Differential phosphorylation of T-47D human breast cancer cell substrates by D1-, D3-, E-, and A-type cyclin-CDK complexes

The cyclin-dependent kinases (CDKs) promote cell cycle transitions in mammalian cells by phosphorylation of key substrates, To characterize substrates of the G(1) and S phase cyclin-CDK complexes, including cyclin D1-CDK4, cyclin D3-CDK4, cyclin D3-CDK6, cyclin E-CDK2, and cyclin A-CDK2, which are largely undefined, we phosphorylated T-47D breast cancer cell nuclear lysates partially purified by ion-exchange chromatography with purified baculovirus expressed cyclin-CDK complexes, A comparison of the substrates that were phosphorylated by the different cyclin D-CDKs revealed some common as well as specific substrates, Hence, cyclin D1-CDK4 specifically phosphorylated a 38-kDa protein while cyclin D3-CDK4 specifically phosphorylated proteins of 105, 102, and 42 kDa, A 24-kDa protein was phosphorylated by both complexes, Cyclin D3-CDK6 exhibited similar substrate preferences to cyclin D3-CDK4, phosphorylating the 105- and 102-kDa proteins but not the 24-kDa protein, Hence, both the cyclin D1 and D3 as well as CDK4 and CDK6 subunits can confer substrate specificity on the overall cyclin D-CDK complex, Cyclin E-CDK2 and cyclin A-CDK2 phosphorylated a greater number of substrates than the cyclin D-CDKs, ranging in size from 10 kDa to over 200 kDa, Twenty-two substrates were common to both complexes, while six were specific for cyclin A-CDK2 and only one protein of 34 kDa was specific for cyclin E-CDK2, These studies indicate that cyclins E and A modulate the specificity of CDK2 and have demonstrated substrates that may be important for the specific roles of these cyclin-CDKs during G(1) and S phase progression, Protein sequencing of one of the cyclin-CDK substrates characterized in this study identified this protein as nucleolin, a previously characterized CDC2 (CDK1) substrate, thus indicating the utility of this approach in identifying cycln-CDK targets, These results show that both the cyclin and CDK subunits can regulate the substrate specificity of the overall cyclin-CDK complex and have demonstrated numerous substrates of D-, E-, and A-type cyclin-CDK complexes potentially involved in regulating transit through the G(1) and S phases of the cell cycle.