Cdk4 activation is dependent on the subunit rearrangement in the complexes

Although several factors have been implicated in the regulation of Cdk4 act ivity, little is known regarding the contributions of cyclin-dependent kina se inhibitors (CKIs) in Cdk4 activation in the mid G1 phase. Using a mouse macrophage cell line (Bac1.2F5), we found that most of Cdk4 bound to p15 wh en cells were in a quiescent state. Following CSF-1 stimulation, Cdk4 bound to cyclin D1 and then to p21, concomitant with the dissociation of p15 fro m the complexes. The activation of Cdk4 correlated well with p21 binding to the complexes, and the majority of active Cdk4 complexes contained p21, Du ring regeneration of mouse liver after partial hepatectomy, Cdk4 activity c oincided precisely with ternary complex formation of cyclin D1/Cdk4/p21. Us ing the baculovirus expression system, we succeeded in reconstituting a cap acity for Cdk4 activation in insect cells, forming an active cyclin D1/Cdk4 /p21 ternary complex. Taken together, it is suggested that p21 and cyclin D 1 act cooperatively as activators of Cdk4 through the release of CKIs of th e INK4 family. (C) 2000 Academic Press.