ASGSB 1998 Annual Meeting Abstracts

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FUNCTIONAL DOMAINS AND THE GENOMIC STRUCTURE OF CHIMERIC CALCIUM/CALMODULIN-DEPENDENT PROTEIN KINASE.   B.W. Poovaiah, Z. Liu, W. Wang, and P.V. Sathyanarayanan. Dept. of Horticulture, Washington State Univ., Pullman, WA.

Biochemical characterization and structure/function studies of chimeric Ca²⁺/calmodulin-dependent protein kinase (CCaMK) have revealed that it has dual modes of regulation by Ca²⁺ and Ca²⁺/calmodulin (Patil et al., Proc. Natl. Acad. Sci. 92: 4897-4901, 1995; Takezawa et al., J. Biol. Chem. 271: 8126-8132, 1996). CCaMK has unique structural features, including a catalytic domain, a calmodulin-binding domain, and a neural visinin-like Ca²⁺-binding domain. The existence of these three features in a single polypeptide distinguishes it from other kinases. The calcium-binding C-terminal region of CCaMK is similar to mammalian neural visinin-like calcium-binding proteins. Calcium binding to the EF hands in the visinin-like domain controls autophosphorylation of CCaMK. The autophosphorylated CCaMK shows maximal kinase activity in the presence of calmodulin (Takezawa et al., J. Biol. Chem. 271: 8126-8132, 1996). The use of deletion mutants of CCaMK lacking EF hands I, II, and III indicated that these calcium-binding sites were crucial for full calcium/calmodulin-dependent kinase activity (Ramachandiran et al., J. Biochem. 121: 984-990, 1997). Site-directed mutants were produced to alter the calcium-binding to each EF hand in the visinin-like domain. The calcium-binding properties of these mutants as well as auto- and substrate phosphorylation are being investigated. The genomic clone of CCaMK was isolated and characterized. It contains 6 introns and the putative promoter region (1.7 kb) of CCaMK has a cAMP regulatory element (CRE2, nucleotides 1696 to 1702) and the consensus sequences for activator protein 2 (AP-2) element (nucleotides 1565 to 1574). The functional significance of these elements and the two regulatory domains (calmodulin-binding domain and the Ca²⁺-binding visinin-like domain) will be discussed. (Supported by NASA grant NAG-10-0061 and NSF grant MCB 96-30337.)

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