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 μM 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 okadaic 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.