing around. Shown with the blue box. - Others bind cell surface proteins. They bind to different ligands on the EC surface. - There is a large variation if you look across. We can hypothesize that They will be binding to different ligands in extracellular space. - Look at everything they have in common. They all have a transmembrane domain. Can send signal from extracellular space to intracellular space into the cytoplasm. - If we look at the cytoplasmic tails of their proteins they all have a common domain, tyrosine kinase domain. - Different signals in the outside but feeding into the tyrosine kinase signal from the inside. There are whole number of these receptors. Table shows some. All of the details in the table is not critical The theme as we look across the information The name of the signaling protein and various growth factors. In response to the signaling there are stimulations of: Stimulate from self-growth Stimulation of proliferation So this is a common theme across the table signaling which promotes cell proliferation and cell growth. This is critical for development. For cells to build body. Critical for cancer as well. Misregulation of these pathways leads to cell overgrowth leading to cancer. BIO230 Page 2 Here is the basic mechanisms of any of the tyrosine receptors are activated. We have an extracellular ligand, and this is bivalent. Has two binding sites. Because it has two binding sites it dimerizes the receptor. This brings the kinase domains together and they phosphorylate each other.. This is called transautophosphorylation. Signal molecule in the outside is inducing the trans auto phosphorylation. Trans means across , one of the pair is phosphorylating the other one. And vice versa. Auto because same receptor. Two copies of the same receptor comes together and phosphorylate each other. Phosphorylation of one another increases their activity and then there will be phosphorylation of other targets along the cytoplasmic tail. There will be docking sites for assembling signal complexes. Inactive RTKs is not completely correct, because there is low activity there. When the ligands binds to them they have very low activity on their own, but when the ligand brings them together to their kinase target they pair and phosphorylate each other. Step 1: initial phosphorylation which involves low level of activity. First step increases the activity of the kinases domain. Which goes from low to high level of activity. With that high activity you get the phosphorylation of the additional sites to build the signaling complex. What's the organization of this complex and mechanism of signaling? Imagine a Scenario where you have a mutant tyrosine kinase receptor with a mutation in the kinase domain. one has the potential to phosphorylate, the other doesn’t. so the signal molecule comes in and diverse it. What would happen? Will this create a signal? A, B or C? A. There would be an inactive complex. We would think NO. but I think this is the answer based on his explanation. Ask from me. B. No response? C. Reduced response We have to think about signaling through copulation of molecules not just as a single pair, a cell does not have two copies of the receptor has only two copies on the surface that binds to the ligand and sends he signal in. there are 1000s of 100s of copies of the receptor on the surface of the cell. We should think about how it's actually functioning in the cell we have Imagine when there are like two copies of the cell and one is mutated and the other one is normal it would from a normal complex. But when to think that there are large populations of molecules that are conducting this activity. Not only as a single signal conductor. there is a thousands of copies it really depends on the ratio of how much of the mutant protein do you have vs. how much of the normal protein you would have. When you think about how it's actually functioning in the cell. Then that is what matters. BIO230 Page 3 If we do move inwards from the activated receptor you can see that when there is extra phosphorylation sites on the dimer it creates docking sites for other proteins. Downstream component sof the signaling pathway. This would create a relay of signals. One of the key domains that would recruits proteins to these receptors is an SH2 domain. SH2 domain or a PTB domain. And the key thing about these domains is that they have a binding site for phosphorylated tyrosine. In the previous slide it’s a phosphate specifically on tyrosine residue. Tyrosine kinase domain it will specifically add phosphate domains on the tyrosine residue and you have protein domains that have a binding site for the phosphorylated tyrosine so it only binds tyrosine when its phosphorylated and doesn’t bind when it's not. So its specifically gets recruited to this complex. They often have binding sites next to it as well which gives them some specificity. Phospho tyrosine is relatively simple residues binding sites next door that binds to a specific sequence of AA residues that can provide some extra specificity for the recruitment. Crystal structure of this. Important thing to remember is that these domains are like the knobs on top of Lego bricks so one protein can have multiple domains and they can come together in different combinations. BIO230 Page 4 We can here that the PDGF receptor is phosphorylation at multiple residues. These are phosphotyrosines. They recruit specific proteins with SH2 domains which is shown here in red. And they binds to these specific phosphotyrosines because it’s a slight different surroundings aa residues. So this SH2 binds to there…etc. There's slight differences in the surrounding AA residues. In addition to contain the SH2 domains the proteins can also contain other domains too. G protein coupled receptors acts through phospholipase C which cleaves the pep 2 and creates DAG and IP3. Q is: this occurred downstream of receptor tyrosine kinase signaling. It can. This protein has a phospholipase domain( another domain the protein) and the particular phospholipase domain has an SH3 domain, so it will response to phosphotyrosines signaling because it gets recruited to that receptor. But phospholipase C gets activated by GPCR and instead have a binding site for G protein. Rather than a binding site for phoshotyrosine. So this is a way of linking up different proteins to different signaling pathways. Additionally the domains present in the domain recruit adapters to recruit proteins that don’t have SH2 domains. So SH3 domain binds to polyrpoline, so that can recruit addiotnal proteins to the local complex. Eg: on how this pathway was found. This pathway was first identified in genetic streams of drosophila. Led to the development if drosophila eye. Its eye is a compound eye. There are 800 ommatidia covering the surface of the eye. Each of them are composed with 8 photoreceptor cells and 12 support cells. You have 800*8 are the photoreceptors crossing the entire surface of the drosophila eye. So the screen was based on the development of the 8 photoreceptor cells from a single epithelial sheet during development. The fact that there is a sequential differentiation of the photoreceptor cells. That is shown at the top where you Start by forming R8 cell.( we are going from 1-8 photoreceptor cells), it induce the formation of R5 and R2, R4 and R3 and R6 and R1 and then R8 signals to R7. This signaling to R7 , the screen revealed, was based on RTK signaling. We are talking about the Developmental pathway which needs R8 to tell to make R7. Special thing about R7 is that its specifically needed to detect UV light. Though as soon as R7 differentiate in response to the signal it expresses the proteins the fly needs to detect UV light. This is a nice way to set up a screen to know if the fly had set up the R7 cell or not. If it hasn’t formed the R7 cell it won't respond to UV, it has, then it has formed R7. Based on intensity to UV light BIO230 Page 5 Screen was formed to identify flies that failed to develop R7 which meant there was a failure in the signaling pathway. They exposed the cells to UV and watched if there was a response or not. When through the screen and one of the first mutants that was identified was seven less. In Drosophila the names of the genes expressed the mutant phenotype. It was called sevenless because it didn’t have receptor cell number 7. The gene that was mutated , was found as the gene that encoded the sevenless proteins which is a RTK. It was shown to the a receptor tyrosine kinase which was specifically expressed in R7 cells. Here is the RTk wihch was mutated in the first line and its expressed in the R7 and not in R8. R8 is on top. R7 is in bottom. In order to activate R 7 you need the protein sevenless. Logic is if you mutate sevenless you will be sevenless. If you mutate sevenless you will be missing R7. you need sevenless to get R7. R8 is communicating with sevenless. Subsequent mutation found was called bride of sevenless. BOSS. BOSS was shown to be the ligand for sevenless, and it was expressed on R8 cells. They Cloned the gene ,made antibodies for it and saw where it was expressed on R8, and observed that it acted as a ligand for the receptor. The ligand receptor pair, through the same screen was able to identify additional component downstream. You also need mutations boss or bride of sevenless to activate sevenless. You also need BIO230 Page 6 They were able to identify additional components downstream. Drk and Sos was identified. Drk is one of the adaptor proteins. You can imagine now that the activated RTK phosphorylate itself, so now it has the phsophotyrosine right there. SH2 domain of Drk binds to that. And because it has SH3 domains on it , it can recruit proteins identified as Sos. So Sos has a polyprolin region which binds to the Sh3 regions which were brought to the complex and then Sos is a GEF, and exchange factor for the small G protein Ras. Here we have GEF which turns on Ras and it then drives downstream signals. This was identified in the drosophila. This has a major implication in cancer development and growth in all animals. You also need Drk, and son of sevenless (Sos) , Drk is downstream if receptor kinase. Downstream if receptor kinase activate Sos. Sos is a GEF for Ras. So Ras in bound to GDP and its inactivated. So Sos helps to remove it and then activate it. Sevenless is a receptor…kinase. Drk is activated in the same scenario. SH2 domain binding to …same scenario. Make sure you know the domains. This is a really good review slide. They have shown you what happens in the R7. You see RAS everywhere. What is Ras? Ras BIO230 Page 7 Ras is the founding member of the Ras superfamily of G proteins. We have talked about the Rab protein and Rho proteins. All of these proteins are switches that can be switched on and off to control the pathway of the cell. Ras is for growth regulation. For the growth of cells. For the division of cells to growth of cells. RAS is a huge superfamily, RAS is a protein. Then there is all these other proteins in the RAS super protein. RAS is particularly interesting because 30% human cancers are RAS. If a molecular switch downstream of RTK. Read slide. It's in close proximity to RTK and can be brought in to proteins. They have Ras that cannot be turned on. Drives the proliferation of cancer. They have a form of Ras that cannot be turned off. RAS is a monomeric GTPase. BIO230 Page 8 What does Ras signal through? Ras activates a mitogen activated protein kinase module. MAP kinase module. When you say mitogen, that is the name for a secreted molecule that promotes cell growth é cell division. Mitogen is secreted, in the extracellular space, bound to the receptor tyrosine kinase and activates Ras. MAP KKK This pathway is given the name mitogen activated protein kinase because it acting downstream of the mitogen. If you look at the base of the pathway, then this is a serine threonine kinase. So it phosphorylate serines and threonines now. And at the base of the pathway it is MAPKK phosphorylating a whole range of proteins that is promoting cell growth and division, changes in cell behavior. Gene expression. Changes in other machinery that changes cell division and growth. MAP K That was the last map kinase. This is a relay of signals. So the molecule upstream is called MAPkk, this is another serine threonin kianse which phosphorylate MAPk. Then we have another MAPKk, we have MAPKKK at the top. So we have three kinases which phosphorylates one another in a relay. So Ras is linked upto MAPKKK and then MAPK is at the bottom that phosphrylates the target. This is an exmaple of relays. It is the same basic signal phosphorylation event which activates the downstream signal. Ras will activate a map kinase kinase kinase. This is going to take ATP and phosphorylate kinase kinase. Once MAP kinase kinase take ATP it's going to phosphorylate kinase. And then activate protein activity, changes in gene expression etc. MAP KKK can be called a MAC? Here we have one of these relays and the key thing is there is known 5 different map kinases cascades can function at the same time in a mammalian. There are at least 12 map kinases and 7 mapkk and 7mapkkk in the genome. When we look at this we can ask how is nonspecific cross talk controlled? 5 different relays, why don’t they criss cross their wires? We can look at yeast and get a really good example of how this works. We are talking about a different receptor at the top in both cases, and we can think of them as TRK as well. Think only about the M
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