Shirish Shenolikar - US grants

These studies will investigate the importance of the regulation of protein phosphatases to the control of cellular function. Functional interactions between cellular protein phosphatases and their regulators which determine the phosphorylation state and the biological activity of physiologically important proteins will be examined. Experiments designed to elucidate the mode of action of protein phosphatase inhibitor-2 to modulate the activity of protein phosphatase-1 will be undertaken. Studies will focus on the significance of the specific protein structure and the role of phosphorylation of inhibitor-2 in the regulation of protein phosphatase-1. Highly specific antibodies to selected primary sequences will be used to establish the structural determinants essential for inhibitor-2 function. Polyclonal and monoclonal antibodies will be used to study the significance of inhibitor-2 phosphorylation in vitro with purified protein kinases, and in vivo in intact cells in response to physiological stimuli. Protein- chemical and immunological analyses will be undertaken to examine the structural and functional relationship of inhibitor-2 to other phosphatase-1 inhibitors. Antibodies will also be used to examine the molecular and functional properties of inhibitor-2 in other tissues. Synthetic oligonucleotides corresponding to predetermined peptide sequences of inhibitor-2 will be used to clone the cDNA for inhibitor-2. The cDNAs will be used to: (1) generate chimeric gene products for the production of site-specific antisera; (2) produce fragments of inhibitor-2 as either fused or unfused proteins for structure-function studies; (3) identify inhibitor-2 mRNA in tissues, and quantitate the mRNA levels under various metabolic conditions; (4) determine the number of genes coding for inhibitor-2. The ultimate goals of these studies is to provide a clearer understanding of (a) the molecular mechanisms involved in the control of protein phosphatase-1 by its protein modulators, (b) the role of inhibitor-2 in the control of cellular dephosphorylation reactions, and (c) the physiological importance of such regulation to the processes which govern cellular function in mammalian tissues.

Most of the actions of cyclic AMP (cAMP) appear to be mediated via the activation of cAMP-dependent protein kinase. cAMP- dependent protein kinase phosphorylates and activates a protein phosphatase inhibitor, termed inhibitor-1, in skeletal muscle. The resulting inhibition of protein dephosphorylation would be predicted to further amplify the cellular response to hormones. The aim of this proposal is to utilize the current developments in molecular cloning to express the active fragment of inhibitor-1 initially in prokaryotic cells, and subsequently in eukaryotic cells to investigate its role in hormonal regulation. This will be achieved by construction of the synthetic gene for the 44 amino- acid minimal active fragment of rabbit skeletal muscle in E. coli. Biochemical characterization of the expressed peptide will be undertaken to establish its functional identity with inhibitor-1. The gene will be subcloned into both phagemid (for site-directed mutagenesis studies) and in eukaryotic vectors (to examine their effect on cellular regulation). The expression of the inhibitor-1 fragment in eukaryotic cells will be examined. Such studies will develop the probes necessary for the detailed investigation of the physiological role of protein phosphatase inhibitor-1 in mammalian cells.

9318101 Nadler This is a request for shared instrumentation to image and quantitate ion levels and pH in physiological studies on living cells. The requested Noran Odyssey laser scanning con focal microscope will enhance the research of five members of the Department of Pharmacol ogy by allowing the use of novel and innovative experimental approaches. The Noran instru ment has been optimized for the imaging of living tissue in real time. It will be used to investigate the following issues: (1) effects of regulatory substances on stimulus evoked sodium and calcium accumulation in glutamate nerve terminals, (2) NMDA receptor plastici ty in kindling and ontogenesis, (3) role of axon sprouting in facilitating or inhibiting seizure discharge and propagation, (4) role of calcium transients in regulating the cell cycle, (5) role of calmodulin in regulating insulin secretion from pancreatic SYMBOL 98 \f "Symbol" cells, (6) mechanism by which excess calmodulin leads to cardiac hypertrophy, (7) effects of glutamate and GABAA recep tor activation on intracellular calcium and chloride levels in hippocampal neurons, (8) effects of growth stimuli on intracellular pH, (9) role of protein phosphatase activity in the regula tion of cellular calcium levels and DNA synthesis, (10) induction of the protein phosphatase inhibitor 1 gene by calcium and (11) initiation of seizures by spontaneous action potential firing in axon terminals. These studies are aimed at understanding a variety of signaling mechanisms fundamental to cellular physiology. They will also help explain changes in cellular physiology and cell cell communication that occur in response to excessive or inju rious stimuli. u ~ -H- ~CAL3118TMP U. ~CRD0F04TMP #U. ~CAL3527TMP b5 ~CRD142ATMP . ~CRD0F38TMP b5 ~MF0E37 TMP E: ~DOC085BTMP s9 ~CAL2701TMP T~; ~CRD216FTMP X~; ~CAL3639TMP y< 9318101 Nadler This is a request for shared instrumentation to image and quantitate ion levels and pH in physiological studi ( 0 & ( !` ! F ( 0 ( 3 Times New Roman Symbol & Arial 1 Courier 9 " h % % = abstract Deseree King, BIR Deseree King, BIR

DESCRIPTION (Adapted from the Applicant's Abstract): Protein phosphorylation regulates many processes in animal cells. Protein kinases and phosphatases coordinately control the phosphorylation state of cellular proteins. Mechanisms that can temporarily dampen counteracting phosphatases can further amplify hormonal signals. We have investigated phosphatase inhibitor-1 (I-1) as a prototype mechanism for direct communication between a protein kinase and a phosphatase. Increases in cAMP levels activate PKA and promote phosphorylation and activation of I-1. This results in the inhibition of a major cellular phosphatase, PP1. Coordination of PKA and PP1 functions by I-1 greatly amplifies the cAMP signal, as demonstrated by expression of activated I-1 in cells which increases their sensitivity to cAMP and prolongs their physiological response. However, the molecular basis for I-1 s function as a PP1 regulator remains poorly understood. The investigators will undertake detailed structure-activity analysis of recombinant human I-1 to define its mode of action. A specific goal of the investigators' studies is to generate forms of I-1 and PP1 with altered regulatory properties. Expression of mutant and wild-type proteins in cells will further define I-1's role in cell signaling. Environmental toxins that inhibit the major cellular phosphatases, including PP1, have not only emphasized the physiological importance of endogenous inhibitors like I-1 but have raised the possibility that I-1 deregulation may contribute to human disease. Defining the functional interactions between I-1 and PP1 will lead to the design of dominate negative reagents that will be used not only to elucidate I-1 s role in normal physiology but reverse deleterious effects on I-1 overexpression. These studies are relevant to targeting phosphatases to modulate hormonal responses and ameliorate metabolic dysfunctions in many human diseases.

DESCRIPTION (provided by applicant): Protein phosphatase-1 (PP1), is a major serine/threonine phosphatase in the mammalian synapse and has been implicated in the control of long-term potentiation (LTP) and long-term depression (LTD) in the hippocamapal neurons, extensively studied models for learning and memory. PP1 association with the actin cytoskeleton underlying postsynaptic membranes is mediated by two isoforms of neuronal-actin-binding proteins (neurabins) that also bind PP1. This positions PP1 to respond rapidly to receptor activation and regulate postsynaptic substrates involved in plasticity. Disruption of the mouse neurabin II gene abolished LTD and PP1 regulation of glutamate receptors but increased the formation of filopodia and spines, which are also required for strengthening and elaborating neural circuits during learning and memory. Our preliminary studies suggest that PP1 recruitment to the neuron-specific isoform, neurabin I, represents a mechanism for inducing filopodia formation. The aims of this proposal is the (1) delineate the molecular basis for PP1 recruitment and regulation by neurabin I; (2) establish the role of neurabin I in the antagonism between PP1 and the growth-factor-activated kinase, p70 s6 kinase, which binds to an adjacent site on neurabin I and inhibits neurite extension; and finally (3) define the physiological importance of recruiting the known PP1 regulator, inhibitor-2, to neurabin I, it role in stabilizing PP1 association with neurabin I and the impact of I-2 phosphorylation-dephosphorylation in regulating the neurabin I-bound phosphatase. Together, these studies will define the importance of neurabin I in PP1 signaling at the actin cytoskeleton and its role in the signal transduction pathways that regulate synaptic plasticity. Elucidating neurabin-mediated PP1 signaling may define the defects in plasticity that contribute to mental retardation, epilepsy and neurodegenerative disease.

DESCRIPTION (provided by applicant) Harnessing the activity of protein kinases and phosphatases is crucial for signal transduction by hormones and growth factors. Inhibitor-1 (I-1) represents the first mechanism identified for hormonal control of protein phosphatases. PKA phosphorylates and activates I-1 to suppress the activity of PP1, a major cellular protein serine/threonine phosphatase and amplify the cellular responses to the second messenger, cyclic AMP. Though identified as a regulator of glycogen metabolism in skeletal muscle, I-1 is widely expressed and has been implicated in many physiological processes. Our studies identified the growth arrest and DNA damage-inducible gene product, GADD34, as a novel protein scaffold that recruits PP1 and I-1 to regulate protein translation. The PP1-binding domain of GADD34 shares structural homology to the protein product of the Herpes Simplex Virus HSV-1 gamma-34.5 gene that generates a phosphatase that dephosphorylates the eukaryotic translation initiation factor, eIF2alpha, and facilitates protein synthesis. Our studies suggest that the PP1/GADD34/I-1 complex is a major eIF2alpha phosphatase in many mammalian tissues and is disassembled in response to cell stress, such as hypoxia and nutrient deprivation. This results in increased eIF2alpha phosphorylation and shut-down of protein synthesis. Persistent phosphorylation of eIF2alpha has also been linked to programmed cell death. The discovery of a mutation in the gene encoding the major human pancreatic eIF2alpha kinase in Wolcott-Rollins (WR) syndrome suggests that eIF2alpha phosphorylation-dephosphorylation controls insulin synthesis and the survival of pancreatic beta-cells. Thus, WR individuals develop early insulin-dependent diabetes and other serious disorders. These and other studies suggest that eIF2alpha phosphorylation functions as an important physiological mechanism for sensing glucose and nutrient availability. Thus, understanding the structure-function and regulation of the eIF2alpha phosphatase should provide new avenues for the treatment of viral infections, diabetes and other human diseases. The Specific Aims of this project are: 1) Utilize genetic and biochemical assays to define the molecular basis for I-1 as a regulator of PP1 function, specifically the dephosphorylation of eIF2alpha and initiation of protein translation. 2) Define the role of GADD34 in the assembly of the mammalian eIF2alpha phosphatase and the transduction of physiological signals that regulate protein synthesis and dictate the switch between cell death and survival.