Further characterize the ribosomal S6 phosphoprotein caused by the phorbol ester tumor promoter 12-O-tetradeanoyl phorbol-13-acetate (TPA). These further studies will involve: a) Determining the maximal number of sites phosphorylated on the S6 molecule in response to TPA treatment. b) Employ peptide mapping of S6 to compare the specific phosphorylated sites on S6 produced by TPA in vivo with those sites phosphorylated in vitro by cGMP-dependent protein kinase, cAMP-dependent protein kinase and the C- kinase characterized by Nishizuka and colleagues. We also intend to det- ermine if TPA treatment will lead to the phosphorylation of ribosomal prot- ein S6 in the mouse skin. Determine whether a number of agents which have been shown to antagonize the tumor promoting effects of phorbol esters also block S6 phosphorylation in vivo and/or in vitro. Agents to be tested initially are: a) Retinoic acid which has been demonstrated to inhibit mouse skin tumor promotion. b) Indo- methacin, the cyclo-oxygenase inhibitor which has been shown to inhibit tumor promoter induced ornithine decarboxylase activity in both mouse epidermis and H35 rat hepatoma cells; and c-e) a series of protease inhib- itors which have been shown to suppress tumor promotion by TPA in vivo c) Tosyl arginyl methyl ester; d) Chloromethylketones TLCK and TPCK; e) the relatively nontoxic protease inhibitor luepeptin. Determine whether TPA can induce the phosphorylation of the ribosomal S6 protein in a cell-free extract to be obtained from cells not previously exposed to TPA. In these experiments gamma-P32-ATP will be supplies as phosphate donor.
If specific aim no. 3 can be4 achieved, the in vitro conditions for S6 phosphorylation by TPA will be refined and the phosphoryl- ation of S6 will be used as an in vitro assay for the purification and characterization of the putative TPA """"""""second messenger"""""""".
| Byus, C V; Fletcher, W H (1988) Direct cytochemical localization of the free catalytic subunit of cAMP-dependent protein kinase. Methods Enzymol 159:236-54 |
| Trevillyan, J M; Perisic, O; Traugh, J A et al. (1985) Insulin- and phorbol ester-stimulated phosphorylation of ribosomal protein S6. J Biol Chem 260:3041-4 |
