Characterization of a winged bean (Psophocarpus tetragonolobus) protein kinase with calmodulin-like domain: regulation by autophosphorylation.
A soluble protein kinase purified from winged bean (Psophocarpus tetragonolobus) shoots, has been assessed as a monomeric enzyme with an approximate M(r) of 60,000 in spite of the presence of two polypeptides of 61 and 58 kDa determined by SDS/PAGE. Immunoblot analyses using either of the two antisera raised individually against the polypeptides, detect both of them in purified preparations and a single larger polypeptide (62 kDa) in freshly prepared tissue homogenates, clearly indicating the likelihood of the doublet being formed from the larger one by proteolysis. Histone H1, syntide 2 and a synthetic myosin light chain-related peptide (MLC-peptide) have been identified as exogenous substrates of the enzyme. Complete Ca(2+)-dependence for substrate phosphorylation, a drastic inhibition of the reaction by a calmodulin (CaM) antagonist which can be partially reversed by a heterologous CaM and direct 45Ca(2+)-binding on blot, form compelling evidence in favour of a CaM-like domain of the enzyme. Both the polypeptides of the purified enzyme undergo intramolecular autophosphorylation on serine residue(s). Unlike the substrate phosphorylation reaction, autophosphorylation is Ca(2+)-independent and is not inhibited by the CaM antagonist. Down-regulation of substrate phosphorylation by auto-phosphorylation, and stimulation of the autophosphorylation by histone H1 and MLC-peptide, are novel regulatory features of the enzyme.
Saha
P
P
Indian Institute of Chemical Biology, Calcutta.
Singh
M
M
D000595:Amino Acid Sequence; D002118:Calcium / Q000494:pharmacology; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D015536:Down-Regulation / Q000502:physiology*; D004533:Egtazic Acid / Q000494:pharmacology; D005476:Fluphenazine / Q000494:pharmacology; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010940:Plant Proteins / Q000302:isolation & purification* / Q000378:metabolism / Q000502:physiology*; D010944:Plants / Q000201:enzymology*; D013379:Substrate Specificity
D002147:Calmodulin; D010940:Plant Proteins; D004533:Egtazic Acid; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D005476:Fluphenazine; D002118:Calcium