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 member of this family was characterized from the groundnut (Arachis hypogea) plant and called GnCDPK (M. DasGupta [1994] Plant Physiol 104: 961-969). GnCDPK specifically uses the myosin light chain synthetic peptide (MLCpep), which is the phosphate-accepting domain of smooth muscle myosin light chains (KKRPQRATSNVFS), as an exogenous substrate under in vitro experimental conditions. In this report we show that GnCDPK undergoes intramolecular autophosphorylation. This self-phosphorylation occurs in threonine residues in a Ca2+-dependent (K0.5 = 0.5 &mgr;M) and calmodulin-independent manner. The kinase activity toward MLCpep and its sensitivity to Ca2+ were unaffected by prior autophosphorylation when measured under saturating ATP concentrations. The role of autophosphorylation in the exogenous substrate MLCpep phosphorylation 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 suboptimal conditions. Prior autophosphorylation completely abolished this lag phase, and a sharp rise of exogenous substrate phosphorylation was seen from the 1st min. Our results suggest that autophosphorylation is a prerequisite for the activation of GnCDPK.
Department of Biochemistry, Ballygunge Science College, Calcutta University, 35 Ballygunge Circular Road, Calcutta 700019, India.
S
Chaudhuri
A
Seal
MD
Gupta