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. 2003 Aug;132(4):2108-15.
doi: 10.1104/pp.103.024273.

Pea DNA Topoisomerase I Is Phosphorylated and Stimulated by Casein Kinase 2 and Protein Kinase C

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Pea DNA Topoisomerase I Is Phosphorylated and Stimulated by Casein Kinase 2 and Protein Kinase C

Narendra Tuteja et al. Plant Physiol. .
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Abstract

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.

Figures

Figure 1.
Figure 1.
In vitro phosphorylation of pea DNA topoisomerase I by endogenous kinases and its dephosphorylation. Pea NE (NE, 3 μg) was used as source of endogenous kinases to phosphorylate recombinant pea DNA topoisomerase I (0.5 μg). The standard kinase reaction was carried out in the presence of Mg2+ and [γ32P]-ATP followed by SDS-PAGE and autoradiography as described in “Materials and Methods.” A, Autoradiogram showing a 100-kD band of pea topoisomerase I phosphorylated with NE (lane 1). Lanes 2 and 3, Reactions with only topoisomerase 1 (without NE) and NE (without topoisomerase I). B, Autoradiogram showing the phosphorylation of topoisomerase I in absence (lane 1) and presence of 0.5, 1.0, and 2.0 mm MgCl2 (lanes 2–4). C, Autoradiogram showing that there is no autophosphorylation of topoisomerase I at 0.5- and 2.0-μg concentrations (lanes 2 and 3). Lane 1 is a standard kinase reaction. D, Autoradiogram showing topoisomerase I phosphorylation (lane 1) is inhibited by calf intestinal alkaline phosphatase (CIAP) and lambda phosphatase (LP) (lanes 2 and 3). E, Autoradiogram showing the phosphorylation of topoisomerase I (lane 1) is inhibited by 3 μm calphostin (lane 3) and 25 μg of heparin (lane 4) and not inhibited even by higher concentration (100 μm) of staurosporine (lane 2).
Figure 2.
Figure 2.
In vitro phosphorylation of pea DNA topoisomerase I by exogenous kinases and its inhibition. The Xenopus laevis CK2 and rat brain PKC were used as source of exogenous kinases to phosphorylate recombinant pea DNA topoisomerase I (0.5 μg). The standard kinase reaction was carried out in presence of Mg2+ and [γ32P]-ATP followed by SDS-PAGE and autoradiography as described in “Materials and Methods.” A, Autoradiogram showing a 100-kD band of pea topoisomerase I phosphorylated with CK2 (lane 2). The phosphorylation reactions were performed with 1, 5, 10, and 25 μg of heparin (lanes 3–6, respectively). Lane 1, Reaction with only topoisomerase (without CK2). B, Autoradiogram showing phosphorylation of topoisomerase I with PKC in the presence of 5, 10, and 20 μg of phorbol myristate acetate (PMA; lanes 2–4, respectively). This phosphorylation is inhibited by 3 μm calphostin (Cal., lane 1) in presence of 20 μg of PMA. C, Concentration curve of calphostin. The PMA (20 μg)-stimulated phosphorylation of topoisomerase I with PKC in the absence (lane 1) and in the presence of 0.2, 0.5, and 1.0 μm calphostin (lanes 2–4, respectively). D, Autoradiogram showing the phosphorylation of topoisomerase I with CK2 (lane 1) in the presence of 2.5 mm spermine (lane 2) and 2.5 mm spermidine (lane 3).
Figure 3.
Figure 3.
Pea topoisomerase I is phosphorylated at Ser residue(s). A, Phospho-amino acid analysis. The phosphorylation of pea topoisomerase I was carried out by CK2 and PKC by standard method. The phosphorylated 32P-labeled topoisomerase I was hydrolyzed in 6 n HCl and finally analyzed by paper chromatography as described in “Materials and Methods.” Lanes 1 and 2, Analysis by CK2 and PKC phosphorylations, respectively. The positions of standard phosphoamino acids visualized by ninhydrin staining are indicated on the right. B, Inhibition of CK2 phosphorylation of pea topoisomerase I by phospho-Ser antibody. Standard CK2 phosphorylation reactions (lanes 1 and 2) were performed in the presence and absence of phospho-Ser antibody (lanes 1 and 2, respectively).
Figure 4.
Figure 4.
A, Coommassie Blue-stained SDS-PAGE showing one band of 70 kD from one of the protein fractions eluted from PMA-affinity column by NaCl and alpha-PMA (lane 2, 0.10 μg). Lane 1 (M), Protein Mr markers. B, Autoradiogram showing the phosphorylation of pea topoisomerase I by PMA-affinity-purified nuclear protein (0.20 μg) in the presence of PMA and calcium. Lanes 1 and 2, Same reaction without the PKC and topoisomerase I, respectively.
Figure 5.
Figure 5.
Stimulation of pea topoisomerase I activity by CK2 (A) and PKC (B) phosphorylation. Supercoiled pBR322 DNA (0.6 μg) was used for each assay. A and B, Lane 1 is the reaction without any protein (only DNA), and lane 2 is the standard topoisomerase I reaction showing the ladder formation. The topoisomerase I used in lane 3 was prephosphorylated with CK2 (A) or PKC (B) before performing the topoisomerase I assay. In lane 3 of A and B, the ladder moved up (stimulation). Lane 4, Control reaction with only CK2 (A) or PKC (B) to show that these preparations do not contain any topoisomerase I activity.

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