Abstract
The Mr = 38,300 polypeptide of the purified recombinant rat DNA polymerase beta served as an excellent substrate for protein kinase C (PKC) in vitro but not for the catalytic subunit of cAMP-dependent protein kinase. The phosphorylation by PKC resulted in inactivation of DNA polymerase beta activity, and recovery was achieved by dephosphorylation with alkaline phosphatase. Since the phosphorylated DNA polymerase beta was retained with use of a single-stranded DNA-cellulose column, inactivation might occur at a site different from that for the DNA binding. Amino acid sequence analysis of the phosphopeptides revealed that the phosphorylated sites were 2 serine residues at positions 44 and 55 from the NH2 terminus, either or both of which might be involved in the catalytic activity of DNA polymerase beta. Thus, the inactivation of the DNA repair enzyme, DNA polymerase beta, by PKC may be an important process in the modification of DNA metabolism in the nucleus through signal transduction processes.
Highlights
DNA repair enzyme, DNA polymerase 8, by Protein kinase C (PKC) may be an important process in the modification of DNA metabolism inthe nucleus through signal transduction processes
Polymerase /3 activity to modify DNA metabolismin the nucleus, we overproduced rat DNA polymerase p with high enzyme activity in Escherichia coli, and a large quantity was purified to apparent homogeneity [14].In the present study, we attempted to phosphorylatein vitro the purified recombinant rat DNA polymerase /3 by PKC; DNA polymerase /3 was inactivated, depending on the extentof phosphorylation
Tant for cell differentiation and proliferation [1].When cells were treated by various growth factors or tumor promoters suchas 12-0-tetradecanoylphorbol-13-acetat(eTPA), PKC functioned in the phosphorylation of a number of proteins
Summary
Tant for cell differentiation and proliferation [1].When cells were treated by various growth factors or tumor promoters suchas 12-0-tetradecanoylphorbol-13-acetat(eTPA), PKC functioned in the phosphorylation of a number of proteins. Enzymes-The catalytic subunit of CAMP-dependent protein kinase was prepared from bovine hearts using the method of Beavo et al [15]. Protein kinase C was prepared from rat brains according to our reported methods [16, 17]. The enzymatic and immunological properties of the recombinant DNA polymerase p were indistinguishable from that purified from rat cells except that the NH,-terminal methionine residue was absent in the jected to modification. Purchased from Toyobo Co., Ltd. Phosphorylation by Protein Kinases-DNA polymerase p MM MgCl,, 25 mM Tris-HCI, pH 7.0, at 25 To quantifythe phosphorylation of the DNA polymerase p or vimentin polypeptide, the reactionwas terminated by adding 0.5 volume of a stock solution containing6%SDS, 6% 2-mercaptoethanol, 15% glycerol, 0.19 M Tris-HC1, pH 6.8. Inactivation of D N A Polymerase /3 by PKC separating and stacking gels contained 10 and 3% acrylamide, re-
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