Biochemical characterization of recombinant CDPK1 from Solanum tuberosum

SANTIN, F; GIAMMARIA, V; ULLOA, RM

Potero de los Funes, San Luis

Congreso; 47th Annual Meeting - Argentine Society for Biochemistry and Molecular Biology; 2011

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

BIOCHEMICAL CHARACTERIZATION OF RECOMBINANT CDPK1 FROM Solanum tuberosum Calcium-dependent protein kinases (CDPKs) are essential calcium sensors in plants. In potato, several CDPK isoforms are present. Particularly, StCDPK1 is expressed during tuber transition and could also participate in hormone transduction pathways. In this work, the biochemical characterization of the recombinant polyhistidine-tagged StCDPK1 (StCDPK1::Hisx6) is presented. Enzymatic activity was assayed using a synthetic peptide (Syntide-2) as substrate. CDPKs typical phosphorylation of histone H1 was also observed. The enzymatic activity was enhanced ten fold in the presence of 1 ìM calcium and was inhibited by the calmodulin inhibitor chlorpromazine (CPZ) and the kinase inhibitor staurosporine (ST). The kinetic parameters differed significantly from those exhibited by other isoforms. This is consistent with the fact that CDPKs are involved in various signal transduction pathways and are differentially expressed among tissues. The recombinant enzyme was capable of calcium-dependent autophosphorylation. This post-translational modification is broadly used by CDPKs as a means of modulating their activity. In silico analysis revealed several phosphorylation sites in the protein sequence. In an attempt to determine the functional autophosphorylation sites and understand the enzyme modulation,2D PAGE analysis of StCDPK1 is being performed.