Biochemical and expression analysis of an Arabidopsis calcium-dependent protein kinase-related kinase

A cDNA clone from Arabidopsis corresponding to a typical CDPK-related protein kinase, AtCRK3, was isolated. AtCRK3 contains all conserved kinase domain, and has N-terminal myristoylation site and C-terminal domain containing apparently degenerate EF-hands. Biochemical analysis showed that the kinase activity of AtCRK3 for both autophosphorylation and substrate phosphorylation was neither enhanced by Ca2+, nor inhibited by EGTA, similar to other CRKs. In addition, the kinase activity of AtCRK3 is not stimulated by any of four Arabidopsis CaM isoforms, which is different from AtCRK1. Further experiments indicated that the Vmax and Km of AtCRK3 were 35.5 nmol min−1 mg−1 and 6.2 μM, respectively when using histone IIIs as substrate. Capillary electrophoresis showed that autophosphorylation of AtCRK3 occurred on threonine residues. These data showed that AtCRK3 was a serine/threonine protein kinase. In situ hybridization showed that AtCRK3 was expressed in productive and vegetative tissues with temporal and spatial changes during Arabidopsis growth and development. This kinase was also induced in response to ABA treatment. Our study suggested that AtCRK3 might be an important signaling component in plant signaling transduction pathway.