Generation of the Ca2(+)-independent form of Ca2+/calmodulin-dependent protein kinase II in cerebellar granule cells.
Conditions that regulate the generation of the Ca2(+)-independent form of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) in cultured rat cerebellar granule cells have been investigated. Under basal conditions, 4-5% of total CaM-kinase II activity, assayed in the presence of Ca2+/CaM, was the Ca2(+)-independent form active in the presence of EGTA. Depolarization with 56 mM K+ produced a transient increase to 9% Ca2+ independence within 15 s followed by a decline to 5-6% at 10 min. The divalent cation ionophore ionomycin elicited 10% Ca2+ independence, which remained elevated. Removal of Ca2+ from the Krebs-Ringer medium reduced basal Ca2+ independence to 1-2% and eliminated the elevation in response to K+ depolarization. Inclusion of 5 microM okadaic acid, a protein phosphatase inhibitor, in the incubation medium potentiated the levels of Ca2(+)-independent activity of CaM-kinase II. Additional studies in granule cell extracts indicated that there were both okadiac acid-sensitive and -insensitive protein phosphatases involved in the reversal of the Ca2+ independence of CaM-kinase II. Phosphopeptide mapping of the CNBr-cleaved 32P-labeled 58-60-kDa subunit of CaM-kinase II revealed that under basal conditions, the kinase contained phosphate in many sites. Conditions that promoted formation of the Ca2(+)-independent form of the kinase increased the 32P incorporation into multiple sites of the kinase. However, there was a good temporal correlation between 32P incorporation into CNBr peptide 1, which contains Thr-287, and generation of the Ca2(+)-independent kinase activity. These results indicate that formation of the Ca2(+)-independent species of CaM-kinase II is dynamically regulated in cerebellar granule cells by Ca2(+)-mobilizing agents and by protein phosphatase activity and is correlated with autophosphorylation of Thr-287.
Fukunaga
K
K
Howard Hughes Medical Institute, Vanderbilt University Medical School, Nashville, Tennessee 37232-0615.
Rich
D P
DP
Soderling
T R
TR
D000818:Animals; D002118:Calcium / Q000494:pharmacology* / Q000502:physiology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology / Q000201:enzymology*; D004988:Ethers, Cyclic / Q000494:pharmacology; D006706:Homeostasis; D015759:Ionomycin / Q000494:pharmacology; D007700:Kinetics; D019319:Okadaic Acid; D010749:Phosphoprotein Phosphatases / Q000378:metabolism; D011188:Potassium / Q000494:pharmacology; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains
D004988:Ethers, Cyclic; D019319:Okadaic Acid; D015759:Ionomycin; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D010749:Phosphoprotein Phosphatases; D011188:Potassium; D002118:Calcium