Generation of the Ca2+-independent form of Ca2+/calmodulin-dependent protein kinase II in cerebellar granule cells

K. Fukunaga, D. P. Rich, T. R. Soderling

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    93 Scopus citations

    Abstract

    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.

    Original languageEnglish (US)
    Pages (from-to)21830-21836
    Number of pages7
    JournalJournal of Biological Chemistry
    Volume264
    Issue number36
    StatePublished - Dec 1 1989

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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