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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM039620-02
Application #
3296762
Study Section
Cellular Biology and Physiology Subcommittee 1 (CBY)
Project Start
1988-02-01
Project End
1993-01-31
Budget Start
1989-02-01
Budget End
1990-01-31
Support Year
2
Fiscal Year
1989
Total Cost
Indirect Cost
Name
Cold Spring Harbor Laboratory
Department
Type
DUNS #
065968786
City
Cold Spring Harbor
State
NY
Country
United States
Zip Code
11724
Okamoto, K; Kamibayashi, C; Serrano, M et al. (1996) p53-dependent association between cyclin G and the B' subunit of protein phosphatase 2A. Mol Cell Biol 16:6593-602
Demetrick, D J; Zhang, H; Beach, D H (1996) Chromosomal mapping of the genes for the human CDK2/cyclin A-associated proteins p19 (SKP1A and SKP1B) and p45 (SKP2). Cytogenet Cell Genet 73:104-7
Casso, D; Beach, D (1996) A mutation in a thioredoxin reductase homolog suppresses p53-induced growth inhibition in the fission yeast Schizosaccharomyces pombe. Mol Gen Genet 252:518-29
Demetrick, D J; Zhang, H; Beach, D H (1996) Chromosomal mapping of the human genes CKS1 to 8q21 and CKS2 to 9q22. Cytogenet Cell Genet 73:250-4
Zhang, H; Kobayashi, R; Galaktionov, K et al. (1995) p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase. Cell 82:915-25
Demetrick, D J; Matsumoto, S; Hannon, G J et al. (1995) Chromosomal mapping of the genes for the human cell cycle proteins cyclin C (CCNC), cyclin E (CCNE), p21 (CDKN1) and KAP (CDKN3). Cytogenet Cell Genet 69:190-2
Demetrick, D J; Zhang, H; Beach, D H (1994) Chromosomal mapping of human CDK2, CDK4, and CDK5 cell cycle kinase genes. Cytogenet Cell Genet 66:72-4
Okamoto, K; Beach, D (1994) Cyclin G is a transcriptional target of the p53 tumor suppressor protein. EMBO J 13:4816-22
Demetrick, D J; Beach, D H (1993) Chromosome mapping of human CDC25A and CDC25B phosphatases. Genomics 18:144-7
Zhang, H; Xiong, Y; Beach, D (1993) Proliferating cell nuclear antigen and p21 are components of multiple cell cycle kinase complexes. Mol Biol Cell 4:897-906

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