Regulatory Factors of Cyclin Functions in the Cell Cycle

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According to the proposed research plan, Hideki Kobayashi, Kyushu University in Japan, carried out the collaboration on the cyclin functions in the cell cycle, with Dr.Tim Hunt at ICRF Clare Hall Laboratories, and Dr.Mark Carrington at Cambridge University, England. Hideki Kobayashi isolated Xenopus cyclin A-binding protein by yeast 2-hybrid screen and identified the novel protein THF4 that binds to the N-terminus of cyclin A and inhibits its degradation selectively. Tim Hunt analyzed its biochemical properties on the cell cycle in Xenopus egg extracts. Mark Carrington has isolated murine homolog of THF4 and identified its binding domain to cyclin A.In summary, the N-terminal domain of Xenopus cyclin A1 was used in a 2-hybrid screen to identify proteins that interact with cyclin A.The major interacting species was THF4, a protein that contains a ubiquitin-like domain in its N-terminus shows a significant homology in the C-terminus of S.cerevisiae Dsk2 and S.porn be dph1. THF4 persists … More in Xenopus cells as a nuclear phosphoprotein and its phosphorylation is induced by cyclin A-dependent kinase. THF4 binds to both embryonic and somatic forms of cyclin A (Al and A2) in vivo and in vitro, but not to B-type cyclins. The N-terminal u1biquitin-like domain of THF4, but not the C-temimal conserved domain, is required for interaction with cyclin A.THF4 requires residues 130-160 of cyclin Al for efficient binding, which do not include the destruction box of cyclin A.The addition of bacterially-expressed THF4 protein to_frog egg extract inhibited the Ca2+-induced degradation of cyclin A, but not that of cyclin B.The injection of THF4 protein into the fertilized egg blocked the embryonic cell division.These results suggest that THF4 is a candidate substrate for cyclin A-dependent kinase and that THF4 may act as a negative regulator of cyclin A degradation in mitosis.These results would give a new insight into cyclin proteolysis in the cell cycle This collaborative research project during three years (1996-1998) was quite successful, and we will continue the collaborative research more. Less