Kun-Liang Guan, PhD - US grants

? DESCRIPTION (provided by applicant): The mechanistic target of rapamycin (mTOR) controls cell growth by integrating a wide range of signals, such as growth factors, nutrients, and stress. mTOR is a conserved protein kinase that forms two complexes, referred to as TORC1 and TORC2, with distinct subunit composition and physiological functions. The physiological function of TORC1 in promoting cell growth is well characterized and inhibitors that target the complex are used clinically to treat several diseases including cancer, transplant rejection, and restenosis. The mechanism of TORC1 regulation is a topic of intense interest in both basic cell biology and translational research. Growth factors and nutrients activate TORC1 to promote cell growth by stimulating anabolism and inhibiting catabolism. Previous studies have investigated TORC1 regulation by growth factors and nutrients. However, the molecular insights of inhibitory signals that regulate TORC1 are ambiguous. Both oxidative stress and hyperosmotic stress rapidly and potently inhibit TORC1. Our preliminary studies indicate that the nemo like kinase (NLK) plays a crucial role in TORC1 inhibition in response to hyperosmotic or oxidative stress. In addition to oxidative and hyperosmotic stress, we observed that protein kinase A (PKA) mediates an inhibitory effect to mTORC1 in response to the second messenger cyclic AMP (cAMP). The long-term goal of this project is to elucidate the molecular mechanisms of TORC1 regulation and the function of TORC1 in cell physiology and disease. In this proposal, we will determine the biological function and molecular mechanism of NLK in mediating the osmotic and oxidative stress to inhibit TORC1. Furthermore, we will investigate the mechanisms of mTORC1 inhibition by PKA and functional cross talk between cAMP and mTORC1. The specific aims for this proposal are: 1. Determine the function of NLK in TORC1 regulation by osmotic and oxidative stress 2. Elucidate the biochemical basis of NLK in TORC1 inhibition by osmotic and oxidative stress 3. Investigate the molecular mechanisms and functional role of TORC1 inhibition by cAMP-PKA

The long term objective of this project is to understand the physiological functions and regulation of protein tyrosine phosphatases (PTPs). Tyrosine phosphorylation plays pivotal roles in regulating cell proliferation, differentiation, and oncogenic transformation. The importance of these regulatory mechanisms, such as the activation of the mitogen activated protein (MAP) kinase, is shown to be conserved from yeast to human. To understand the physiological function and regulation of PTPs, the budding yeast Saccharomyces cerevisiae is chosen as the model system because both mechanism and molecular genetic methods can be readily applied in this organism. Preliminary studies from our laboratory demonstrate that PTP2 and PTP3 have overlapping functions in the mating pheromone signal transduction and are required for meiosis and sporulation. PTP2/PTP3 may be involved in the recovery of mating by directly inactivating the pheromone responsive FUS3/KSS1 MAP kinases. The first aim is to understand the molecular mechanisms of PTPs in regulation of mating pheromone signal transduction. The physiological functions of PTP2/PTP3 in regulation of the FUS3/KSS1 MAP kinases will be elucidated. Both genetic and biochemical experiments will be performed to demonstrate that FUS3/KSS1 are the direct in vivo substrates of PTP2/PTP3. Functional relationship among PTPs will be tested by combinatorial disruptions. New regulators of the pheromone activated MAP kinase pathway will be isolated and characterized. The second specific aim is to study the physiological functions and regulation of PTP2 and PTP3 in meiosis and sporulation. The requirements of PTP2/PTP3 for meiosis/sporulation demonstrate that tyrosine phosphorylation plays an essential role in the regulation of this process. The role of PTP2/PTP3 in meiotic initiation and commitment will be determined. The significance of PTP2/PTP3 for either ploidy or nutritional control of sporulation will be elucidated. In vivo, substrates whose dephosphorylation is essential for sporulation will be identified and characterized using both biochemical and molecular genetic approaches. Experiments in this proposal aim to establish the relationships between phosphatases, their substrates, and the biological functions they carry out by focusing on the molecular mechanisms of PTP2 and PTP3 in regulation of the mating pheromone signal transduction and the meiosis/sporulation processes. The completion of this proposal will provide important information of PTP in cellular regulation and build a valuable model for the functions and regulation of PTP in higher eukaryotes.

DESCRIPTION (Adapted from the applicant's abstract): The overall goal of this proposal is to understand the signal transduction mechanisms of the Ras protooncogene. It is well established that Ras acts as a molecular switch existing between GDP- and GTP- bound forms. The GTP-bound Ras is active, and directly binds to and regulates downstream effector molecules while GDP-bound Ras is thought to be inactive. Recent evidence suggests that GDP-bound Ras may also have signaling functions rather than being an inactive form as predicted by the current model. The investigator has found that smgGDS (small GTPase GDP dissociation stimulator), which is an activator for several small GTPases, specifically binds to GDP-bound but not GTP-bound H-Ras. The main goal of this proposal is now to demonstrate that GDP-bound Ras has an active role in signal transduction. The focus is to establish that GDP-bound Ras regulates the activities of smgGDS and to demonstrate that smgGDS functions as a physiological downstream effector for GDP-bound Ras. The specific aims are to examine: 1) Interaction between smgGDS and Ras-GDP; 2) Regulation of smgGDS activity by Ras; and 3) Role of smgGDS in Ras-GDP signal transduction

[unreadable] DESCRIPTION (provided by applicant): Tuberous sclerosis complex (TSC) is a genetic disease caused by mutations in either the TSC1 or TSC2 tumor suppressor gene. TSC is characterized by hamartoma formation in a wide range of tissues with the most severe clinical problems in brain and kidney. The TSC2 tumor suppressor protein has a conserved GTPase activating protein (GAP) domain and plays a key role in regulation of protein synthesis and cell size. The major cellular function of TSC2 is to decrease the phosphorylation of S6K (ribosomal S6 kinase) and 4EBP1 (eukaryotic initiation factor 4E binding protein), possibly by inhibiting mTOR (mammalian target of rapamycin). Rheb (Ras homology enriched in brain) is a Ras family small GTPase whose physiological function was previously unknown. Recently, it has been demonstrated that TSC2 directly stimulates the GTP hydrolysis of Rheb. Furthermore, Rheb can stimulate phosphorylation of both S6K and 4EBP1. These observations suggest that Rheb may function between TSC2 and mTOR to regulate cell growth. The long term objectives of this project are to elucidate the physiological functions of Rheb and to demonstrate that Rheb acts downstream of TSC2 and upstream of mTOR to regulate cell growth, cell size, and cell survival. The specific aims for this proposal are: [unreadable] 1. To perform biochemical and functional characterizations of TSC2 GAP and Rheb GTPase activity. [unreadable] 2. To investigate the mechanism of Rheb in mTOR regulation. [unreadable] 3. To elucidate the function of Rheb in TSC2 signaling, cell size control, cellular energy response, and cell survival. [unreadable] 4. To demonstrate Rheb as a key cellular target of FTI (famesyl transferase inhibitor) in cell growth inhibition. [unreadable] 5. To identify and characterize Rheb interacting proteins. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Tuberous sclerosis complex (TSC) is a genetic disease characterized by hamartoma formations in a wide range of tissues. Mutation in either the TSC1 or TSC2 tumor suppressor gene is responsible for TSC. TSC1 and TSC2 play a major role in the regulation of cell growth and cell size through control of protein synthesis. The TSC1/TSC2 complex functions upstream of the mammalian target of rapamycin (mTOR) and inhibits mTOR function. These observations suggest that TSC1/TSC2 are key regulators of cell growth and tumor formation. Protein synthesis is regulated by multiple intracellular and extracellular signals, such as mitogenic growth factors, nutrient sufficiency, and cellular energy levels. Cellular energy starvation results in inhibition of both protein synthesis and cell growth. However, the molecular mechanisms coordinating cellular energy level and cell growth is not well understood. Recent studies from our laboratory have indicated that TSC2 plays a major role in coordinating cellular energy levels and cell growth. The major goal of this proposal is to study the function and regulation of TSC2 in the cellular energy response. Completion of this proposal will address the fundamental cell biology question of the coordination between cell growth and cellular energy levels and will provide an exciting mechanism of how multiple signaling pathways are integrated at the molecular level. Results from this project will reveal a molecular basis for tumorigenesis induced by disregulation of the Wnt pathway. The following specific aims will be addressed. 1. To determine the function of GSK3 and Wnt in the TSC-mTOR-S6K pathway. 2. To determine whether AMPK and GSK3 collaboratively phosphorylate and regulate TSC2 under energy starvation conditions. 3. To elucidate the physiological functions of TSC2 phosphorylation by AMPK and GSK3 in cellular energy response. 4. To investigate the mechanism of TSC2 in energy starvation-induced cell death.

DESCRIPTION (provided by applicant): The Hippo tumor suppressor pathway functions to limit tissue growth and organ size by inhibiting proliferation and inducing apoptosis. Dysregulation of the Hippo pathway contributes to tumorigenesis. The key downstream effectors of the Hippo pathway are the transcription co-activator YAP, which is phosphorylated and inhibited by the Hippo pathway kinase Lats. YAP overexpression and hyperactivation are found in human cancers. Extensive studies have identified many intracellular proteins that modulate the Hippo pathway. However, key questions regarding the extracellular signals and cell surface receptors for the Hippo pathway have not been addressed. We recently discovered that G-protein coupled receptors (GPCR) and their cognate ligands regulate the Hippo pathway. GPCR modulates many intracellular signaling molecules including protein kinase A (PKA) and protein kinase C (PKC). PKA is activated by cAMP, a second messenger that is elevated by stimulation of Gs-coupled receptor. PKC is activated by diacylglycerol that is also a second messenger elevated by Gq/11- coupled receptors. Both PKA and PKC are involved in a wide range of cellular regulation, including gene expression and cell growth. Our preliminary studies reveal that PKA and PKC potently modulate YAP. PKA inhibits YAP by increasing phosphorylation while PKC activates YAP by inducing dephosphorylation. The long- term goal of this project is to elucidate the mechanism of YAP regulation by PKA and PKC, to understand the regulation and function of the Hippo-YAP pathway in cell growth, organ size, tumorigenesis and cancer metastasis, and to provide potential therapeutic targets for cancer treatment.

DESCRIPTION (provided by applicant): The Ras family small GTPases are molecular switch involved in controlling a wide range of cellular activities, including proliferation, growth, morphology, migration, intracellular trafficking, nuclear import/export. Post-translational modifications of the C-terminal CAAX motif are important for membrane association and biological functions of many Ras family proteins. Of particular importance is the isoprenylation of C-terminal cysteine in the CAAX motif. Isoprenylation is also present in many other proteins and is essential for their cellular functions. GCN2 is a multi-domain protein kinase involved in nutrient sensing and translation regulation. Our preliminary studies indicate that GCN2 affects the post-translational modifications of Ras and Rheb. The major goal of this proposal is to determine the molecular mechanism of GCN2 in regulation of the post-translational modifications and biological functions of the Ras family GTPases. Biochemical, cell biological, and genetic approaches will be used to achieve our goals. PUBLIC HEALTH RELEVANCE: Ras is the most frequently mutated oncogene in human cancer. The Ras family GTPases regulate normal cell growth but dysregulation can cause diseases, such as cancer. Our goal is to understand how Ras function is regulated. These studies may lead to new therapeutic treatment for cancer.

DESCRIPTION (provided by applicant): The mammalian target of rapamycin (mTOR) is a central growth controller. Recent studies have elucidated a conserved signaling pathway consisting of TSC1/TSC2-Rheb-mTOR. TSC1 and TSC2 are two tumor suppressor genes mutated in the tuberous sclerosis. The TSC1/TSC2 complex functions as a GTPase activating protein (GAP) to inhibit the Rheb small GTPase, which is a potent activator of mTOR. This signaling pathway integrates a wide range of extracellular and intracellular signals to regulate cell growth. mTOR activity is rapidly and dramatically regulated by the availability of cellular energy and amino acids. Previous studies have established that the TSC-mTOR pathway plays a critical role in the coordination between cell growth and nutrient availability at the cellular level. The major focus of this proposal is to investigate the function of TSC- Rheb-mTOR pathway in organismal energy balance and to elucidate the mechanism of this pathway in regulation of leptin signaling, appetite control, and energy expenditure. We will use mouse genetics and cell biological techniques to achieve these goals. The specific aims for this proposal are: Aim 1. To determine the function of TSC1 in POMC neurons in appetite and metabolic control and obesity Aim 2. To determine the function of TSC1 in AGRP/NPY neurons in regulation of appetite Aim 3. To elucidate the mechanism of mTOR activation in inducing leptin resistance and hyperphagia Aim 4. To determine the effect of low mTOR activity on food intake, metabolism, obesity, leptin sensitivity, and resistant to high fat diet-induced obesity. PUBLIC HEALTH RELEVANCE The TSC-mTOR pathway plays a major role in hormonal and nutritional signals to regulate cell growth. This proposal will investigate the function of TSC-mTOR in affecting leptin signaling and appetite control. The information generated from this project will provide new insights into to appetite regulation, obesity, and diabetes.

DESCRIPTION (provided by applicant): The Hippo tumor suppressor pathway plays a crucial role in regulating organ size by inhibiting cell proliferation and promoting apoptosis, and limitin stem/progenitor cell self- renewal and expansion. The YAP/TAZ transcription co-activators are the major downstream effectors of the Hippo pathway. Despite extensive studies, upstream signals regulating the Hippo pathway are unknown. Currently, no extracellular ligand or cell surface receptor has been identified to regulate the mammalian Hippo-YAP. Our preliminary studies have discovered that lysophosphatidic acid (LPA) and sphingosine 1- phosphophate (S1P) are important signaling molecules that regulating the Hippo pathway. LPA and S1P act through their respective G-protein coupled receptors (GPCRs) to activate YAP. Both LPA and S1P have been implicated in cancer development and metastasis. Notably, activating mutations of Gq/11 are frequently found (83%) in uveal melanoma, which is the most common intraocular tumor in the eye with strong propensity of metastasis into the liver. Our preliminary study showed that active Gq/11 potently stimulates YAP activity. The major goals of this proposal are to investigate the mechanism of Hippo-YAP regulation by GPCR and to determine the functional significance of YAP/TAZ activation in the biology of LPA, S1P and other extracellular signals. Moreover, we will investigate the pathophysiological function of YAP/TAZ activation in the development of uveal melanoma and aim to provide scientific basis for treatment of this disease.

DESCRIPTION (provided by applicant): The mammalian target of rapamycin, mTOR (also known as mechanistic target of rapamycin) controls cell growth. Dysregulation of mTOR has been implicated in many human diseases and drugs inhibiting mTOR have been approved for cancer treatment. mTOR forms two distinct structural and functional complexes, mTORC1 and mTORC2. mTORC1 is the rapamycin sensitive target and plays a major role in cell growth by promoting protein synthesis and anabolism, and inhibiting autophagy. The precise regulation of mTORC1 is critically important for cell growth and organism development. mTORC1 activity is controlled by cellular energy levels, nutrients, and growth factors. Amino acids have emerged as a major signal for mTORC1. The Rag family GTPases has been identified as key mediators relaying amino acid signals to mTORC1 activation. However, the physiological function of Rag GTPases and their role in mTORC1 regulation in vivo are largely unknown. The major goal of this proposal is to determine the in vivo function of Rag GTPases in mTORC1 regulation and physiology using genetically altered mouse models. In addition, we have discovered that amino acids can still activate mTORC1 even when the RagA and RagB genes are deleted. We will investigate the Rag-independent amino acid signaling to mTORC1 activation.

? DESCRIPTION (provided by applicant): Understanding the basic mechanisms of cell growth regulation and how alteration of such regulatory networks leads to cancer is fundamentally important for understanding cancer development and designing new strategies for treating cancer. The mTORC1 and Hippo signaling pathways are two major pathways that control cell growth and tissue/organ homeostasis. mTORC1 is a central cell growth controller which promotes cell growth by stimulating biosynthesis and inhibiting autophagy. The Hippo tumor suppressor pathway limits tissue and organ size by inhibiting proliferation and stimulating apoptosis. Dysregulation of either pathway contributes to human cancer. As such, mTORC1 inhibitors have received FDA approval for cancer treatment and there is an intensive effort in searching for drugs that can target the Hippo pathway for cancer indication. Previous works from the PI have revealed the molecular mechanisms of mTORC1 regulation by growth factors and cellular energy status via AKT and AMPK, respectively. Amino acids are arguably the most important stimuli of mTORC1. The PI has also identified Rag GTPases as critical mediators of mTORC1 activation by amino acids. Despite rapid progress in the field, key issues in amino acid signaling to mTORC1, such as the nature of amino acid sensors, remain to be solved. One major goal of this R35 proposal is to elucidate the molecular mechanism of mTORC1 activation by amino acids. The Hippo pathway is an exciting emerging field. The PI's group has made key contributions in establishing the major framework of the Hippo pathway, including identification of upstream signals, the biochemical mechanism of YAP regulation, and demonstration of the YAP-TEAD transcription module. However, fundamental issues such as regulation of core Hippo pathway components, molecular basis of YAP in promoting oncogenesis, and physiological signals that control organ size are key open questions that have yet to be answered. The overall mission of this R35 proposal is to obtain a comprehensive molecular understanding of the mTORC1 and Hippo pathways under normal physiological conditions and to elucidate how dysregulation of these pathways contributes to tumorigenesis.

Abstract Uveal melanoma is the most common ocular tumor in adults. It harbors genetic mutations very different from those seen in cutaneous melanoma. Liver metastasis is common in uveal melanoma and contributes to the very poor prognosis with average survival of several months. Currently there is no treatment for metastatic uveal melanoma. Activating mutations in GNAQ and GNA11 are the most important cancer drivers, as approximately 80% of uveal melanomas have mutations in either GNAQ or GNA11. Our recent studies have shown that the mutant GNAQ/11 potently activates the YAP oncoprotein, which is a key component of the Hippo tumor suppressor pathway. Moreover, the elevated YAP activity is essential for tumor growth of uveal melanoma cells containing an activating mutation in GNAQ/11. Besides GNAQ/11, mutations in BAP1, SF3B1, and EIF1AX are also frequently observed in uveal melanoma in a mutually exclusive manner. However, a mechanistic understanding of these genes in uveal melanoma and their functional interactions are largely unknown. The major goals of this project are to determine the contributions of the genes commonly mutated in uveal melanoma and their functional interaction in promoting tumorigenesis of uveal melanoma. We also aim to characterize and validate potential therapeutic targets and tools for treatment of uveal melanoma.