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CHIMERIC CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE: ROLE OF THE NEURAL VISININ-LIKE DOMAIN IN REGULATING AUTOPHOSPHORYLATION AND CALMODULIN AFFINITY. P.V.Sathyanarayanan, C.R.Cremo, W.F.Siems and B.W.Poovaiah. Washington State University, Pullman, WA-99164.

Calcium/calmodulin-modulated proteins have been implicated in playing a role in the transduction of the gravity signal. Previously, we reported the expression of chimeric Ca²⁺/calmodulin dependent protein kinase (CCaMK) in roots (J. Biol. Chem, 274:12001-8,1999). CCaMK is characterized by a serine-threonine kinase domain, an autoinhibitory domain, a calmodulin (CaM) 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. 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 CaM, the equilibrium binding constants of CCaMK were measured by fluorescence emission anisotropy using dansylated CaM. 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 CaM affinity regulated by Ca²⁺-stimulated autophosphorylation mediated through the visinin-like domain is a new regulatory mechanism for CCaMK activation and CaM-dependent protein kinases (J. Biol. Chem, in press). Using MALDI-TOF mass spectrometry, threonine 267 was identified as the Ca²⁺-stimulated autophosphorylation site of CCaMK. 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. These molecular switches may help this kinase in transducing calcium transients into phosphorylation signals in plant roots.

 (Supported by NASA and NSF.)