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 (Km = 13 microM; 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 microM; 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).
Colbran
R J
RJ
Department of Molecular Physiology and Biophysics, Vanderbilt University School of Medicine, Nashville, Tennessee 37232.
Soderling
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000494:pharmacology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000378:metabolism* / Q000494:pharmacology; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004533:Egtazic Acid / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D007700:Kinetics; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000378:metabolism*; D010766:Phosphorylation; D010768:Phosphoserine / Q000378:metabolism*; D010769:Phosphothreonine / Q000378:metabolism*; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats; D012694:Serine / Q000031:analogs & derivatives*; D013912:Threonine / Q000031:analogs & derivatives*
D002147:Calmodulin; D010446:Peptide Fragments; D010769:Phosphothreonine; D010768:Phosphoserine; D013912:Threonine; D012694:Serine; D004533:Egtazic Acid; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium