Pea DNA topoisomerase I is phosphorylated and stimulated by casein kinase 2 and protein kinase C.
DNA topoisomerase I catalyzes the relaxation of superhelical DNA tension and is vital for DNA metabolism; therefore, it is essential for growth and development of plants. Here, we have studied the phosphorylation-dependent regulation of topoisomerase I from pea (Pisum sativum). The purified enzyme did not show autophosphorylation but was phosphorylated in an Mg(2+)-dependent manner by endogenous protein kinases present in pea nuclear extracts. This phosphorylation was abolished with calf intestinal alkaline phosphatase and lambda phosphatase. It was also phosphorylated by exogenous casein kinase 2 (CK2), protein kinase C (PKC; from animal sources), and an endogenous pea protein, which was purified using a novel phorbol myristate acetate affinity chromatography method. All of these phosphorylations were inhibited by heparin (inhibitor of CK2) and calphostin (inhibitor of PKC), suggesting that pea topoisomerase I is a bona fide substrate for these kinases. Spermine and spermidine had no effect on the CK2-mediated phosphorylation, suggesting that it is polyamine independent. Phospho-amino acid analysis showed that only serine residues were phosphorylated, which was further confirmed using antiphosphoserine antibody. The topoisomerase I activity increased after phosphorylation with exogenous CK2 and PKC. This study shows that these kinases may contribute to the physiological regulation of DNA topoisomerase I activity and overall DNA metabolism in plants.
Tuteja
Narendra
N
International Centre for Genetic Engineering and Biotechnology, Aruna Asaf Ali Marg, P.O. Box 10504, New Delhi 110 067, India. narendra@icgeb.res.in
Reddy
Malireddy Kodandarami
MK
Mudgil
Yashwanti
Y
Yadav
Badam Singh
BS
Chandok
Meena Rani
MR
Sopory
Sudhir Kumar
SK
D047390:Casein Kinase II; D004264:DNA Topoisomerases, Type I / Q000737:chemistry / Q000378:metabolism*; D004789:Enzyme Activation; D018532:Pisum sativum / Q000201:enzymology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000302:isolation & purification / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*
D047390:Casein Kinase II; D017346:Protein Serine-Threonine Kinases; D011493:Protein Kinase C; D004264:DNA Topoisomerases, Type I