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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



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



Journal of Biological Chemistry 265(19): 11213-11219



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 (Km = 13 .mu.M; Vmax = 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 (Km = 23 .mu.M; Vmax = 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).

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Accession: 007074435

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PMID: 2162839


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