The protein products of a number of retroviral oncogenes and growth factor receptors are protein tyrosine kinases. The key question is how the signal produced by tyrosine kinase activity is transduced into changes in cellular biochemistry leading to the transformed state. We have studied the phosphorylation of serine residues of ribosomal protein S6 as one biochemical event amenable to analysis that is stimulated by virtually all known protein tyrosine kinases. We have stimulated S6 kinase activity from insulin-treated oocytes, purified it to homogeneity, and shown it to be a substrate for activation by phosphorylation by another kinase known as microtubule-associated protein, or MAP kinase. MAP kinase itself is activated by phosphorylation on tyrosine and threonine residues by yet another kinase in the pathway known as MAP kinase activator. A major objective of this grant is to purify the activator to homogeneity, clone the gene for it, and determine the mechanisms of activation of the activator by insulin and other agents which work through - activation of tyrosine kinase activity. Other studies will evaluate oncogene and proto-oncogene interactions in the pathway of signalling that leads to activation of MAP kinase and S6 kinase. In particular, there appear to be important interactions between the v-src oncogene, which encodes a tyrosine kinase, the Harvey Ras oncogene which encodes a GTP binding protein, and the c- mos proto- oncogene which encodes a serine/threonine protein kinase activity. Each of these agents is capable of eliciting the activation of MAP kinase and S6 kinase by pathways that are either dependent or independent of one another. Using the Xenopus oocyte and egg system, as well as extracts from these cells, the action of these oncogenes will be studied singly and in combination in terms of the activation of MAP kinase and S6 kinase enzymes. Finally, the substrate specificity of the S6 kinase enzyme itself will be evaluated using site-directed mutagenesis to mutate particular domains of the enzyme involved in structure/function relationships. The enzyme is unusual in having two complete kinase domains, and the main focus of this work will be mutation of residues in one of the other kinase domain that may lead to changes in protein kinase activity and ability to be activated by MAP kinase. By continuing to characterize each step of the signalling pathway leading to S6 phosphorylation and working backwards up the pathway towards the tyrosine kinase activity of the insulin receptor, the complete regulation of this pathway of signal transduction should be elucidated.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
2R01DK028353-12
Application #
3228769
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1981-03-01
Project End
1996-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
12
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Colorado Denver
Department
Type
Schools of Medicine
DUNS #
065391526
City
Aurora
State
CO
Country
United States
Zip Code
80045
Silverman, Eran; Frodin, Morten; Gammeltoft, Steen et al. (2004) Activation of p90 Rsk1 is sufficient for differentiation of PC12 cells. Mol Cell Biol 24:10573-83
Eyers, Patrick A; Maller, James L (2004) Regulation of Xenopus Aurora A activation by TPX2. J Biol Chem 279:9008-15
Tunquist, Brian J; Maller, James L (2003) Under arrest: cytostatic factor (CSF)-mediated metaphase arrest in vertebrate eggs. Genes Dev 17:683-710
Tunquist, Brian J; Eyers, Patrick A; Chen, Lin G et al. (2003) Spindle checkpoint proteins Mad1 and Mad2 are required for cytostatic factor-mediated metaphase arrest. J Cell Biol 163:1231-42
Eyers, Patrick A; Erikson, Eleanor; Chen, Lin G et al. (2003) A novel mechanism for activation of the protein kinase Aurora A. Curr Biol 13:691-7
Tunquist, Brian J; Schwab, Markus S; Chen, Lin G et al. (2002) The spindle checkpoint kinase bub1 and cyclin e/cdk2 both contribute to the establishment of meiotic metaphase arrest by cytostatic factor. Curr Biol 12:1027-33
Maller, James L; Schwab, Markus S; Gross, Stefan D et al. (2002) The mechanism of CSF arrest in vertebrate oocytes. Mol Cell Endocrinol 187:173-8
Qian, Y W; Erikson, E; Taieb, F E et al. (2001) The polo-like kinase Plx1 is required for activation of the phosphatase Cdc25C and cyclin B-Cdc2 in Xenopus oocytes. Mol Biol Cell 12:1791-9
Schwab, M S; Roberts, B T; Gross, S D et al. (2001) Bub1 is activated by the protein kinase p90(Rsk) during Xenopus oocyte maturation. Curr Biol 11:141-50
Maller, J L; Schwab, M S; Roberts, B T et al. (2001) The pathway of MAP kinase mediation of CSF arrest in Xenopus oocytes. Biol Cell 93:27-33

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