Different CDKs initiate different cell cycle phases by phosphorylating specific proteins. It is now clear that rather than phosphorylating a small number of proteins that in turn initiate a certain cell cycle stage, CDKs phosphorylate a myriad of substrates, thereby directly initiating all aspects of a given cell cycle phase. Analysis of a small number of substrates has provided examples that show how phosphorylation by mitotic CDKs mediates many of the early events of mitosis: chromosome condensation, formation of the mitotic spindle, and disassembly of the nuclear envelope. We discuss these events in detail in the sections that follow.

In recent years, systematic efforts to identify all CDK substrates have been initiated. The challenge in identifying the substrates of a particular kinase is to distinguish that kinase’s phosphorylation events from those carried out by other kinases. A breakthrough in understanding which proteins are targets of CDKs was facilitated by the engineering of a CDK mutant that can use an analog of ATP that is not bound by other kinases. This ATP analog has a bulky benzyl group attached to N₆ of the adenine, which makes the analog too large to fit into the ATP-binding pocket of wild-type protein kinases. However, the ATP-binding pocket of the mutant CDK was modified to accommodate this N₆-benzyl ATP analog. Consequently, only the mutant CDK can use this ATP analog as a substrate for transferring its γ phosphate to a protein side chain. When the N₆-benzyl ATP analog with a labeled γ phosphate was incubated with cell extracts containing a recombinant mitotic CDK with the altered ATP-binding pocket, multiple proteins were labeled. In yeast, this procedure identified most of the known CDK substrates plus more than 150 additional yeast proteins. Similar approaches have also been used in mammalian cells to identify CDK substrates. For example, a hunt for substrates of the S phase cyclin A-CDK2 complex revealed 180 potential substrates. These substrates are currently being analyzed for their functions in cell cycle processes.