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

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).