Functional analysis and regulation of chimeric calcium/calmodulin dependent protein kinase

Chimeric Ca2+/calmodulin dependent protein kinase (CCaMK) is a serine-threonine protein kinase with three Ca2+ binding EF hands in the C-terminal visinin-like domain. CCaMK also has a calmodulin-binding domain that is involved in the interaction with Ca2+/calmodulin complex during the activation of kinase. Using EF-hand deletions in the visinin-like domain, we found that the visinin-like domain regulated Ca2+-stimulated autophosphorylation of CCaMK. To investigate the effects of Ca2+-stimulated autophosphorylation on the interaction with calmodulin, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated calmodulin. Binding was eight-fold tighter after Ca2+-stimulated autophosphorylation. This shift in affinity did not occur in CCaMK deletion mutants lacking Ca2+-stimulated autophosphorylation. A variable calmodulin affinity regulated by Ca2+-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and calmodulin-dependent protein kinases.; Our experiments demonstrate the existence of two functional molecular switches in a protein kinase regulating the plant kinase activity, namely a visinin-like domain acting as a Ca2+-triggered switch and a CaM-binding domain acting as an autophosphorylation triggered molecular switch.; The Ca2+-stimulated autophosphorylation site of CCaMK was identified by MALDI-TOF (Matrix Assisted Laser Desorption Ionization Time Of Flight) mass spectrometry. T267 was confirmed as the CaU-stimulated autophosphorylation site by post source decay (PSD) experiments and by site directed mutagenesis. Analysis of Ca2+-stimulated autophosphorylation with increasing concentration of CCaMK indicates that the Ca2+-stimulated phosphorylation occurs by an intermolecular mechanism.; The Ca2+-dependent autophosphorylation and Ca2+ /Calmodulin-dependent substrate phosphorylation showed a complex non-linear response to different Ca2+ concentrations. This suggests that chimeric Ca2+/calmodulin-dependent protein kinase from plants is able to decode amplitude-dependent Ca2+ signals into distinct amounts of kinase activity. The time-dependent loss of activity or self-inactivation due to autophosphorylation is another property of CCaMK. Self-inactivation is dependent on reaction pH and ATP concentration. Inactivation of enzyme resulted in the formation of sedimentable enzyme due to self-association. When observed under a transmission electron microscope, the autophosphorylated kinase revealed particles that clustered into branched complexes.