Previous studies have shown that p34SEI-1, also known as TRIP-Br1, is involved in cell cycle regulations by interacting with a number of important proteins including CDK4. However, the detailed mechanism and structural basis of the interaction remains to be determined. We report the use of in vitro studies to address these problems. First, it was shown that p34 SEI-1 binds to CDK4 directly, and the binding does not compete directly with p16. In the presence of p16, a quaternary complex is formed between p34SEI-1, CDK4, cyclin D2, and p16. Second, it was found that p34SEI-1 activates the kinase activity of CDK4 at lower concentrations (reaching the maximum at 500 nM) but inhibits the same activity at higher concentrations, implying that p34SEI-1-mediated CDK4 activation is dose-dependent. Again, the effects of p34SEI-1 and p16 are independent of each other. Third, it was shown that p34SEI-1 possesses a LexA-mediated transactivation activity. Finally, a set of truncation mutants were used to dissect the structural elements responsible for the different functions of p34SEI-1. The results indicate that the fragment 30-160 can bind, activate, and inhibit CDK4; the fragment 30-132 can bind and activate but does not inhibit CDK4, while the fragment 30-88 cannot bind, activate, or inhibit but retains the LexA-mediated transactivation activity.
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