Autophosphorylation-dependent activation of a calcium-dependent protein kinase from groundnut

Ca2+-dependent protein kinases (CDPKs) containing a calmodulin-like domain integrated in their primary sequence are present primarily in plants. A mem ber of this family was characterized from the groundnut (Arachis hypogea) p lant and called GnCDPK (M. DasGupta [1994] Plant Physiol 104: 961-969). GnC DPK specifically uses the myosin light chain synthetic peptide (MLCpep), wh ich is the phosphate-accepting domain of smooth muscle myosin light chains (KKRPQRATSNVFS), as an exogenous substrate under in vitro experimental cond itions. In this report we show that GnCDPK undergoes intramolecular autopho sphorylation. This self-phosphorylation occurs in threonine residues in a C a2+-dependent (K-0.5 = 0.5 mu M) and calmodulin-independent manner. The kin ase activity toward MLCpep and its sensitivity to Ca2+ were unaffected by p rior autophosphorylation when measured under saturating ATP concentrations. The role of autophosphorylation in the exogenous substrate MLCpep phosphor ylation reaction was reinvestigated at low ATP concentrations. A pronounced lag time of 1 to 2 min, followed by a linear increase of activity for 7.5 min, was seen in the initial rate of MLCpep phosphorylation under such subo ptimal conditions. Prior autophosphorylation completely abolished this lag phase, and a sharp rise of exogenous substrate phosphorylation was seen fro m the 1st min. Our results suggest that autophosphorylation is a prerequisi te for the activation of GnCDPK.