CaMK-II inhibition reduces cyclin D1 levels and enhances the association of p27kip1 with Cdk2 to cause G1 arrest in NIH 3T3 cells.
The calmodulin-dependent protein kinase-II (CaMK-II) inhibitor KN-93 has been shown to reversibly arrest mouse and human cells in the G1 phase of the cell cycle [Tombes, R. M., Westin, E., Grant. S., and Krystal, G. (1995) Cell Growth Differ. 6, 1073-1070; Rasmussen, G., and Rasmussen, C. (1995) Biochem. Cell Biol. 71, 201-207]. The stimulation of Ca(2+)-independent (autonomous) CaMK-II enzymatic activity, a barometer of in situ activated CaMK-II, was prevented by the same KN-93 concentrations that cause G1 phase arrest. KN-93 caused the retinoblastoma protein pRB to become dephosphorylated and the activity of both cdk2 and cdk4, two potential pRb kinases, to decrease. Neither the activity of p42MAP kinase, an early response G1 signaling molecule, nor the phosphorylation status or DNA-binding capability of the transcription factors serum response factor and cAMP responsive element-binding protein was altered during this G1 arrest. The protein levels of cyclin-dependent kinase 2 (cdk2) and cdk4 were unaffected during this G1 arrest and the total cellular levels of the cdk inhibitors p21cip1 and p27kip1 were not increased. Instead, the cdk4 activity decreases resulting from KN-93 were the result of a 75% decrease in cyclin D1 levels. In contrast, cyclin A and E levels were relatively constant. Cdk2 activity decreases were primarily the result of enhanced p27kip1 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 kinetics of recovery from cell cycle arrest were similar to those reported for other G1 phase blockers. These results suggest a mechanism by which G1 Ca2+ signals could be linked via calmodulin-dependent phosphorylations to the cell cycle-controlling machinery through cyclins and cdk inhibitors.
Morris
T A
TA
Massey Cancer Center, Medical College of Virginia, Virginia Commonwealth University, Richmond 23298-0230, USA.
DeLorenzo
R J
RJ
Tombes
R M
RM
D016475:3T3 Cells; D000818:Animals; D001596:Benzylamines / Q000494:pharmacology*; D042846:CDC2-CDC28 Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000037:antagonists & inhibitors*; D018797:Cell Cycle Proteins; D002455:Cell Division / Q000187:drug effects*; D017362:Cyclic AMP Response Element-Binding Protein / Q000378:metabolism; D019938:Cyclin D1 / Q000378:metabolism*; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D016193:G1 Phase; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000378:metabolism*; D009687:Nuclear Proteins / Q000378:metabolism; D010766:Phosphorylation; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D016160:Retinoblastoma Protein / Q000037:antagonists & inhibitors; D026362:Serum Response Factor; D013449:Sulfonamides / Q000494:pharmacology*; D014157:Transcription Factors / Q000378:metabolism; D025521:Tumor Suppressor Proteins; D000096926:Benzenesulfonamides
D001596:Benzylamines; D042846:CDC2-CDC28 Kinases; D054732:Calcium-Calmodulin-Dependent Protein Kinase Type 2; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D018797:Cell Cycle Proteins; D017362:Cyclic AMP Response Element-Binding Protein; D019938:Cyclin D1; D051357:Cyclin-Dependent Kinase 2; D050760:Cyclin-Dependent Kinase Inhibitor p27; D018844:Cyclin-Dependent Kinases; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D008869:Microtubule-Associated Proteins; D009687:Nuclear Proteins; D017346:Protein Serine-Threonine Kinases; D016160:Retinoblastoma Protein; D026362:Serum Response Factor; D013449:Sulfonamides; D014157:Transcription Factors; D025521:Tumor Suppressor Proteins; C493961:Cdkn1b protein, mouse; C072105:KN 93; D000096926:Benzenesulfonamides; C495893:Cdk2 protein, mouse