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

K. Fukunaga, D. P. Rich, Thomas Soderling

Research output: Contribution to journalArticle

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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 - 1989
Externally publishedYes

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Calcium-Calmodulin-Dependent Protein Kinase Type 2
Phosphoprotein Phosphatases
Phosphotransferases
Okadaic Acid
Depolarization
Phosphopeptides
Ionomycin
Egtazic Acid
Ionophores
Divalent Cations
Cell Extracts
Rats
Phosphates
Peptides

ASJC Scopus subject areas

  • Biochemistry

Cite this

Generation of the Ca2+-independent form of Ca2+/calmodulin-dependent protein kinase II in cerebellar granule cells. / Fukunaga, K.; Rich, D. P.; Soderling, Thomas.

In: Journal of Biological Chemistry, Vol. 264, No. 36, 1989, p. 21830-21836.

Research output: Contribution to journalArticle

Fukunaga, K. ; Rich, D. P. ; Soderling, Thomas. / Generation of the Ca2+-independent form of Ca2+/calmodulin-dependent protein kinase II in cerebellar granule cells. In: Journal of Biological Chemistry. 1989 ; Vol. 264, No. 36. pp. 21830-21836.
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