Studies on the regulatory domain of Ca2+/calmodulin-dependent protein kinase II. Functional analyses of arginine 283 using synthetic inhibitory peptides and site-directed mutagenesis of the α subunit

Y. L. Fong, T. R. Soderling

    Research output: Contribution to journalArticle

    22 Scopus citations

    Abstract

    The regulatory role of Arg283 in the autoinhibitory domain of Ca2+/calmodulin-dependent protein kinase II was investigated using substituted inhibitory synthetic peptides and site-directed mutation of the expressed kinase. In the synthetic peptide corresponding to the autoinhibitory domain (residues 281-309) of Ca2+/calmodulin-dependent protein kinase II, substitution of Arg283 by other residues increased the IC50 values of the peptides in the following order: Arg << Lys << Gln << Glu. Site-directed mutations of Arg283 to glutamic acid and glutamine in the kinase α subunit cDNA were transcribed and translated in vitro. The expressed enzymes had the same total kinase activities, determined in the presence of Ca2+/CaM, but the Glu283 mutant had a slightly higher Ca2+-independent kinase activity (5.46 ± 0.88%) compared to the wild-type Arg283 (1.86 ± 0.71%) and the Gln283 mutant (2.15 ± 0.60%). When the expressed kinases were subjected to limited autophosphorylation on ice to monitor generation of the Ca2+-independent activity, the Arg283 kinase attained maximal Ca2+-independent activity (about 20%) within 30 s, whereas the Gln283 and Glu283 mutants attained maximal Ca2+-independence only after about 40 min of autophosphorylation. The results indicate that Arg283 is a very important determinant for the regulatory autophosphorylation of Thr286 that generates the Ca2+-independent activity but is not essential for the other multiple autophosphorylations within Ca2+/calmodulin-dependent protein kinase II, and that Arg283 is only one of several important residues for the inhibitory potency of the autoinhibitory domain.

    Original languageEnglish (US)
    Pages (from-to)11091-11097
    Number of pages7
    JournalJournal of Biological Chemistry
    Volume265
    Issue number19
    StatePublished - Jul 20 1990

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology

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