KEY CONCEPT QUESTIONS IN SIGNAL TRANSDUCTION

Signal Transduction - Part 2 Key Concepts - Receptor tyrosine kinases control cell metabolism and proliferation Growth factor signaling through Ras Mutated cell signaling genes in cancer cells are called oncogenes Insulin signaling through PI-3 kinase - TNF receptors activate protein complexes that control cell death and survival TNF receptors transmit signals through formation of adaptor complexes Cell survival and cell death are opposing pathways KEY CONCEPT QUESTIONS IN SIGNAL TRANSDUCTION: What biochemical mechanisms mediate short term and long term signaling responses? Why are defects in signal transduction pathways a common cause of metabolic disease? Biochemical Applications of Signal Transduction: Gleevec is an enzyme inhibitor of the signaling protein Bcr-Abl kinase and is used to treat chronic myelogenous leukemia. The success of Gleevec in cancer therapy has led to the development of new drugs designed to inhibit specific signaling pathways. Receptor Tyrosine Kinases Control Cell Metabolism And Proliferation After G protein coupled receptors (GPCRs), the next most abundant receptor class in the human genome is the receptor protein tyrosine kinases which have three distinguishing characteristics. First, receptor tyrosine kinases transmit extracellular signals by ligand-activation of an intrinsic tyrosine kinase function encoded in the cytoplasmic tail of the receptor. Second, activation of the tyrosine kinase activity requires receptor dimerization which is often stimulated, or stabilized, by ligand binding. Third, autophosphorylation of tyrosine residues within the receptor creates phosphotyrosine docking sites for signaling proteins that establish a relay signal between the receptor and a downstream phosphorylation cascade. Figure 1 illustrates these key features. Figure 1. 1 of 9 pages

Growth factor signaling through Ras The presence of phosphotyrosine residues on the EGF receptor cytoplasmic tail creates a protein docking, surface for intracellular adaptor proteins containing a protein motif called a Src homology domain, or SH2 domain. The adaptor protein Grb2 binds to activated EGF receptor. The SH2 domain is a ~100 amino segment that is highly conserved amongst adaptor signaling proteins and functions to selectively bind phosphotyrosine residues with high affinity. The structure of SH2 domain proteins includes a phosphotyrosine binding pocket and a separate specificity pocket that recognizes a nearby amino acid on the receptor. The docking sites for three SH2 domain containing signaling proteins are illustrated in figure 2. Figure 2. With Grb2 in place, the EGF receptor is primed to transmit the signal downstream to a molecular switch controlled by a small GTPase protein called Ras. However, Grb2 does not interact directly with Ras, but rather it binds through its SH3 domain (another protein docking site) to a guanine nucleotide exchange factor (GEF) called SOS. GEF proteins such as SOS activate Ras through stimulation of the GDP-GTP exchange reaction as shown in figure 3. In this regard, SOS is functioning like the cytoplasmic domain of a G coupled protein receptor which activates the GDP- GTP exchange reaction in G subunits. The activation of Ras by SOS is normally short-lived because GTPase activating proteins (GAP) such as RasGAP bind to Ras and stimulate GTP hydrolysis. Moreover, phosphotyrosinespecific phosphatases inactivate the EGF receptor to cause disassembly of the Grb2 adaptor complex. The Ras activity "cycle" depends on the relative activities of GEFs and GAPs as shown in figure 3. Figure 3. How does Ras pass this transient signal downstream to ensure that the intended cell proliferation response is enacted? The answer is through the stable activation of a phosphorylation cascade mediated by a trio of related kinases which transmit the Ras signal to target proteins in the 2 of 9 pages

nucleus. All three of these kinases belong to the MAP kinase family and have names that reflect their initial characterization. As shown in figure 4, the MAP kinase activated by Ras in the EGF signaling pathway is called Raf, also known as MAP kinase kinase kinase for reasons that will become clear shortly. Raf is a 74 kda serine/threonine kinase that is recruited to the membrane by activated Ras where it can then be activated by phosphorylation. Src is one of several membrane-associated Raf activators that have been identified and shown to be a key signaling protein. Figure 4 illustrates the role of Grb2 in connecting EGF receptor to SOS via SH2 and SH3 domains. Figure 4. Once activated, Raf phosphorylates the next kinase in the pathway called MEK (MAP kinase kinase) on two specific serine residues (S218 and S222) as shown in figure 4. The primary target of MEK in the EGF signaling pathway is the third kinase in the trio called ERK, which was originally named mitogen activated protein (MAP) kinase because it phosphorylates proteins in response to cell proliferation signals such as EGF. ERK phosphorylates serine residues on transcription factors 3 of 9 pages

such as Elk which forms a heterodimer with another transcription factor called SRF. The Elk-SRF complex regulates genes involved in controlling cell proliferation in response to EGF signaling. Mutated cell signaling genes in cancer cells are called oncogenes Some of the first cancer-related genes to be isolated were found by identifying human DNA fragments obtained from tumor samples that conferred high proliferation rates on cells grown in culture. Since tissue culture methods use media containing calf serum, a rich source of growth factors, many of the oncogenes identified (oncology is the study of cancer), were those that interfered with feedback inhibition of growth factor signaling pathways. These activated cancer genes were all found to contain gain of function mutations. Two examples of common Ras activating mutations are a glycine to valine mutation at codon 12 (G12V), and a glutamine to arginine mutation at codon 61 (Q61R). As shown in figure 5A, these Ras mutations disrupt the intrinsic GTPase activity and thereby prevent Rasmediated signaling from being turned off even in the absence of growth factor. Activating Ras mutations account for more than 50% of pancreatic cancers and up to 25% of all solid tumor cancers. Figure 5B shows the types of G12 Ras mutations in pancreatic cancers. Figure 5B. Figure 5A. Insulin signaling through PI-3 kinase We have already seen how glucagon, the I am hungry hormone, increases blood glucose levels by signaling through a G protein coupled receptor to activate adenylate cyclase to generate the second messenger cyclic AMP. We now examine how insulin, the I just ate hormone, signals glucose uptake through a receptor protein tyrosine kinase. Glucagon and insulin are both produced by the pancreas, albeit by different cell types within this organ, and are always present in the blood at a ratio that reflects their rate of secretion in response to blood glucose levels. The mature insulin receptor is an α 2 β 2 heterotetramer consisting of cross-linked α and β chains connected by disulfide bridges. The α chains form the extracellular ligand binding domain and the β chains encode the intracellular protein tyrosine kinase function. Biochemical characterization of the insulin receptor suggests that insulin binding to α subunits causes a conformational change in the receptor that stimulates the protein tyrosine kinase activity in the β subunits by blocking an inhibitory function of the subunits. Once the β subunits are phosphorylated, the tyrosine kinase activity of the receptor is stimulated ten-fold and a binding surface is created for a family of proteins called insulin receptor substrate (IRS) proteins. As 4 of 9 pages

Bioc 460 - Dr. Miesfeld, Spring 2008 shown in figure 6, association of IRS proteins with the activated β subunit of the insulin receptor leads to their phosphorylation on tyrosine residues and subsequent activation. Figure 6. Phosphotyrosine residues on IRS proteins serve as docking sites for other signaling proteins which stimulate two major pathways as shown in figure 6. One branch of the insulin signaling pathway involves IRS activation of the MAP kinase pathway through the same Grb2-SOS-Ras module we saw with the EGF receptor. The other major branch of the insulin pathway involves IRS mediated activation of phosphoinositide-3 kinase (PI-3K) through SH2 domains that bind to phosphotyrosines on IRS. Figure 7. How does activation of PI-3K signaling by insulin lower serum glucose levels? Before we can answer this question, we first need to see what chemical reaction is catalyzed by PI-3 kinase. The structure of human PI-3 kinase containing the inhibitor staurosporine bound in the enzyme active site is shown in figure 7. As shown in figure 8, recruitment PI-3 kinase to the plasma membrane by IRS proteins stimulates phosphorylation of phosphatidylinositol 4,5-bisphosphate (PIP2) to produce phosphatidylinositol 3,4,5-triphosphate (PIP3) which remains in the plasma membrane and serves as a docking site for signaling proteins containing pleckstrin homology (PH) domains. Figure 8. Figure 9. 5 of 9 pages

Remember from lecture 22 that PIP 2 is also the substrate for phospholipase C-β which catalyzes a hydrolysis reaction to produce DAG and IP 3 (see figure 21 on page 11 of lecture 22). As shown in figure 9, PH domains are highly conserved protein structural motifs that serve to anchor signaling proteins to the plasma membrane. Much like SH2 binding to phosphotyrosines on receptor tyrosine kinases, PH domain binding to PIP 3 in the plasma membrane creates a plug and socket interaction that directs downstream signaling events through a protein recruitment mechanism (high localized concentration of kinase substrates). Two proteins in the insulin signaling pathway that contain PH domains are phosphoinositoldependent kinase (PDK1), and AKT, a serine-threonine kinase originally identified as an oncogene gene (cancer gene) in the Akt8 murine retrovirus. AKT is also known as protein kinase B (PKB) because it shares amino acid homology with protein kinase A and protein kinase C. As shown in figure 10, both PDK1 and AKT bind to PIP 3 leading to PDK1 phosphorylation of AKT and subsequent dissociation of AKT from PIP 3. Once Akt leaves the membrane, it is free to phosphorylate numerous protein targets throughout the cytoplasm. Figure 10. 6 of 9 pages

Insulin receptor activation in muscle and adipose cells stimulates glucose uptake and storage in the form of glycogen through three mechanisms: 1) AKT-mediated phosphorylation and inactivation of glycogen synthase kinase (GSK) 2) Activation of protein phosphatase 1 (PP1) by insulin-stimulated protein kinase (ISPK) 3) Localization of the glucose transporter GLUT4 to the plasma membrane by mechanisms involving both the insulin receptor tyrosine kinase and AKT. By increasing the intracellular concentration of glucose through GLUT4-mediated uptake, and at the same time stimulating the incorporation of glucose into glycogen through activation of glycogen synthase (and inhibition of GSK), insulin signaling results in net glucose import into cells. Again, don't get lost in the details of insulin control of glycogen metabolism as we will examine this in more detail in lecture 35. However, you should understand two components of this signaling pathway, 1) the mechanism of insulin receptor activation of AKT (PI-K3 mediated formation of PIP3 and recruitment of PDK1), and 2) the three mechanims by which insulin stimulates glucose uptake and storage in the form of glycogen (see list above). TNF receptors activate protein complexes that control cell death and surival We will now look at a different type of signal Figure 11. integration, one that involves activation of a single receptor that stimulates two intracellular pathways with opposing cellular responses; one leading to cell survival and the other resulting in cell death (apoptosis). Apoptosis is a Greek word meaning to fall off, like leaves falling off of a tree. When cells die by apoptosis, they break into pieces called "apoptotic bodies" which are engulfed by surrounding cells to clean up the debris. Tumor necrosis factor (TNF) is an inflammatory cytokine that was initially discovered as a signaling molecule that induces apoptosis in some cancer cells through binding to a trimeric membrane receptor. TNF signaling at the cell surface initiates two or more intracellular pathways as illustrated in figure 11. TNF receptors transmit signals through formation of adaptor complexes The primary mechanism of signal transduction through TNF receptor family members is adaptor complex formation through protein structural modules. The TNF R1 receptor is activated by TNFα and contains an 80 amino acid structural motif in the cytoplasmic tail called a Death Domain (DD) which serves as a docking site for other DD containing proteins. As shown in figure 12, the TNF signaling pathway bifurcates at the level of TRADD binding to the receptor. 7 of 9 pages

Figure 12. Cell survival and cell death are opposing pathways As shown in figure 12, FADD binding to procaspase 8 stimulates an autocleavage reaction which results in the formation of active caspase 8 and subsequent cleavage and activation of downstream caspases resulting in cell death. The cell survival pathway is controlled by binding of a protein called TRAF2 to sequences in TRADD which then recruit the NFkB-inducing kinase (NIK) to the complex. Importantly, cells live or die depending on the expression levels of the signaling proteins FADD and caspase 8 (apoptosis), relative to the protein levels of TRAF2, RIP and NFkB (survival pathway). Bottom-line: the fate of the cell rests in the relative abundance (and activities) of proteins in two separate, but inter-related, signaling pathways. 8 of 9 pages

ANSWERS TO KEY CONCEPT QUESTIONS: The two primary biochemical mechanisms that mediate short term and long term signaling responses are phosphorylation cascades and regulated gene expression, respectively. Rapid responses to first messenger signals are achieved by enzymatic reactions that regulate target proteins through covalent modification mechanisms. The most common modifications are phosphorylation and dephosphorylation reactions which can quickly alter the activity of a target protein. Moreover, reversal of the activating signal is achieved by the same mechanisms. Long term signaling responses are the result of altered rates of gene expression which lead to increased or decreased rates of transcription and are associated with developmental processes or signaling by intracellular receptors. Defects in signal transduction are common causes of metabolic disease because cell signaling pathways are interdependent and disruption one component can alter many pathways. Cell signaling pathways are critical to metabolic regulation and cell homeostasis. Alterations in receptor activation or any number of downstream signaling events can be detrimental to cell regulation. Diabetes and cancer are two examples in which defects in signaling pathways cause pleitropic effects. Many types of pharmaceutical drugs are designed to target membrane receptors. 9 of 9 pages