Calcium/calmodulin-independent autophosphorylation sites of calcium/calmodulin-dependent protein kinase II. Studies on the effect of phosphorylation of threonine 305/306 and serine 314 on calmodulin binding using synthetic peptides

R. J. Colbran, T. R. Soderling

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    112 Scopus citations

    Abstract

    Two synthetic peptides containing the previously identified calmodulin (CaM)-binding domain of Ca2+/CaM-dependent protein kinase II (CaM-kinase II) (residues 296-309, Payne, M.E., Fong, Y.-L., Ono, T., Colbran, R.J., Kemp, B.E., Soderling, T.R., and Means, A.R. (1988) J. Biol. Chem. 263, 7190-7195) were phosphorylated by Ca2+/CaM-independent forms of the kinase. In the presence of EGTA, CaMK-(290-309) was phosphorylated exclusively on threonine residues (K(m) = 13 μM; V(max) = 211 nmol/min/mg). When the phosphorylated product was analyzed by reversed-phase high performance liquid chromatography (HPLC) two radioactive peaks were resolved. The first peak contained CaMK-(290-309) phosphorylated on Thr306, whereas the second peak contained CaMK-(290-309) phosphorylated on Thr305. However, under the same conditions CaMK-(294-319) was phosphorylated predominantly (approximately 70%) on serine residues (K(m) = 23 μM; V(max) = 99 nmol/min/mg) and HPLC analysis revealed a single major radioactive peak predominantly (more than 90%) phosphorylated at Ser314. Phosphorylation of both peptides was completely blocked in the presence of Ca2+ and a stoichiometric amount of CaM. Samples of each phosphorylated peptide were tested for CaM-binding ability by two procedures and compared to the nonphosphorylated peptides. Phosphorylation of either Thr305 or Thr306 greatly reduced the interaction between CaMK-(290-309) and CaM, whereas phosphorylation of Ser314 did not affect the ability of CaMK-(294-319) to bind CaM. These results indicate that Thr305 and/or Thr306 may be the Ca2+/CaM-independent autophosphorylation site(s) responsible for the loss of ability of CaM-kinase II to bind and be activated by Ca2+/CaM (Hashimoto, Y., Schworer, C.M., Colbran, R.J., and Soderling, T.R., J. Biol. Chem. 262, 8051-8055).

    Original languageEnglish (US)
    Pages (from-to)11213-11219
    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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