A previous study has suggested that the autoinhibitory domain of Ca2+/calmodulin-dependent protein kinase II (CaM-kinase II) may contain an α-helical structure, which is important for the proper orientation of inhibitory residues to interact with the catalytic domain (Smith, M. K., Colbran, R. J., Brickey, D. A., and Soderling, T. R. (1992) J. Biol. Chem. 267, 1761-1768). The present study was designed to test the importance of the secondary structure in the autoinhibitory domain (residues 281-302) by site- specific mutagenesis of selected residues to prolines. Single mutants C289P, C289A, A295P, and the double mutant C289P/A295P were expressed using the baculovirus/Sf9 cell system and purified on CaM-Sepharose. The single mutants had specific activities (7-12 μmol/min/mg) and eluted from gel permeation chromatography (600-650 kDa) identical to wild-type kinase. Since the double mutant had a very low specific activity and eluted as a mixture of a large aggregate and proteolyzed monomer, it was not characterized further. Only the C289P mutant exhibited enhanced Ca2+-independent activity (5-12% of total activity) prior to autophosphorylation. When autophosphorylation was performed in the absence of Ca2+/CaM at 5°C, only the C289P mutant showed a significant increase in Ca2+-independent activity. This autophosphorylation of Thr286 in the absence of Ca2+/CaM has not been observed with wild-type kinase or any other autoinhibitory domain mutant we have characterized. This result suggests that Thr286, the autophosphorylation site responsible for Ca2+-independent activity, may not be available for autophosphorylation in the wild-type kinase or the other mutants because of structural restrictions due to the secondary structure in this region. This structural restraint is presumably disrupted by the binding of Ca2+/CaM or by insertion of a proline residue.
|Original language||English (US)|
|Number of pages||6|
|Journal||Journal of Biological Chemistry|
|State||Published - Aug 12 1994|
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology