Functional determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II: Role of His282 and multiple basic residues

M. K. Smith, R. J. Colbran, D. A. Brickey, T. R. Soderling

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    Important determinants in the autoinhibitory domain of calcium/calmodulin-dependent protein kinase II (CaMK-II), corresponding to residues 281-302 of the kinase α-subunit sequence, were identified. Replacement of Thr286 with Ala (CaMK-(281-302 Ala286)) had no effect on either the potency (IC50 = 2 μM) or inhibitory mechanism (competitive with ATP) using the catalytic fragment of CaMK-II. Single replacement of charged residues in CaMK-(281-302, Ala286) identified His282, Arg283, Lys291, Arg297, and Lys298 as important determinants (>10-fold increase in IC50) for potent inhibition of CaMK-II. Glu285, Asp288, Lys291, Arg296, and Lys300 were not as essential (<4-fold change in IC50) for potent CaMK-II inhibition. Replacement of either Arg283, Lys291, or Arg297, and Lys298 with Ala did not alter the ATP-competitive mechanism of inhibition although the K(i) values increased 16-530-fold. However, replacement of His282 with Ala decreased the IC50 by 20-fold and altered the mechanism of inhibition to noncompetitive with respect to ATP. The non-protonated form of His282 was functionally active since decreasing the pH from 7.5 to 5.5 increased the IC50 of CaMK-(281-302, Ala286) almost 20-fold. Histidine protonation also appeared to disrupt the autoinhibitory domain of intact forms of CaMK-II since preincubation of non-proteolyzed rat brain CaMK-II with calcium/calmodulin (in the absence of ATP) at pH 5.5 generated up to 16% calcium-independent activity when assayed at pH 5.5. Similarly, the level of calcium-independent activity of a baculovirus-expressed Asp286 mutant CaMK-II ((D286)mCaMKα) increased to almost 80% calcium independence when assayed at pH 5.5 compared to only 20% when assayed at pH 7.5. The levels of calcium-independent activity of both the (D286)mCaMKα (at pH 5.5 and 7.5) and the rat brain CaMK-II (at pH 5.5) were sensitive to the concentrations of both ATP and peptide substrate (syntide-2) in the assays. These data suggest that the basic residues Arg283, Lys291, Arg297, and Lys298 are important for potent inhibition of CaMK-II and that the non-protonated form of His282 may play a unique role in the ATP-directed mechanism of inhibition by the CaMK-II autoinhibitory domain.

    Original languageEnglish (US)
    Pages (from-to)1761-1768
    Number of pages8
    JournalJournal of Biological Chemistry
    Issue number3
    StatePublished - 1992

    ASJC Scopus subject areas

    • Biochemistry
    • Molecular Biology
    • Cell Biology


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