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.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK041077-04
Application #
3241677
Study Section
Physiological Chemistry Study Section (PC)
Project Start
1988-12-01
Project End
1993-11-30
Budget Start
1991-12-01
Budget End
1992-11-30
Support Year
4
Fiscal Year
1992
Total Cost
Indirect Cost
Name
University of Virginia
Department
Type
Schools of Medicine
DUNS #
001910777
City
Charlottesville
State
VA
Country
United States
Zip Code
22904
Santiago, J; Sturgill, T W (2001) Identification of the S6 kinase activity stimulated in quiescent brine shrimp embryos upon entry to preemergence development as p70 ribosomal protein S6 kinase: isolation of Artemia franciscana p70S6k cDNA. Biochem Cell Biol 79:141-52
Smith, J A; Poteet-Smith, C E; Lannigan, D A et al. (2000) Creation of a stress-activated p90 ribosomal S6 kinase. The carboxyl-terminal tail of the MAPK-activated protein kinases dictates the signal transduction pathway in which they function. J Biol Chem 275:31588-93
Smith, J A; Poteet-Smith, C E; Malarkey, K et al. (1999) Identification of an extracellular signal-regulated kinase (ERK) docking site in ribosomal S6 kinase, a sequence critical for activation by ERK in vivo. J Biol Chem 274:2893-8
Poteet-Smith, C E; Smith, J A; Lannigan, D A et al. (1999) Generation of constitutively active p90 ribosomal S6 kinase in vivo. Implications for the mitogen-activated protein kinase-activated protein kinase family. J Biol Chem 274:22135-8
Malarkey, K; Coker, K J; Sturgill, T W (1998) Ribosomal S6 kinase is activated as an early event in preemergence development of encysted embryos of Artemia salina. Eur J Biochem 251:269-74
Reardon, D B; Dent, P; Wood, S L et al. (1997) Activation in vitro of somatostatin receptor subtypes 2, 3, or 4 stimulates protein tyrosine phosphatase activity in membranes from transfected Ras-transformed NIH 3T3 cells: coexpression with catalytically inactive SHP-2 blocks responsiveness. Mol Endocrinol 11:1062-9
Dent, P; Wang, Y; Gu, Y Z et al. (1997) S49 cells endogenously express subtype 2 somatostatin receptors which couple to increase protein tyrosine phosphatase activity in membranes and down-regulate Raf-1 activity in situ. Cell Signal 9:539-49
Rani, C S; Wang, F; Fuior, E et al. (1997) Divergence in signal transduction pathways of platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) receptors. Involvement of sphingosine 1-phosphate in PDGF but not EGF signaling. J Biol Chem 272:10777-83
Jelinek, T; Dent, P; Sturgill, T W et al. (1996) Ras-induced activation of Raf-1 is dependent on tyrosine phosphorylation. Mol Cell Biol 16:1027-34
Dent, P; Reardon, D B; Wood, S L et al. (1996) Inactivation of raf-1 by a protein-tyrosine phosphatase stimulated by GTP and reconstituted by Galphai/o subunits. J Biol Chem 271:3119-23

Showing the most recent 10 out of 34 publications