Conditional binding to and cell cycle-regulated inhibition of cyclin- dependent kinase complexes by p27(Kip1)

Mammalian cultures primarily regulate cell cycle traverse during G₁. For progression through G₁ and commitment to DNA synthesis, the activity of a family of proteins, the cyclin-dependent kinases (cdks), is required. There are two primary regulatory portions of G₁: (a) the G₀-G₁ transition, which allows entry into G1; and (b) the G₁-S transition, promoting entry to DNA synthesis and commitment to cell division. In the present manuscript, we provide evidence for cross-talk between these two cell cycle transitions. Extracts prepared from quiescent mouse mammary epithelial cells are shown to act in a dominant manner to specifically inhibit the histone H1 kinase activity of preformed/active cdk2, cdk4, cyclin A, or cyclin E complexes from G₁-S cell extracts. The inhibitory activity arises as cells enter quiescence and decreases once cultures are stimulated to begin G₁ traverse and endogenous cdk activity becomes evident. This activity is associated with the regulated binding of the cdk inhibitor p27(Kip1) to cyclin A/cdk2 kinase complexes upon mixing of the extracts. Removal of p27(Kip1) from the quiescent cell extract specifically abolishes the inhibitory effect. The inhibitory activity and p27(Kip1) binding in vitro depend on incubation of the extracts at physiological temperature or the presence of a reducing agent. The results suggest an interplay between the acquisition of quiescence, cdk activity, and G₁ traverse.