Cyclins and cyclin-dependent kinases (Cdks) are interacting cell cycle regulatory proteins. Assembly of distinct cyclin-Cdk complexes characterizes specific stages Of the cell cycle, and activation of those complexes is essential for cell cycle progression. For example, complexes containing the D-type cyclins and Cdk4, and ones containing cyclin E and Cdk2 are both necessary and rate-limiting for completion of G1, whereas the cyclin B-Cdc2 complex is essential for mitosis. Although substantial progress is being made in understanding how the cyclins and Cdks are regulated by mitogenic and anti-mitogenic signals, relatively little progress has been made in understanding how these proteins promote the execution of downstream cell cycle events. In other words, a major limitation of current cell cycle research is that essential substrates of the cyclin-dependent kinases, with few exceptions, have not been identified. The general goal of this proposal is to identify and characterize essential Cdk substrates, particularly those necessary for the G1 and S phase. We have used in vitro mutagenesis to map the domains in cyclin E that are essential for G1 cyclin function, but are not involved in assembly or activation of the cyclin E-Cdk2 complex. These domains are likely to be necessary for targeting the active cyclin ECdk2 complex to its essential substrates. We propose to exploit this discovery to identify downstream targets of cyclin-Cdk2 complexes. The specific goals of this proposal are to: I) map the protein interaction domains in human G1 cyclins that are necessary for G1 cyclin function (but not involved in Cdk binding or activation); 2) identify proteins that interact with human G1 cyclins, using cyclins mutated in specific interaction domains to discriminate between physiologically significant and insignificant interactions; 3) determine whether these interacting proteins are essential Cdk substrates and/or perform other functions necessary for cyclin activity.
