LECTURE TITLE: DATE: TIME: PROFESSOR: TRANSCRIBER: PHONE:

Receptor tyrosine kinase signaling: a moving target for therapeutic intervention November 19, 2009 The previous author made a 9:30-10:25 AM Todd Miller Di Fan (510)384-1467

Lecture Number: 82 Version: draft EMAIL:di.fan@hsc.stonybrook.edu

PDF le. I followed it as the lecture progressed. The order of the slides changed slightly but the content did not. Any questions, contact me, Nicholas Barasch, at 917-861-0855.

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Objectives:

*Recount the regulatory steps through which signals are propagated *To describe how the signaling events lead to specific cellular responses *Describe the relationship between pathway activity and various pathological conditions.

Background
Receptor tyrosine kinases were identified through the study of growth factors o Growth factors were found in the 1950s as factors necessary for the growth and proliferation of cells in culture-without it, cells would not divide and grow. o Receptors were cloned starting in the 1980s ! They were seen to have tyrosine kinase domains that closely resembled the kinase domain of Src Uncontrolled tyrosine kinase signaling can cause cancer o Evidence for point mutations in tyrosine kinases found in tumors

I. Architecture of receptor tyrosine kinases (RTKs)

Catalytic domains are the intracellular parts of the receptors that contain the tyrosine kinase activity o Have homology to other tyrosine kinases (such as Src) and all other protein kinases as well o All tyrosine kinase domains have the same structure-a N terminal to bind ATP and a C terminus to bind substrate o The kinase activity of RTKs is absolutely essential for their function ! Evidence: mutations involving the Lys residues conserved in the ATP binding sites of the RTKs would still allow the receptors to assemble at the cell surface and bind ligands, but they would be unable to transmit signals A single hydrophobic transmembrane !-helix attaches the receptor to the membrane o Hydrophobic sequence of about 25 amino acids or so o The helix passes through membrane only once (unlike GPCRs which thread through the membrane 7 times) Diverse extracellular domains of the receptors contain the ligand-binding sites o Some conserved structural elements are mixed and matched in the extracellular regions of the RTKs, which confers specificity for the different ligands ! The extracellular region allows us to group ligands into different families (see diagram on the next page for some of the different families and conserved structural elements) Quaternary structures of RTKs o Large, mostly monomeric proteins with two exceptions: ! Insulin receptor family has an !2"2 structure Exist as tetramers in the unstimulated and stimulated states ! Met receptor family has an !" structure

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LECTURE TITLE: DATE: TIME: PROFESSOR: TRANSCRIBER: PHONE:

Receptor tyrosine kinase signaling: a moving target for therapeutic intervention November 19, 2009 9:30-10:25 AM Todd Miller Di Fan (510)384-1467

Lecture Number: 82 Version: draft EMAIL:di.fan@hsc.stonybrook.edu

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II. Ligand binding and activation

Activation of receptor TK o When a cell is stimulated with growth factor, within seconds (or at least within a minute), there is a rapid rise in tyrosine phosphorylation o This is a transient event: the increase in phosphorylation goes down after about a minute or so o Later, after about 15-30 minutes, begin to get changes in gene expression First thing that happens after ligand binds to the receptor is that the RTK dimerizes o Most of the RTK are monomeric when in an unactivated state, but they dimerize when activated o This dimerization can happen in response to a bivalent ligand, but it is thought that even monomeric ligands that bind to a single polypeptide still promote dimerization o This is not true for the insulin receptor family since they exist as a tetramer when unactivated After dimerization, the RTKs autophosphorylate (one molecule phosphorylates the other = intermolecular or trans phosphorylation). This autophosphorylation happens in two regions: 1) Within the catalytic domain in a region called the activation loop ! This phosphorylation often increases the kinase activity 2) At the C terminus, where the autophosphorylation sites provide docking sites for SH2 domains o Recall that autophosphorylation happens when one molecule in the dimer phosphorylates the other, so it is an intermolecular or trans phosphorylation ! Evidence for intermolecular phosphorylation: If you take a mutant form of a kinase that is dead (ex. it lacks the Lys needed for ATP binding) and introduce it into cells, you can show that it gets phosphorylated by its partner in the dimer ! The activation loop phosphorylation serves the same purpose as the Tyr 416 phosphorylation on Src

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LECTURE TITLE: DATE: TIME: PROFESSOR: TRANSCRIBER: PHONE:

Receptor tyrosine kinase signaling: a moving target for therapeutic intervention November 19, 2009 9:30-10:25 AM Todd Miller Di Fan (510)384-1467

Lecture Number: 82 Version: draft EMAIL:di.fan@hsc.stonybrook.edu

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Mechanism for kinase domain activation using the insulin receptor as a model oInsulin receptor tyrosine kinase has been studied by x-ray crystallography and the structure has been found in the active and inactive states !In unactivated state, the activation loop (shown in green in the figure below) blocks the enzymes active site, which is where the substrates want to bind. Therefore, the substrates have blocked access to active site as a result of this activation loop. !When the activation loop is phosphorylated, a large conformational change causes it to move out of the way of the active site, enabling substrates to bind !In the case of the insulin receptor family members, this involves three phosphorylations, on Y1158, 1162, 1163. Dimerization is a common mechanism for RTK activation o Several of the RTKs can heterodimerize with other proteins o This is most clear in the case of the epidermal growth factor (EGF) family of RTKs ! EGF receptor family has 4 members that are sometimes called ErbB1-4, and sometimes called HER1-4 (ErbB1 is the same thing as HER1, etc.) ! The family members can homodimerize (ex. ErbB1-ErbB1) or there exists a whole different set of heterodimers (ex. ErbB1-ErbB2) o Heterodimers can have different signaling properties than homodimers ! This is an example of what is called combinatorial signaling: because the different combinations can signal differently, there is an increased diversity of signals possible in this family ! Makes it a lot tougher to study this since you cant really distinguish homodimers and heterodimers with current experimental systems ! Some ligands are specific for specific heterodimers

III. RTK Autoinhibition

A variety of mechanisms have evolved to keep RTKs at low activity in their basal state and defects in these mechanisms can lead to cancers. Some of the parts of RTKs involved in autoinhibition include: o Extracellular region of some RTKs (ex. EGFR) ! Extracellular region of EGFR has 4 domains: domains I and III are involved in ligand binding (growth factors) while domains II and IV are engaged in autoinhibition (an intramolecular interaction that inhibits receptor activation) The binding of EGF to domains I and III extends the extracellular structure of the EGFR In the extended conformation, a part of domain II can interact with its partner in another domain II to make the dimer interface for signaling Defects in this dimerization interface lead to constitutive activation of EGFR oJuxtamembrane region !A region between the membrane and the kinase that, in several RTK families, inhibits the kinase catalytic domain (ex. PDGF receptor) !In many cases, there are tyrosines within the

Printed 11/19/09 9:29 AM

LECTURE TITLE: DATE: TIME: PROFESSOR: TRANSCRIBER: PHONE:

Receptor tyrosine kinase signaling: a moving target for therapeutic intervention November 19, 2009 9:30-10:25 AM Todd Miller Di Fan (510)384-1467

Lecture Number: 82 Version: draft EMAIL:di.fan@hsc.stonybrook.edu

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juxtamembrane region that are involved in inhibitory contacts and when those tyrosines are phosphorylated, it breaks apart the interaction with the juxtamembrane region and leads to kinase activation ! Mutation in Juxtamem region can cause stromal tumors o C-terminal sequence ! Fold back into the kinase domain and probably block substrate binding to the kinase ! Ex. Tie2 receptor tyrosine kinase **All of these are mechanisms to keep the kinase activity of the receptor low until a stimulus is provided to the cell**

IV. Binding and phosphorylation of cellular substrates.

After the kinase is activated, the phosphorylation of RTKs in their C-terminal regions causes cellular proteins to be recruited to the vicinity of the RTK oThis occurs because of specific interactions between the SH2 domains of those proteins and the phosphorylation sites on the RTKs !Recall that different SH2 domains prefer different sequences around phosphotyrosine, so you can have a group of different cellular proteins recruited to the vicinity of the membrane because they have specific interactions with the sequences at the different autophosphorylation sites Thus, activation of RTK creates a large multiprotein signaling complex near the membrane Three of the most common SH2 domains recruited to the RTK are PI3-kinase, GTPase-activating protein and phospholipase C-# Three different ways the signal can be promoted by the recruitment of the SH2 containing proteins: 1) Can cause proteins that were cytoplasmic to be recruited to the membrane (see panel A. in figure below) ! Ex: PI3K is recruited to the membrane by virtue of its SH2 domains docking onto a RTK. Its substrate, PIP2, lies in the membrane and it generates PIP3, another membrane phopsholipid. PIP3 production in the membrane then recruits two kinases: PKB and PDK1 When they are recruited to PIP3, PKB and PDK1 are activated which leads to protection of cell from apoptosis All of these events have to !

Printed 11/19/09 9:29 AM

LECTURE TITLE: DATE: TIME: PROFESSOR: TRANSCRIBER: PHONE:

Receptor tyrosine kinase signaling: a moving target for therapeutic intervention November 19, 2009 9:30-10:25 AM Todd Miller Di Fan (510)384-1467

Lecture Number: 82 Version: draft EMAIL:di.fan@hsc.stonybrook.edu

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happen at the membrane because the substrates for these enzymes are membrane lipids. This only happens because of the initial recruitment of PI3K to the membrane because of its SH2 domains 2)In some of SH2 containing proteins, a conformational change occurs when the SH2 domain binds !Ex: PI3K is inactive in the cytoplasm as a result of the interaction between its regulatory subunit (containing the SH2 domains) with its p110 catalytic subunit. !When PI3K is recruited to the RTK and the SH2 domains become engaged with phosphotyrosines, and the catalytic activity p110 becomes activated 3) Through PLC-#, which is a subset of the PLC enzymes ! It contains two SH2 domains ! It is recruited to the RTK, the RTK phosphorylates and activates the PLC-# ! Thus PLC-# finds itself near its substrate (the membrane phospholipid), cleaves it, and promotes signaling Note: These mechanisms are not mutually exclusive. For example, PLC-# is activated not just by phosphorylation but also because it was initially recruited to the membrane.

V. Recruitment of adaptor proteins

Another class of proteins recruited to RTKs are adaptor proteins oThese are proteins that have no enzymatic function and consist entirely of small modular signaling domains oTheir function is to act as glue to bring together two other proteins because of protein-protein interactions oGrb2 is an adaptor protein with one SH2 domain and two SH3 domains !Was identified as the homolog of the C. elegans protein Sem5 (C. elegans is a roundworm that is used in signaling studies. It is a common occurrence these days that a mammalian signaling protein is first identified as a homolog of a signaling protein identified genetically in some other organism.) ! Binds to a protein called son of sevenless (SOS), so called because it was found in a RTK pathway called sevenless Identified in Drosophila Is a guanine nucleotide exchange factor (GEF) What does Grb2/SOS complex do in terms of mitogenic signaling? o SOS is a GEF specific for the small G-protein called Ras ! Ras is very important in cell signaling and is mutated in a high proportion of human cancers ! Ras cycles between an active GTP-bound state and an inactive GDP-bound state, and GEF (which in this case is the SOS) has the activity of exchanging the GDP for GTP

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LECTURE TITLE: DATE: TIME: PROFESSOR: TRANSCRIBER: PHONE:

Receptor tyrosine kinase signaling: a moving target for therapeutic intervention November 19, 2009 9:30-10:25 AM Todd Miller Di Fan (510)384-1467

Lecture Number: 82 Version: draft EMAIL:di.fan@hsc.stonybrook.edu

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In cancers, the hyperactive mutants of Ras are locked in the GTP bound state

Note: The tyrosine phosphorylation events and activation of Ras are relatively short-lived events at the plasma membrane that happen within minutes. To send the signals for the cell to grow or divide, these quick events have to be translated to longer-lasting events and must ultimately get to nucleus to affect changes in gene expression. The relay system comes from a set of Serine/Threonine kinases: the MAP kinase cascade.

VI. The MAP kinase cascade

Active Ras protein triggers a cascade of Ser/Thr phosphorylations that will ultimately end up changing the activity of gene regulatory proteins oThe generic terms for these Ser/Thr kinases are MAPkinase-kinase-kinase (MAPKKK), MAP kinase-kinase (MAPKK), and MAP-kinase (MAPK) oIn yeast and mammals there are a number of these cascades going on !Parallel kinase cascades respond to different sorts of stimuli !In the pathway of cell proliferation in response to growth factors, the relevant proteins are Raf (a MAPKKK), Mek (a MAPKK), and ERK (a MAPK) !Activated Ras increases the membrane localization and activation of the MAPKKK, which will then phosphorylate its downstream MAPKK, which will then phosphorylate the downstream MAPK ! When activated, MAPKs can dimerize and translocate to the nucleus, where they have a number of targets ! The MAPKs specifically phopshorylate gene regulatory proteins, a major one of which is Elk1 Elk1 is a transcription factor that can control transcription of a early genes that can control the transcription of other proteins number which can drive the cell into the S phase These kinase cascades dont happen because of random collisions between kinases and substrates, but instead there exist scaffolding proteins in both yeast and mammals that appear to tether together the components of the MAPK signaling cascade o For example, a scaffolding protein called Ste5 can bind an upstream activator (analogous to Ras), the MAPKKK, the MAPKK, and the MAPK ! All of these are tethered together so that it is easy for one protein to phosphorylate the next protein o This not only increases efficiency of the process, but it also reduces possibility of cross activation of different kinases

Printed 11/19/09 9:29 AM

LECTURE TITLE: DATE: TIME: PROFESSOR: TRANSCRIBER: PHONE:

Receptor tyrosine kinase signaling: a moving target for therapeutic intervention November 19, 2009 9:30-10:25 AM Todd Miller Di Fan (510)384-1467

Lecture Number: 82 Version: draft EMAIL:di.fan@hsc.stonybrook.edu

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VII. RTK inhibition in cancer

A number of RTKs are found to be mutated in human cancers Strategies to target hyperactive RTKs o Make blocking agents or antibodies that will prevent the ligand from binding to the extracellular part of the receptor ! Ex. Herceptin is a humanized monoclonal antibody that targets the extracellular domain of ErbB2, a member of the Erb family of RTKs that is overexpressed in breast cancer o Tyrosine kinase inhibitors: inhibit the kinase domain o Farnesyltransferase inhibitors !Ras has to be farnesylated to be targeted to the membrane, and Ras needs to be targeted to the membrane to be activated !By inhibiting the farneyltransferase, Ras will be unable to be targeted to the membrane and thus unable to be activated oRAF and MEK inhibitors are also being tested Biochemical considerations that go into development of one of these agents: oLow-MW compounds are much easier to make than something like a humanized monoclonal antibody !Easier to make and easier to characterize when they are made oSmall compounds are more likely to penetrate into tumors and to cross cell membranes if they are hydrophobic enough oTarget molecule should be druggable ! Should be vulnerable to attack ! Typically it needs to bind tightly and specifically to the inhibitor o Specificity is a key consideration in developing signaling inhibitors: kill one molecule and leave others alone o Best bet in terms of druggability are enzymes, because they have a well defined active site and sometimes there is a structure already known for the active site Most kinase inhibitors target ATP binding sites, since it is the easiest and most well defined binding pocket to target for kinase inhibitors. o Two examples of drugs approved by FDA in the past few years are the EGFR inhibitors Tarceva and Iressa ! These are built from a common chemical skeleton, which is the part that binds the ATPbinding site ! Structures on the skeleton are elaborated on to make them better drugs (ex. to make them better at crossing the membrane, to provide specificity for the EGFR, etc.) ! They wedge into the ATP binding site and prevent ATP binding and as a consequence block enzyme activity ! Gleevec (the Abl inhibitor from the last lecture) also binds to the ATP binding site An interesting development on the EGFR inhibitors is that in the first clinical trials of these agents, only about 10% of the

Printed 11/19/09 9:29 AM

LECTURE TITLE: DATE: TIME: PROFESSOR: TRANSCRIBER: PHONE:

Receptor tyrosine kinase signaling: a moving target for therapeutic intervention November 19, 2009 9:30-10:25 AM Todd Miller Di Fan (510)384-1467

Lecture Number: 82 Version: draft EMAIL:di.fan@hsc.stonybrook.edu

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stonybrook school of medicine 2013 note service

patients showed any kind of response to the drug o Several of the 10%, however, were helped quite a bit ! Example: a large reduction was seen in the size of a lung mass in the patient on the right after 6 weeks of Iressa treatment o About 4 years later, the EGFRs from patients were sequenced, and it was found that almost all of the people who responded to Iressa had specific mutations in the EGFR gene ! A specific subset of EGFR point mutations activated the enzyme, but also made it more vulnerable to Iressa ! This shows the benefits of tumor sub-classification: if we can identify what kind of mutation patients have in the EGFR, we will know whether or not they will respond to the drug Iressa o The bad news is that even though some patients respond well to Iressa tend to relapse because resistance develops to the drug ! The figure to the right shows patient relapse after 2 years of Iressa treatment as a result of a second site mutation, specifically, a Threonine to Methionine mutation that prevents Iressa binding o This highlights the possibility of using multidrug therapy (Iressa plus something else) to knock out the activity of the EGFR

Clicker Questions
1. What takes place during the activation of RTK? a. Release of autoinhibitory interactions b. Autophosphorylation c. Dimerization d. Recruitment of cellular proteins containing SH2 domains e. All of the above 2. Which protein acts as a GEF for Ras? a. Grb2 proteins b. RGS proteins c. EGF receptor d. SOS e. Src 3. _____ increase the specificity and efficiency of MAP kinase cascades a. GEFs b) RGS c) Scaffolding proteins d) Tyrosine kinase inhibitors e) PH domains The previous people did a great job--I only made some minor changes and added only 1 more clicker question. -Di
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