CAMK-II INHIBITION REDUCES CYCLIN D1 LEVELS AND ENHANCES THE ASSOCIATION OF P27(KIP1) WITH CDK2 TO CAUSE G1 ARREST IN NIH 3T3 CELLS

The calmodulin-dependent protein kinase-II (CaMK-II) inhibitor KN-93 h as been shown to reversibly arrest mouse and human cells in the G1 pha se of the cell cycle [Tombes, R.M., Westin, E., Grant, S., and Krystal , G. (1995) Cell Growth Differ. 6, 1073-1070; Rasmussen, G,, and Rasmu ssen, C. (1995) Biochem. Cell Biol. 71, 201-207]. The stimulation of C a2+-independent (autonomous) CaMK-II enzymatic activity, a barometer o f in situ activated CaMK-II, was prevented by the same KN-93 concentra tions that cause G1 phase arrest. KN-93 caused the retinoblastoma prot ein pRB to become dephosphorylated and the activity of both cdk2 and c dk4, two potential pRb kinases, to decrease. Neither the activity of p 42(MAP kinase), an early response G1 signaling molecule, nor the phosp horylation status or DNA-binding capability of the transcription facto rs serum response factor and cAMP responsive element-binding protein w as altered during this G1 arrest. The protein levels of cyclin-depende nt kinase 2 (cdk2) and cdk4 were unaffected during this G1 arrest and the total cellular levels of the cdk inhibitors p21(cip1) and p27(kip1 ) were not increased. Instead, the cdk4 activity decreases resulting f rom KN-93 were the result of a 75% decrease in cyclin D1 levels. In co ntrast, cyclin A and E levels were relatively constant. Cdk2 activity decreases were primarily the result of enhanced p27(kip1) association with cdk2/cyclin E, All of these phenomena were unaffected by KN-93's inactive analog, KN-92, and were reversible upon KN-93 washout. The ki netics of recovery from cell cycle arrest were similar to those report ed for other G1 phase blockers, These results suggest a mechanism by w hich G1 Ca2+ signals could be linked via calmodulin-dependent phosphor ylations to the cell cycle-controlling machinery through cyclins and c dk inhibitors. (C) 1998 Academic Press.