Regulatory interactions of the calmodulin-binding, inhibitory, and autophosphorylation domains of Ca2+/calmodulin-dependent protein kinase II.
Two peptide analogs of Ca2+/calmodulin-dependent protein kinase II (CaMK-(peptides)) were synthesized and used to probe interactions of the various regulatory domains of the kinase. CaMK-(281-289) contained only Thr286, the major Ca2+-dependent autophosphorylation site of the kinase (Schworer, C. M., Colbran, R. J., Keefer, J. R. & Soderling, T. R. (1988) J. Biol. Chem. 263, 13486-13489), whereas CaMK-(281-309) contained Thr286 together with the previously identified calmodulin binding and inhibitory domains (Payne, M. E., Fong, Y.-L., Ono, T., Colbran, R. J., Kemp, B. E., Soderling, T. R. & Means, A. R. (1988) J. Biol. Chem. 263, 7190-7195). CaMK-(281-309), but not CaMK-(281-289), bound calmodulin and was a potent inhibitor (IC50 = 0.88 +/- 0.7 microM using 20 microM syntide-2) of exogenous substrate (syntide-2 or glycogen synthase) phosphorylation by a completely Ca2+/calmodulin-independent form of the kinase generated by limited proteolysis with chymotrypsin. This inhibition was completely relieved by the inclusion of Ca2+/calmodulin in excess of CaMK-(281-309) in the assays. CaMK-(281-289) was a good substrate (Km = 11 microM; Vmax = 3.15 mumol/min/mg) for the proteolyzed kinase whereas phosphorylation of CaMK-(281-309) showed nonlinear Michaelis-Menton kinetics, with maximal phosphorylation (0.1 mumol/min/mg) at 20 microM and decreased phosphorylation at higher concentrations. The addition of Ca2+/calmodulin to assays stimulated the phosphorylation of CaMK-(281-309) by the proteolyzed kinase approximately 10-fold but did not affect the phosphorylation of CaMK-(281-289). A model for the regulation of Ca2+/calmodulin-dependent protein kinase II is proposed based on the above observations and results from other laboratories.
Colbran
R J
RJ
Howard Hughes Medical Institute, Vanderbilt University Medical School, Nashville, Tennessee 37232.
Fong
Y L
YL
Schworer
C M
CM
Soderling
T R
TR
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D001921:Brain / Q000201:enzymology; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002147:Calmodulin / Q000378:metabolism*; D007700:Kinetics; D009842:Oligopeptides / Q000138:chemical synthesis / Q000378:metabolism; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011494:Protein Kinases / Q000378:metabolism*; D051381:Rats
D002147:Calmodulin; D009842:Oligopeptides; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases