Purification and characterization of a PMA-stimulated kinase and identification of PMA-induced phosphorylation of a polypeptide that is dephosphorylated by low temperature in Brassica juncea.
Heterologous classical protein kinase C (cPKC) rat polyclonal antibodies showed presence of PKC homolog in Brassica juncea seedlings. It was purified to homogeneity by ammonium sulfate precipitation, diethyl amino ethyl (DEAE)-Sephacel, gel-filtration chromatography, and preparative gel electrophoresis. PKC-like kinase activity was fractionated into three distinct peaks after DEAE-Sephacel chromatography. The kinase activity was associated with a 55 kDa polypeptide. It was calcium dependent and lipids (phosphatidylserine, PS and oleyl acetylglycerol, OAG) stimulated it further, suggesting it to be a classical type protein kinase C. This was further confirmed by the stimulation of the kinase activity by phorbol 12-myristate 13-acetate (PMA) (a diacylglycerol, DAG analog) and its inhibition by H-7 (a general kinase inhibitor) and staurosporine (a PKC specific inhibitor). Histone was the preferred substrate over casein and BSA. Phosphoamino acid analysis showed it to be a serine/threonine kinase. Western blotting with the purified polypeptide showed an immunopositive 55 kDa polypeptide. In search of the substrate for the kinase in vitro phosphorylation was done in presence of kinase inhibitors. PKC-dependent phosphorylation was observed which was inhibited by PKC inhibitors (H-7 and staurosporine) and enhanced by PKC activator (PMA). Low temperature induced dephosphorylation of the same polypeptide. Direct involvement of PKC-dependent phosphorylation in early LT signaling is indicated for the first time.
Deswal
Renu
R
Plant Molecular Physiology and Biochemistry Laboratory, Department of Botany, University of Delhi, Delhi 10007, India. deswalr@hotmail.com
Chowdhary
Gopal K
GK
Sopory
Sudhir K
SK
D002118:Calcium / Q000378:metabolism; D003080:Cold Temperature; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000378:metabolism; D009149:Mustard Plant / Q000201:enzymology* / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D010766:Phosphorylation; D011493:Protein Kinase C / Q000187:drug effects / Q000302:isolation & purification* / Q000378:metabolism*; D036226:Seedlings / Q000201:enzymology / Q000378:metabolism; D013379:Substrate Specificity; D013755:Tetradecanoylphorbol Acetate / Q000378:metabolism*
D004791:Enzyme Inhibitors; D010743:Phospholipids; D011493:Protein Kinase C; D008274:Magnesium; D013755:Tetradecanoylphorbol Acetate; D002118:Calcium