Plant chimeric Ca2+/calmodulin-dependent protein kinase -: Role of the neural visinin-like domain in regulating autophosphorylation and calmodulin affinity

Chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin-binding domain and a neural visinin-like domain with three EF-hands. The neural visinin-like Ca2+-binding domain at the C-terminal end of the CaM-binding domain makes CCaMK unique among all the known calmodulin-dependent kinases. Biological functions of the plant visinin-like proteins or visinin-like domains in plant proteins are not well known. 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 8-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 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.