Regulation of Cyclin D1/Cdk4 Complexes by Calcium/Calmodulin-dependent Protein Kinase I

Abstract

The selective inhibitor of the muitifunctional calcium/ calmodulin-dependent kinases (CaMK), KN-93, arrests a variety of cell types in G1. However, the biochemical nature of this G1 arrest point and the physiological target of KN-93 in G1 remain controversial. Here we show that in WI-38 human diploid fibroblasts KN-93 reversibly arrested cells in late G1 prior to detectable cyclin-dependent kinase 4 (cdk4) activation. At the KN-93 arrest point, we found that cyclin D1/cdk4 complexes had assembled with p21/p27, accumulated in the nucleus, and become phosphorylated on Thr-172, yet were relatively inactive. Additional examination of cdk4 complexes by gel filtration analysis demonstrated that, in late G1, cyclin D1-containing complexes migrated toward lower molecular weight (Mr) fractions and this altered migration was accompanied by the appearance of two peaks of cdk4 activity, at 150-200 and 70 kDa, respectively. KN-93 prevented both the activation of cdk4, and this shift in cyclin D1 migration and overexpression of cyclin D1/cdk4 overcame the KN-93 arrest. To determine which multifunctional CaMK acts in G1, we expressed kinase-deficient forms of CaMKI and CaMKII. Overexpression of kinase-deficient CaMKI, but not CaMKII, prevented cdk4 activation, mimicking the KN-93 arrest point. Therefore, we hypothesize that KN-93 prevents a very late, uncharacterized step in cyclin D/cdk4 activation that involves CaMKI and follows complex assembly, nuclear entry, and phosphorylation.