Mutational analysis of Ca(2+)-independent autophosphorylation of calcium/calmodulin-dependent protein kinase II.
Previous studies with synthetic peptides indicate that residues 290-309, corresponding to the calmodulin (CaM)-binding domain of Ca2+/CaM-dependent protein kinase II interact with the catalytic core of the enzyme as a pseudosubstrate (Colbran, R. J., Smith, M. K., Schworer, C. M., Fong, Y. L., and Soderling, T. R. (1989) J. Biol. Chem. 264, 4800-4804). In the present study, we attempted to locate the pseudosubstrate motif by generation or removal of potential substrate recognition sequences (RXXS/T) at selected positions using site-directed mutagenesis. Based on previous results, Arg297, Thr305/306, and Ser314 were selected as key residues. Single mutations such as N294S, K300S, A302R, A309R, and R311A were expressed, purified, and characterized. Several of the mutants exhibited decreased binding of and activation by CaM, not surprising since the mutations were within the CaM-binding domain. None of the mutants exhibited enhanced Ca(2+)-independent kinase activity toward exogenous substrate, but the K300S and N294S mutants showed a significant enhancement in the rate and stoichiometry of 32P incorporation during Ca(2+)-independent autophosphorylation. Using two-dimensional peptide mapping and phosphoamino acid analyses, enhanced phosphorylation of the introduced Ser residue was demonstrated in the K300S mutant but not in the N294S mutant. This specific Ca(2+)-independent autophosphorylation of Ser300 is consistent with the hypothesis that Arg297 may occupy the P (-3) position in a pseudosubstrate autoinhibitory interaction with the catalytic core in the nonactivated state of the kinase.
Mukherji
S
S
Vollum Institute, Oregon Health Sciences University, Portland 97201, USA.
Soderling
T R
TR
D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D002118:Calcium / Q000502:physiology*; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000032:analysis / Q000378:metabolism*; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010449:Peptide Mapping; D010766:Phosphorylation
D000596:Amino Acids; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D002118:Calcium