Nuclear import of Cdk/cyclin complexes: Identification of distinct mechanisms for import of Cdk2/cyclin E and Cdc2/cyclin B1

Reversible phosphorylation of nuclear proteins is required for both DNA rep lication and entry into mitosis, Consequently, most cyclin-dependent kinase (Cdk)/cyclin complexes are localized to the nucleus when active. Although our understanding of nuclear transport processes has been greatly enhanced by the recent identification of nuclear targeting sequences and soluble nuc lear import factors with which they interact, the mechanisms used to target Cdk/cyclin complexes to the nucleus remain obscure; this is in part becaus e these proteins lack obvious nuclear localization sequences. To elucidate the molecular mechanisms responsible for Cdk/cyclin transport, we examined nuclear import of fluorescent Cdk2/cyclin E and Cdc2/cyclin B1 complexes in digitonin-permeabilized mammalian cells and also examined potential physic al interactions between these Cdks, cyclins, and soluble import factors. We found that the nuclear import machinery recognizes these Cdk/cyclin comple xes through direct interactions with the cyclin component. Surprisingly, cy clins E and B1 are imported into nuclei via distinct mechanisms. Cyclin E b ehaves like a classical basic nuclear localization sequence-containing prot ein, binding to the alpha adaptor subunit of the importin-alpha/beta hetero dimer. In contrast, cyclin B1 is imported via a direct interaction with a s ite in the NH2 terminus of importin-beta that is distinct from that used to bind importin-alpha.