Isolation of Proteins Interacting with the Cyclin D1-CDK6 Complex from Normal and Tumorigenic Human Breast Cells Using a Novel Yeast Three-Hybrid System.
Annual rept. 1 Jul 97-30 Jun 98
NORTH CAROLINA UNIV AT CHAPEL HILL
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A hallmark of many tumor cells is their ability to continuously cycle under conditions where normal cells would be proliferating at a reduced rate. Transitions through different cell cycle stages are primarily regulated by a family of closely related protein kinases, the cyclin-dependent kinases CDKs, presumably by phosphorylating critical cellular proteins Cyclin D1 is a proto-oncogene found to be amplified in 15-20 of human breast cancer cases. Cyclin D1, through its association with the catalytic subunit CDK6 and CDK4, controls G1 cell cycle progression, presumably by phosphorylating a substrate proteins whose function is involved in controlling cell growth and whose activity is critically regulated by the cyclin D1 - dependent kinases in G1. Here I describe the development of a yeast three-hybrid system that expresses two separate genes from the same plasmid and potentially supports the formation of a ternary complex in the yeast cell. Using this system, I have screened a human keratinocyte cDNA library to assay the ability of the three-hybrid system to identify proteins known to interact with CDK6cyclin D1. In addition to the proteins known to interact with this complex p130 and p21, several novel interactions were detected, some of which were phosphorylated by the complex in vitro.
- Medicine and Medical Research