In preliminary experiments we have identified, by means of monoclonal antibodies, a 34kD protein in human cells that is homologous to the cdc2+/CDC28 cell cycle control protein of the yeasts, S. pombe and S. cerevisiae. In yeast, this protein kinase plays a central role in regulating the passage of cells into both DNA synthesis and nuclear division. This research proposal outlines four immediate experimental objectives that are directed towards understanding the role and significance of p34 in mammalian cells. 1) Isolation of the human gene that encodes p34. 2) Introduction into tissue culture cells of (a) monoclonal antibodies against p34 (b) """"""""anti-sense"""""""" RNA or oligonucleotides directed against the p34 gene (c) known dominant alleles of the yeast cdc2 gene or its human counterpart and (d) introduction of the wild-type cdc2+ gene or its human homolog into existing temperature sensitive tissue culture cell lines that have a cell division cycle defect to test for rescue of the mutant phenotype. The objective of these experiments is to discover whether the p34 plays the same role in human cells a does the cdc2+/CDC28 protein in yeast. 3) Investigation of the intracellular localization of p34 in tissue culture cells. 4) Assessment of the abundance, phosphorylation state and the biochemical activity of the p34 in different states of growth, during the phases of the cell cycle and following cellular transformation by known oncogenes. Funding is requested for a five year period. A long term objective is to test whether p34 in a substrate for any known protein kinase, including oncogene products. In addition, we will seek substrates of the p34 protein kinase. This will involve not only direct experiments on mammalian cells, but will also draw upon our on-gong studies on the control of the cell division in S. pombe. For example, any protein identified as a substrate of the cdc2+ protein kinase in yeast will be sought in mammalian cells.
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