Regulation of cyclin D1/Cdk4 complexes by calcium/calmodulin-dependent protein kinase I.
The selective inhibitor of the multifunctional calcium/calmodulin-dependent kinases (CaMK), KN-93, arrests a variety of cell types in G(1). However, the biochemical nature of this G(1) arrest point and the physiological target of KN-93 in G(1) remain controversial. Here we show that in WI-38 human diploid fibroblasts KN-93 reversibly arrested cells in late G(1) 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 G(1), cyclin D1-containing complexes migrated toward lower molecular weight (M(r)) 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 G(1), 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.
Kahl
Christina R
CR
Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710, USA.
Means
Anthony R
AR
D000256:Adenoviridae / Q000235:genetics / Q000378:metabolism; D001596:Benzylamines / Q000494:pharmacology; D015153:Blotting, Western; D001973:Bromodeoxyuridine / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002460:Cell Line; D002465:Cell Movement; D002467:Cell Nucleus / Q000378:metabolism; D002850:Chromatography, Gel; D019938:Cyclin D1 / Q000096:biosynthesis*; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases / Q000096:biosynthesis* / Q000378:metabolism; D004247:DNA / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005347:Fibroblasts / Q000378:metabolism; D005434:Flow Cytometry; D016193:G1 Phase; D006801:Humans; D008856:Microscopy, Fluorescence; D010766:Phosphorylation; D011233:Precipitin Tests; D011485:Protein Binding; D011518:Proto-Oncogene Proteins; D013449:Sulfonamides / Q000494:pharmacology; D013997:Time Factors; D000096926:Benzenesulfonamides
D001596:Benzylamines; D001973:Bromodeoxyuridine; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D019938:Cyclin D1; D051358:Cyclin-Dependent Kinase 4; D018844:Cyclin-Dependent Kinases; D004247:DNA; D004791:Enzyme Inhibitors; D011518:Proto-Oncogene Proteins; D013449:Sulfonamides; C425780:KN 92; C072105:KN 93; D000096926:Benzenesulfonamides; C495900:CDK4 protein, human