Thomas W. Sturgill - US grants

A complete understanding of the biochemical pathways used for insulin signalling is essential for understanding pathogenesis of diabetes mellitus and designing new therapies. We identified a serine/threonine kinase, termed MAP kinase, which is activated by insulin prior to S6 kinase in 3T3- L1 cells. Extensive use will be made of a procedure we developed for rapid isolation and stabilization of activated MAP kinase, resolved from other kinases and phosphatases. We are focusing on the following questions: 1. Analysis of Mechanisms of Regulation of MAP Kinase Activity by Insulin. Considerable effort will go into analyzing the role of phosphorylation in this regulation. (a) Inactivation of activated MAP kinase by phosphatases will be studied in vitro. (b) Inactivation of MAP kinase (isolated from 32PO4 -labelled cells) by phosphatase will be correlated with changes in phosphoamino acid content. (c) Different strategies for reactivating MAP kinase (previously inactivated by phosphatase) by tyrosine specific kinases and/or Ser/thr kinases will be investigated. (d) The relatedness of MAP kinase and the 40 kDa substrate for mitogen stimulated tyrosine phosphorylation will be tested. 2. Analysis of Cellular Functions of MAP Kinase. In this regard, most of our attention will be focused on expanding our collaborative investigation, of the possible involvement of MAP kinase in a mitogenic cascade for activation of S6 kinase. (a) Activation of S6K II inactivated by serine- specific phosphatases by MAP kinase will be studied in vitro. (b) The phosphorylation sites in S6 KII for MAP kinase will be studied by tryptic peptide mapping. (c) Activation of MAP kinase in oocytes stimulated with insulin will be studied as a precondition for involvement of MAP kinase in S6 KII activation in Xenopus oocytes stimulated with insulin. (d) In vivo phosphorylation of S6 KII by MAP kinase will be tested by comparison of tryptic peptide maps of S6 KII isolated from oocytes stimulated with insulin to S6 KII phosphorylated in vitro with MAP kinase. 3. Further Characterization and Purification of MAP Kinase. (a) Attempts will be made to provide additional proof that the 40 kD a phosphoprotein is the kinase by renaturation of the band eluted form SDS gels, azido-ATP labelling, and immunoprecipitation of MAP kinase activity and labelled ppp40 with anti-pY IgG. (b) A precursor for MAP kinase will be sought. (c) Methods will be developed for isolation of sufficient MAP kinase from a bulk source for microsequencing for production of anti-peptide antibodies and nucleic acid probes.

A complete understanding of the biochemical pathways used for insulin signalling is essential for understanding pathogenesis of diabetes mellitus and designing new therapies. We identified a serine/threonine kinase, termed MAP kinase, which functions in an insulin-stimulated protein kinase cascade to mediate at least some of the cellular actions of insulin, including its hallmark-activation of glycogen synthase. To define the upstream pathways for activation of MAP kinase, we are focusing on the following questions, utilizing both biochemical and molecular genetic approaches. 1. Characterization of cDNAs for MAP Kinase Kinase (MAPKK). We have isolated and sequenced a cDNA (K28) that encodes a MAPKK. In addition, the strategy for molecular cloning allowed isolation of additional clones at least one of which (K5) encodes an additional MAPKK related protein. Analysis of the clones will be completed and all distinct MAPKK homologs therein characterized. 2. Utilization of MAPKK cDNAs to generate reagents needed for mechanistic studies. Development of several reagents and procedures are necessary preliminaries for critical biochemical investigations of the upstream pathways: recombinant MAPKK(s), anti-MAPKK antibodies, site-directed mutants of MAPKKs. 3. Analysis of Mechanism(s) of activation of MAPKK by insulin. Characterization of insulin-stimulated phosphorylation of MAPKK in intact cells. Determination of the basis of MAPKK protein heterogeneity. Identification and purification of insulin-stimulated activator(s) of MAPKK by reactivation assays. Assessment of nuclear targeting of MAPKK. 4. Investigation of the role of p21 ras in activation of MAPKK by insulin. Several studies point to p21 ras as an upstream component of the MAP kinase pathway and will be pursued by: development of an in vitro system for activation of MAP kinase by p21 ras in mammalian cells and utilization of the system to identify and purify the target protein for p21 ras responsible for activation of MAPKK. 5. Examination of possible association/interaction of upstream components of MAP kinase pathway with Insulin Receptor Substrate 1 (IRS-1) (collaborative with Morris White, Joslin Diabetes Center) Tyrosine phosphorylated IRS-1 is hypothesized to be a 'docking' protein for signal transducing proteins, and therefore selective examination of associations of IRS-1 with components of the MAP kinase pathway will be made.

DESCRIPTION (provided by applicant): This proposal is to study regulation and function of MAP kinase-activated protein kin ses (MAPKAPKs), which are important, expanding, and understudied components in the MAP kinase cascades. One key role of MAPKs is to control gene expression. MAPKs and MAPKAPKs collaborate in transcription, translation, and stabilization. Understanding this collaboration is vital to design of therapies in many diseases because MAP (mitogen-activated protein) kinase (MAPK) cascades are activated by extracellular signals (growth factors, hormones, cytokines) and intracellular signals (cellular stresses and checkpoints) as part of interconnected cascades of enzyme activations and inhibitions controlling metabolism, gene expression, and growth and development. Abnormal functions of the pathways is involved in cancer, proliferative complications of diabetes, and genetic disorders. There are three principal MAPKs (ERK, JNK, and p38 MAPK) but there are many isoforms. MAP kinases select specific targets for regulation, including members of a diverse group called collectively MAPKAPKs which extend the cascade. MAPKAPKs are related to each other by their C-terminal domains independent of whether they have one kinase domain (MNKs, MAPKAPK2) or two domains (RSKs, MSKs). Aim one is determine how MAP kinases bind and regulate their specific MAPKAP kinase targets. Aim two is to study the cellular functions of MAPKAPKs by surveying the mammalian proteome for phosphorylations inducible by MAPKAPKs. Aim three is to design and use recombinant activated COOH-terminal domains (CTDS) of RSKs and MSKs to characterize their CTD kinase activity independently of the NTD domain, and to characterize nuclear RSKs and MSKs by extraction and chromatography. Aim four is to characterize the activation, enzymatic properties, and functions of MAPKAP kinase-like enzymes (Rcklp, Rck2p) in yeast, a model eukaryote, using combined biochemical and genetic approaches.

DESCRIPTION (provided by applicant): mTOR is a protein kinase that signals in pathways involved in the control of cell growth and proliferation. mTOR function is stimulated by both insulin/growth factors and certain amino acids;however, just how the essential kinase activity is controlled in cells is a mystery. Inappropriate activation of mTOR leads to insulin resistance, a contributing factor in the pathogenesis of type 2 diabetes. mTOR inhibitors are used as immunosuppressants, and trials are underway to evaluate their efficacy in treating cancer. Clearly, there is a need to understand better the control and function of mTOR. Two mTOR signaling complexes were recently discovered. mTORC1 contains mTOR, mLST8 (homologous to G protein beta subunits), and raptor, which binds substrates phosphorylated by mTOR. Rapamycin inhibits mTORC1, which probably mediates most of the functions now attributed to mTOR. mTORC2 is not inhibited by rapamycin, and the downstream targets of mTORC2 are largely unknown. mTORC2 contains mTOR, mLST8, and pianissimo, a protein originally implicated in the control of adenylate cyclase. We have searched for new mTOR interacting proteins by yeast two hybrid screening and by performing mass spectrometric (MS) analyses of proteins that immunoprecipitate with mTOR. In investigating an interaction involving eIF3, a key translation initiation factor, we discovered a dramatic rapamycin-sensitive action of insulin to stimulate the association of eIF3 and eIF4G. Aim 1 is to investigate this novel and potentially important effect of insulin. Aim 2 is to investigate the function and control of mTORC1 and mTORC2. We will test the hypothesis that insulin increases mTORC1 activity by increasing substrate binding to raptor. Based on preliminary findings, we also propose to test the hypothesis that insulin promotes formation of mTORC2, to identify the phosphorylation sites in pianissimo, and to investigate connections between mTORC2 and cAMP production. Aim 3 is to identify new upstream effectors and downstream targets of mTORC1 and mTORC2. We will investigate novel candidate mTOR-interacting proteins identified by MS. Finally, to understand better the mechanisms involved in mTORC2, we propose to identify pianissimo-interacting proteins.