Philip Stork - US grants

The long term goals are to study the regulation of neuronal transmission in the central nervous system and to understand the role of pharmacological agents in modifying neuronal transmission. The current proposal to clone the catecholamine uptake transmitter reflects to the application of basic science to clinical issues . The importance of catecholamine uptake transporters is demonstrated by the impact in our society of specific inhibitors of these proteins, namely cocaine, amphetamines, and tri-cyclic antidepressants. 'Me stimulatory action and abuse potential is directly related to their ability to bind and block catecholamine uptake at the presynaptic nerve terminals. To study the pharmacology of cocaine and to understand the molecular basis for its medical effects, it is necessary to isolate and characterize the transporter proteins themselves. Molecular cloning of catecholamine transporters is the most direct way to gain access to this class of transporters. Their ability to bind inhibitors with high affinity and to concentrate catecholamines within a cell make these proteins ideally suited to molecular cloning by expression in heterologous cells. This application presents a cloning strategy employing radiolabelled ligands and substrates to isolate the cDNA encoding a catecholamine transporter following its expressing in COS cells.

Tissue-specific proteolytic cleavage of prosomatostatin generates two biologically active peptides, somatostatin-14 (SS-14) and somatostatin- 28 (SS-28). These two peptides are thought to activate distinct receptors in the brain, pituitary, pancreas, and GI tract. Because somatostatin regulates the functions of so many target tissues, unraveling the complexities of somatostatinergic signaling is a major problem in neuroendocrinology. We, and others, have recently identified and isolated cDNAs encoding two distinct somatostatin receptors: SSR1 and SSR2. These cDNAs provide the best evidence for the existence of distinct functional subtypes of somatostatin receptors and additionally supply the means to determine the physiological significance of this receptor multiplicity. One of the goals of this proposal is to determine whether these receptor subtypes display different affinities for SS-14 and SS-28. Both somatostatin peptides elicit relatively complex biological responses, by activating or inhibiting multiple signal transduction pathways. For example, alterations in adenylyl cyclase activity, potassium efflux, and calcium influx all contribute to the ability of SS-14 to inhibit prolactin secretion from pituitary cells. A second goal of this proposal is to determine whether these different biological actions of somatostatin are mediated by distinct receptor subtypes and G protein signaling pathways. Perhaps the most unique action of somatostatin is its ability to inhibit tumor cell growth. This antineoplastic action of somatostatin may be mediated through its stimulation of a protein tyrosine phosphatase. We have identified a novel G protein pathway used by somatostatin to stimulate a protein tyrosine phosphatase (PTP) activity in human tumor cells. Our third goal is to characterize the components of the pathway that mediate this effect. In conclusion, somatostatin participates in a unique signaling system capable of activating at least four separate G protein-coupled pathways. Prosomatostatin and the family of somatostatin receptors and effectors can serve as a paradigm for how a single neuropeptide system mediates a wide variety of biological effects. Through the coordinated expression of specific ligands, receptors, G proteins, and effectors somatostatin is able to increase the range and scope of its actions. The specificity of the interactions between ligands, receptors, G proteins and effectors governs the signaling pathway utilized in the cell. We have isolated the necessary reagents and have developed the technical tools to begin to understand the molecular rules that govern the interactions. Understanding these rules will facilitate the development of therapeutic strategies designed to discriminate among the many effects of somatostatin.

THIS IS A SHANNON AWARD PROVIDING PARTIAL SUPPORT FOR THE RESEARCH PROJECTS THAT FALL SHORT OF THE ASSIGNED INSTITUTE'S FUNDING RANGE BUT ARE IN THE MARGIN OF EXCELLENCE. THE SHANNON AWARD IS INTENDED TO PROVIDE SUPPORT TO TEST THE FEASIBILITY OF THE APPROACH; DEVELOP FURTHER TESTS AND REFINE RESEARCH TECHNIQUES; PERFORM SECONDARY ANALYSIS OF AVAILABLE DATA SETS; OR CONDUCT DISCRETE PROJECTS THAT CAN DEMONSTRATE THE PI'S RESEARCH CAPABILITIES OR LEAD ADDITIONAL WEIGHT TO AN ALREADY MERITORIOUS APPLICATION. THE APPLICATION BELOW IS TAKEN FROM THE ORIGINAL DOCUMENT SUBMITTED BY THE PRINCIPAL INVESTIGATOR. Cell growth and differentiation is tightly regulated by a signaling pathway known as the MAP kinase cascade. This cascade is activated in the majority of human cancers, through the action of specific oncogenes and culminates in the phosphorylation of the MAP kinases (also called ERKs). The proto-oncogene ras (p21) is the most commonly activated oncogene in human tumors and turns on MAP kinases through the stimulation of upstream components of this cascade. Recently a second parallel signaling pathway called the stress-activated protein kinase cascade has been identified. This pathway is activated by a variety of extracellular stresses including radiation and chemotherapeutic agents and like the MAP kinase cascade is also activated by the proto-oncogene ras. We propose that the stress-activated protein kinase cascade triggers apoptosis (programmed cell death) and that this pathway is inactivated by a novel family of MAP kinase phosphatases (MKPs) that are rapidly induced during mitogenesis. We will demonstrate the actions of MKPs on JNKs using biochemical techniques following their expression in transformed cell lines. We propose that the pathophysiological response to JNK activation (programmed cell death) can be blocked through dephosphorylation by MKPs. We will examine the induction of apoptosis in MKP overexpressing cells to demonstrate MKPs ability to block apoptoic responses. The inhibition of MKPs should activate latent stress-activated pathways and induce apoptosis in tumor cells. These studies will establish that MKPs are potential targets for novel phosphatase inhibitors that, by inactivating MKPs, will potentiate the apoptoic action of other anti- cancer treatments.

Angiotensin II (AII) is a key regulator of cardiovascular homeostasis controlling vascular tone, endocrine secretion and cellular growth. Its actions are mediated through specific subtypes of the AII receptor, AT1 and AT2. Both are seven transmembrane receptors coupled to intracellular signaling pathways through heterotrimeric G proteins. The AT2 receptor is highly expressed in fetal tissues but shows a restricted distribution in the adult, suggesting a role in tissue growth and differentiation. AII action on cellular growth and DNA synthesis have been well established in cultured rat vascular smooth muscle cells. In this proposal, we wish to examine the role of MAP kinase-dependent signaling pathways in AII's growth effects. We will determine the receptor subtypes utilized by AII and the magnitude and kinetics of activation of MAP kinases and related protein kinases by AII in these cells and will determine the requirement of MAP kinase activation in AII's growth effects. Using DNA-mediated gene transfer, we will also determine the mechanism by which MAP kinase is activated by AII. AII is a potent transcriptional activator of MAP kinase phosphatases (MKPs). MKPs selectively regulate both MAP kinases and stress-activated protein kinases. We will determine whether AII's induction of MKPs dictates the specificity of AII signaling. We will also examine AII's actions in a developmentally regulated model of VSMC growth, the pig. All does not induce DNA synthesis in adult porcine VSMCs but does stimulate growth in neonatal VSMCs. We will determine whether these developmental differences in AII's developmental effects in the fetal cardiovascular system.

Studies on the growth of endocrine cells have identified two major pathways of regulating proliferation. These pathways are initiated by growth factor stimulation of receptor/tyrosine kinases and hormonal activation of G protein-coupled receptors. Recent data in our laboratory demonstrate that these pathways intersect at multiple levels. For example, hormones that elevate intracellular levels of cyclic adenosine monophosphate (cAMP) have a wide range of cell type-specific effects on cell growth and differentiation. MAP kinase activation mediates the mitogenic response to growth factors in a number of cell types including endocrine cells. We propose that cAMP's mitogenic actions require MAP kinase activation. We have identified a novel pathway mediating cAMP signals in neuroendocrine cells. We show data that one substrate of PKA, the GTPase Rap1, is an activator of MAP kinase in neuroendocrine cells and is activated by cAMP in these cells, and we propose that Rap1 dictates cAMP's activation of MAP kinase in these cells. We have identified the proximal target of Rap activation: the MAP kinase B-Raf. B-Raf is expressed in a subset of cells including neuronal and endocrine cells. We propose that its expression dictates cAMP's ability to activate MAP kinase in neuroendocrine cells. We will examine the mechanism of the activation of B-Raf by Rap and determine whether this pathway mediates cAMP's cell type-specific activation of MAP kinase. Specifically, we will test the hypothesis that Rap1 is a selective activator of B-Raf (Specific Aim 1), and will determine whether Rap1 mediates the actions of cAMP on MAP kinase in B-Raf-expressing cells (Specific Aim 2). Lastly, we will determine whether other physiological signals can activate this novel pathway (Specific Aim 3).

DESCRIPTION (Adapted from applicant's abstract): Activity-dependent regulation of neuronal function is initiated in large part via depolarization-induced calcium influx. Neuronal calcium has been shown to activate a number of intracellular kinase cascades to regulate processes such as gene transcription, synaptic plasticity and cell survival. Many of these activity-dependent functions are shared by the neurotrophin family of growth factors suggesting a common signaling pathway. Two pathways, the cAMP independent protein kinase (PKA) cascade and the extracellular signal regulated kinase (ERK) cascade have been implicated as potential targets for neuronal calcium-mediated signaling. Recent data in the applicant's laboratory have identified a novel signaling pathway downstream of NGF and cAMP signaling. This pathway involves PKA-dependent activation of the Ras-related small G protein, Rap1, and subsequent activation of a neuronal-specific ERK cascade. The major hypothesis of this proposal is that stimulation of this Rap-dependent ERK cascade is a target for activity-dependent calcium influx. This proposal utilizes membrane depolarization of both the PC12 neuronal cell line and hippocampal neurons to mimic activity-dependent regulation of calcium influx. The applicant will test the hypothesis that the small G protein Rap1 participates in the depolarization-mediated activation of ERKs. Recent studies have shown that neuronal Rap1 is activated by multiple second messengers. Therefore, the applicant further proposes to examine the possibility that, like neurotrophic signaling, PKA is required for neuronal calcium activation of Rap1. Finally, the applicant will also test the hypothesis that activation of Rap1 can couple calcium influx to two nuclear actions of ERKs: the nuclear translocation of ERKs themselves and CREB-dependent transcription. The work outlined in this grant will impact many areas of neuroscience and will point to Rap1 as a confluence point of neuronal signals. Given the well documented role for PKA and CREB in these long-term changes that accompany neuronal activity, it is likely that these studies in PC12 cells will identify the importance of Rap-dependent pathways in the long-term changes. The applicant believes that the experiments outlined in this proposal may ultimately impact more general research on synaptic plasticity, long-term potentiation (LTP) and neuronal survival.

One of the major functions of hormone action is the regulation of cell growth. Hormone action is initiated upon hormone binding to specific receptors that couple extracellular signals to intracellular ones. For most hormones, this is achieved via heterotrimeric G proteins which are composed of two components, alpha (Galpha) and beta-gamma (Gbeta-gamma) that may function independently to trigger intracellular signals. It is now clear that G proteins can regulate cell growth by activating intracellular phosphorylation cascades that link hormones to the MAP kinase cascade. The mechanisms by which G proteins activate MAP kinase ERK are poorly understood. It is thought that one of the major pathways from G proteins to MAP kinase is via signals from Gbeta-gamma to Ras, a small G protein that activates the MAP kinase kinase kinase Raf-1. In contrast to this prevailing view, we have identified three novel potential pathways by which Galpha can regulate ERK. Two of these involve the activation of Galpha-s, a G protein linked to the stimulation of adenylyl cyclase to increase intracellular cAMP. The action of cAMP on cellular proliferation and activation is cell-type specific. We propose that these actions depend on the selective activation of MAP kinase kinase kinases B-Raf and inhibition of Raf-1. We propose to test whether hormones that activate Galpha-s also regulate ERKs in this way. We have also shown that Galpha-i and Galpha-s can also regulate ERKs. These subunits are well known for their sensitivity to pertussis toxin, but the intracellular signals they regulate are not well established. We propose that all three G alpha subunits, Galpha-s, Galpha-i, and Galpha-o, regulate ERKs via a novel small G protein Rap1. The goal of this proposal is to elucidate the molecular mechanisms by which Galpha regulates Rap l and to determine the consequence of this regulation on ERK. In four specific aims, we propose experiments designed to examine each potential mechanism. Specific Aim 1 examines Galpha-s activation of ERK through its actions on Rap l via cAMP and PKA and the Raf isoform B-Raf. Specific Aim 2 focuses on Galpha-s inhibition of ERK signaling via Rap 1's antagonism of Ras. Specific Aim 3 will extend our findings to a model of oocyte maturation in Xenopus. Finally, Specific Aim 4 explores a novel mechanism of ERK regulation by Galpha-i and Galpha-o through their interaction with Rap 1 GAP, a selective inhibitor or Rap 1.

DESCRIPTION (Provided by the Applicant): A productive T lymphocyte response to antigen requires the activation of two signaling pathways, involving signals generated by the interactions between the T-cell receptor (TCR) with antigenic peptide presented on antigen-presenting cells (APCs) and the signal mediated by the binding of the accessory receptor CD28 with its ligand B7. Although the requirement for CD28 co-stimulation has been the subject of intensive and extensive investigation, the molecular nature of this co-stimulatory signal is unknown. Some models have identified distinct kinase cascades initiated by either the ICR or CD28, while other models have focused on the convergence of TCR/CD28 signals on particular kinase cascades. One pathway which can mediate the synergistic responses that characterize CD28 co-stimulation is the MAP kinase (ERK) cascade. The activation of the MAP kinase ERK following CD28 co-stimulation is required for IL-2 production and proliferation of responding T lymphocytes. ERK activation in T lymphocytes is regulated by two antagonistic small G proteins: Ras and Rap1. Ras activation is required for ERK activation, while Rap1 antagonizes Ras signaling. Antigen recognition by T-cells in the absence of CD28 co-stimulation is characterized by impaired ERK activation and both decreased IL-2 production and diminished proliferation. This functional unresponsiveness results in the inability to respond to subsequent co-stimulatory signals and is termed clonal anergy. Rap1 is constitutively activated in certain states of I cell anergy or this unresponsiveness may account for the diminished ERK activity and decreased IL-2 production seen in anergic T-cells. In this proposal, we will test the hypothesis that Rap1 is activated by ICR ligation in normal I cells and consequently limits I cell activation through the ICR in the absence of co-stimulation. In addition, we will test the hypothesis that CD28 co-stimulation achieves increased ERK activation, IL-2 production and proliferation by blocking Rap1 activation.

One of the major functions of hormone action is the regulation of cell growth. Hormone action is initiated upon hormone binding to specific receptors that couple extracellular signals to intracellular ones. For most hormones, this is achieved via heterotrimeric G proteins which are composed of two components, alpha (Galpha) and beta-gamma (Gbeta-gamma) that may function independently to trigger intracellular signals. It is now clear that G proteins can regulate cell growth by activating intracellular phosphorylation cascades that link hormones to the MAP kinase cascade. The mechanisms by which G proteins activate MAP kinase ERK are poorly understood. It is thought that one of the major pathways from G proteins to MAP kinase is via signals from Gbeta-gamma to Ras, a small G protein that activates the MAP kinase kinase kinase Raf-1. In contrast to this prevailing view, we have identified three novel potential pathways by which Galpha can regulate ERK. Two of these involve the activation of Galpha-s, a G protein linked to the stimulation of adenylyl cyclase to increase intracellular cAMP. The action of cAMP on cellular proliferation and activation is cell-type specific. We propose that these actions depend on the selective activation of MAP kinase kinase kinases B-Raf and inhibition of Raf-1. We propose to test whether hormones that activate Galpha-s also regulate ERKs in this way. We have also shown that Galpha-i and Galpha-s can also regulate ERKs. These subunits are well known for their sensitivity to pertussis toxin, but the intracellular signals they regulate are not well established. We propose that all three G alpha subunits, Galpha-s, Galpha-i, and Galpha-o, regulate ERKs via a novel small G protein Rap1. The goal of this proposal is to elucidate the molecular mechanisms by which Galpha regulates Rap l and to determine the consequence of this regulation on ERK. In four specific aims, we propose experiments designed to examine each potential mechanism. Specific Aim 1 examines Galpha-s activation of ERK through its actions on Rap l via cAMP and PKA and the Raf isoform B-Raf. Specific Aim 2 focuses on Galpha-s inhibition of ERK signaling via Rap 1's antagonism of Ras. Specific Aim 3 will extend our findings to a model of oocyte maturation in Xenopus. Finally, Specific Aim 4 explores a novel mechanism of ERK regulation by Galpha-i and Galpha-o through their interaction with Rap 1 GAP, a selective inhibitor or Rap 1.

The hypothesis to be tested in this proposal is that modifying the cell number of a developing organ can have consequences on the organ's function during adult life. We will focus our attention on the developing heart. This choice is based on the ability of cardiac cell number to be regulated pathophysiologically during development. A major hypothesis of this Program Project Grant is that intrauterine stresses can modify the program governing cell growth in adult life. For the heart, this is often associated with the stimulation of cardiomyocyte hyperplasia and an alteration in adult heart function. A corollary of this hypothesis is that alterations in cell number can affect cardiac function. This will be tested using genetically modified animal models of cardiac function. Initial studies will determine the program of cell growth in the heart (Specific Aim 1). Subsequent Aims will test the hypothesis that modifying ERK function will have consequences on cell growth during cardiac development, and on cardiovascular function in the adult. In Specific Aim 1 we will determine the critical period when ERK activity and proliferative signals pathways are maximal during organogenesis of the heart. In Specific Aim 2 we will introduce these modifiers of ERK signaling into whole animal models in such a way that their expression limited to the cardiomyocyte. In Specific Aim 3 we will determine whether these modifiers have pathophysiological consequences of the altered developmental processes on heart function during he lifetime of the animal.

DESCRIPTION (Provided by the Applicant): A productive T lymphocyte response to antigen requires the activation of two signaling pathways, involving signals generated by the interactions between the T-cell receptor (TCR) with antigenic peptide presented on antigen-presenting cells (APCs) and the signal mediated by the binding of the accessory receptor CD28 with its ligand B7. Although the requirement for CD28 co-stimulation has been the subject of intensive and extensive investigation, the molecular nature of this co-stimulatory signal is unknown. Some models have identified distinct kinase cascades initiated by either the ICR or CD28, while other models have focused on the convergence of TCR/CD28 signals on particular kinase cascades. One pathway which can mediate the synergistic responses that characterize CD28 co-stimulation is the MAP kinase (ERK) cascade. The activation of the MAP kinase ERK following CD28 co-stimulation is required for IL-2 production and proliferation of responding T lymphocytes. ERK activation in T lymphocytes is regulated by two antagonistic small G proteins: Ras and Rap1. Ras activation is required for ERK activation, while Rap1 antagonizes Ras signaling. Antigen recognition by T-cells in the absence of CD28 co-stimulation is characterized by impaired ERK activation and both decreased IL-2 production and diminished proliferation. This functional unresponsiveness results in the inability to respond to subsequent co-stimulatory signals and is termed clonal anergy. Rap1 is constitutively activated in certain states of I cell anergy or this unresponsiveness may account for the diminished ERK activity and decreased IL-2 production seen in anergic T-cells. In this proposal, we will test the hypothesis that Rap1 is activated by ICR ligation in normal I cells and consequently limits I cell activation through the ICR in the absence of co-stimulation. In addition, we will test the hypothesis that CD28 co-stimulation achieves increased ERK activation, IL-2 production and proliferation by blocking Rap1 activation.

A major hypothesis of this Program Project Grant is that intrauterine stresses can modify the program governing cell growth in adult life by dysregulating proliferative/hypertrophic signals within the heart. Understanding the signaling pathways that couple intrauterine stresses to cardiac growth are directly relevant to the overall goal of this PPG. We propose that selectively manipulating the proliferative and hypertrophic signals within the developing cardiomyocyte will provide an animal model of pathophysiological consequences of altered cardiac cell growth. We will establish genetic models in mice to test this hypothesis. The hypothesis to be tested in this proposal is that modifying hypertrophic and proliferative pathways converge to mediate the growth response in the heart. Studies by Kent Thornburg and co-workers have shown a requirement for extracellular signal-regulated kinase (ERK) and phosphoinositol-3 kinase (PI3-K) in the hyperplastic response of sheep myocardial cells to IGF. Recent studies suggest that PI3-K and ERK signals can also converge on hypertrophic signals via the mTOR pathway that regulates protein translation and growth. Importantly this pathway is also influenced by PI3-K and ERK signaling pathways. One of the major targets of mTOR action, the ribosomal p70 S6 kinase (S6K), is activated by both by PI3-K and ERK signaling pathways. We propose that activation of the mTOR pathway is sufficient for hypertrophy, and that both mTOR and ERKs are necessary for full hypertrophic response to developmental stresses. We predict that constitutive activation of the PI3-K/mTOR cascade is sufficient for cardiac hypertrophy and that both PI3-K/mTOR and ERK pathways are necessary for cardiac hypertrophy. This will be tested in three specific aims using genetically modified animal models of cardiac function.

DESCRIPTION (provided by applicant): This proposal examines the physiological function of a novel class of cAMP-regulated proteins, the exchange proteins activated by cAMP (Epacs) and tests the novel hypothesis that each isoform (Epac1 and ERpac2) carries out distinct physiological functions in the cell. This family of proteins binds cAMP directly and represents the major intracellular target of cAMP other than PKA itself. Our studies of Epac activation of small G protein Rap1 have demonstrated that subcellular localization of exchangers can dictate the choice of effectors utilized by the G proteins they activate (Wang et al. 2006;Liu et al. 2008). We have identified distinct targeting domains in both Epac 1 and Epac2 called Ras association (RA) domains (Liu et al. 2008). We propose that these domains allow specific small G proteins to link to Epac1 and Epac2, respectively, to the activation of distinct subcellular pools of Rap1. We propose that the RA domains of Epac1 and Epac2 link cAMP signaling to the small G proteins, Ran and Ras, respectively. This can explain how Ran couples to Rap1 (via the Epac1 exchanger) and how Ras can couple to Rap1 (via the Epac2 exchanger). This model and its physiological consequences for both Epac1 and Epac2 are examined in this proposal. We have discovered that Epac1 binds to the small G protein Ran at the nuclear pore. We will test the hypothesis that the RA domain of Epac1 couples Ran-GTP to Rap1 to regulate the transport of proteins in and out of the nucleus. This represents the first link of Ran to intracellular signaling cascades and identifies a novel function for both Epac1 and Rap. The significance of this finding is that the nuclear translocation of proteins via the Ran cycle governs cell growth and differentiation, and its dysregulation is a hallmark of many cancers. We show that Epac1 regulates the nuclear functions of the oncoprotein 2-catenin. We propose that this and related functions of Epac1 may explain some of the anti-proliferative effects of cAMP. In contrast to Epac1, we propose that Epac2 binds to activated Ras at the plasma membrane to link cAMP and Ras to Rap1 activation. This model of Epac2 function as a coincidence detector for signals through Ras and cAMP has significant implications for diabetes, as Epacs are now known to be targets of the sulfonylureas. We propose here for the first time a model that Ras and cAMP signals converge on Epac2 to potentiate insulin release. If true, this would establish an innovative approach to diabetes involving combining Ras activators with both insulinomimetic hormones and the sulfonylurea drugs. PUBLIC HEALTH RELEVANCE: Pharmacological therapeutics target the cyclic AMP (cAMP) signaling pathway more than any other pathway. The guanine nucleotide exchanger for the small G protein Rap called Epac is a new member of the cAMP signaling pathways and is directly activated by cAMP binding. This proposal tests the hypotheses that Epac promotes insulin release in both healthy and diabetic patients, and regulates the entry of proteins into the nucleus of normal and cancer cells through the non-overlapping actions of the two Epac isoforms, Epac1 and Epac2.

DESCRIPTION (provided by applicant): Nearly all human pancreatic adenocarcinomas (PACs) are caused by oncogenic (activating) mutations in the KRas gene. There are no treatments that reverse the actions of activated KRas mutants. The consequence of activating KRas mutations is the activation of a protein called Raf. Of the two major isoforms of Raf (C-Raf and B-Raf), C-Raf is the principle target of oncogenic Ras mutants in most cancers. A major function of KRas is to position C-Raf for activating phosphorylations. Our approach is to identify novel protein kinases that are required for KRas-dependent phosphorylation of C-Raf. There are two essential Ras-dependent phosphorylations of C-Raf, one on serine 338 (S338) and another on tyrosine 341 (Y341). These two sites within C-Raf are constitutively phosphorylated in KRas-mutant PACs, and are both required for the Ras-dependent cell proliferation. The tyrosine kinase Src is the only kinase that has been proposed to carry out the phosphorylation of Y341. A number of candidate kinases have proposed to carry out the phosphorylation of S338. We show preliminary pharmacological and molecular data challenging both of these models of C-Raf activation, and propose that both kinases have yet to be identified. We propose to identify the two kinases responsible for each of these phosphorylations by conducting biochemical screens using a panel of kinase inhibitors in a set of human PAC cell lines. We will use a functional siRNA screen to determine whether the identified S388 and Y341 kinases regulate KRas-dependent proliferation of PAC cell lines. The significance of this proposal is that the identification of novel serine and tyrosine kinases required for Ras-induced cell proliferation wil provide new therapeutic targets for inhibiting the growth of pancreatic adenocarcinomas. Despite the significance of discovering a new target for Ras-mutant cancers, this approach has not yet been attempted. The identification of new targets will contribute greatly to the field by providing new avenues for drug discovery, translational research, and ultimately novel therapeutics. Inhibition of either of these new targets will be specifically detrimental to the tumr cells, as they block signaling from oncogenic Ras to C-Raf while sparing signaling from normal Ras to B-Raf. This has the potential for increased drug tolerance and a higher therapeutic index.