Calcium-stimulated autophosphorylation site of plant chimeric calcium/calmodulin-dependent protein kinase

The existence of two molecular switches regulating plant chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK), namely the C-terminal visinin-like domain acting as Ca2+-sensitive molecular switch and calmodulin binding domain acting as Ca2+-stimulated autophosphorylation-sensitive molecular switch, has been described (Sathyanarayanan, P. V., Cremo, C. R., and Poovaiah, B. W. (2000) J. BioL Chem. 275, 30417-30422). Here we report the identification of Ca2+-stimulated autophosphorylation site of CCaMK by matrix-assisted laser desorption ionization time of flight-mass spectrometry. Thr(267) was confirmed as the Ca2+-stimulated autophosphorylation site by post-source decay experiments and by site-directed mutagenesis. The purified T267A mutant form of CCaMK did not show Ca2+-stimulated autophosphorylation, autophosphorylation-dependent variable calmodulin affinity, or Ca2+/calmodulin stimulation of kinase activity. Sequence comparison of CCaMK from monocotyledonous plant (lily) and dicotyledonous plant (tobacco) suggests that the autophosphorylation site is conserved. This is the first identification of a phosphorylation site specifically responding to activation by second messenger system (Ca2+ messenger system) in plants. Homology modeling of the kinase and calmodulin binding domain of CCaMK with the crystal structure of calcium/calmodulin-dependent protein kinase 1 suggests that the Ca2+-stimulated autophosphorylation site is located on the surface of the kinase and far from the catalytic site. Analysis of Ca2+-stimulated autophosphorylation with increasing concentration of CCaMK indicates the possibility that the Ca2+-stimulated phosphorylation occurs by an intermolecular mechanism.