Inactivation of DNA polymerase beta by in vitro phosphorylation with protein kinase C

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.     

Phosphorylation of keratin intermediate filaments by protein kinase C, by calmodulin-dependent protein kinase and by cAMP-dependent protein kinase.

Keratins, constituent proteins of intermediate filaments of epithelial cells, are phosphoproteins containing phosphoserine and phosphothreonine. We examined the in vitro phosphorylation of keratin filaments by cAMP-dependent protein kinase, protein kinase C and Ca2+/calmodulin-dependent protein kinase II. When rat liver keratin filaments reconstituted by type I keratin 18 (molecular mass 47 kDa; acidic type) and type II keratin 8 (molecular mass 55 kDa; basic type) in a 1:1 ratio were used as substrates, all the protein kinases phosphorylated both of the constituent proteins to a significant rate and extent, and disassembly of the keratin filament structure occurred. Kinetic analysis suggested that all these protein kinases preferentially phosphorylate keratin 8, compared to keratin 18. The amino acid residues of keratins 8 and 18 phosphorylated by cAMP-dependent protein kinase or protein kinase C were almost exclusively serine, while those phosphorylated by Ca2+/calmodulin-dependent protein kinase II were serine and threonine. Peptide mapping analysis indicated that these protein kinases phosphorylate keratins 8 and 18 in a different manner. These observations gave the way for in vivo studies of the role of phosphorylation in the reorganization of keratin filaments.     

DNA topoisomerase I phosphorylation in murine fibroblasts treated with 12-O-tetradecanoylphorbol-13-acetate and in vitro by protein kinase.

The phosphorylation of DNA topoisomerase I in quiescent murine 3T3-L1 fibroblasts treated with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (TPA) was characterized by in vivo labeling with [32P] orthophosphate and immunoprecipitation with a scleroderma anti-DNA topoisomerase I autoantibody. DNA topoisomerase I phosphorylation was stimulated 4-fold by 2 h of TPA treatment (TPA at 100 ng/ml maximally enhanced phosphorylation). Purified DNA topoisomerase I was phosphorylated in vitro in a Ca2+ and phospholipid-dependent fashion by types I, II, and III protein kinase C. The phosphorylation reaction was stimulated by TPA and had an apparent K(m) of 0.4 microM. DNA topoisomerase I was phosphorylated in vivo and in vitro predominantly at serine. The major tryptic phosphopeptides from DNA topoisomerase I in TPA-treated fibroblasts and phosphorylated by protein kinase C comigrated in thin-layer electrophoresis. The half-life of incorporated phosphate on DNA topoisomerase I was 40 min in both TPA-treated and control cells. These results suggest that phosphorylation is a mechanism for activating DNA topoisomerase I in fibroblasts treated with TPA and that protein kinase C functions in the phosphorylation.     

Phosphorylation of a gelatin-binding protein from L6 myoblasts by protein kinase C

A gelatin-binding glycoprotein from L6 rat myoblasts, designated gp46, was shown to be phosphorylated in vivo. This phosphorylation was increased slightly (18%) by phorbol ester treatment of L6 suggesting protein kinase C involvement. Purified gp46 could be phosphorylated in vitro with protein kinase C, but not by the catalytic subunit of cAMP-dependent protein kinase. Comparison of the phosphotryptic peptide maps of in vitro and in vivo labeled gp46 suggested that in vivo phosphorylation of gp46 may be mediated by protein kinase C.     

Phosphatidylinositol-dependent activation of DNA polymerase alpha

DNA polymerase alpha was activated in vitro by cAMP-independent, phospholipid-dependent, protein kinase catalytic subunit. Of the phospholipids examined, phosphatidylinositol showed the greatest potential for interaction with protein kinase and ATP to activate DNA polymerase alpha in vitro. DNA polymerase alpha was directly activated by phosphorylated phosphatidylinositol in the absence of protein kinase and ATP. Activation of DNA polymerase alpha as a function of phosphorylation was demonstrated using 32P-ATP as the phosphate donor. In vitro treatment of the enzyme with phosphatidylinositol produced Linweaver-Burk plots showing noncompetitive kinetics of enzyme activation, suggesting that activation occurs prior to binding of the enzyme to DNA template/primer. These data indicate that DNA polymerase alpha may be activated in vitro in the presence of protein kinase, ATP, and phosphatidylinositol, and suggest that phosphorylation of the enzyme may constitute an intracellular mechanism of enzyme activation.     

Exposure of HeLa DNA polymerase alpha to protein kinase C affects its catalytic properties

Protein kinase C (Ca2+/phospholipid-dependent protein kinase) purified from rat brain or endogenous to cell-free extracts from HeLa cells stimulates, by a factor of 2-3, HeLa DNA polymerase alpha but not beta or gamma. Monoclonal antibody to the kinase prevents the stimulation, and monoclonal antibody to human DNA polymerase alpha neutralizes the enhanced activity. Reduced DNA polymerase alpha activity is obtained from noncycling HeLa cells and this activity has lower fidelity when copying synthetic primer-templates than that obtained from log phase cultures. After exposure to the kinase, the fidelities and activities of the polymerase from both sources increase by 2- to 3-fold. This improved accuracy is not accompanied by the appearance of triphosphatase or DNase activities. Exposure to the protein kinase reduces the Km for activated DNA and for poly(dA-dT) but not for dNTPs. Moreover, the Vmax for activated DNA but not for poly(dA-dT) is increased approximately 2- to 3-fold. These alterations suggest a role for protein phosphorylation in modulating DNA polymerase alpha.     

Involvement of protein kinase A in the phosphorylation of spermatidal protein TP2 and its effect on DNA condensation.

Rat spermatidal protein TP2 is rich in serine residues and has several potential sites for phosphorylation by different protein kinases. Recombinant TP2 is phosphorylated upon incubation in vitro with salt extract of testicular sonication resistant nuclei (SRN) (representing elongating and elongated spermatids). The major phosphorylation sites were localized to the C-terminal, V8 protease-derived, fragment (residues 87-114). Phosphorylation experiments with the wild type and different site-specific mutants of TP2 revealed that serine 109 and threonine 101 are the phosphorylation sites. Phosphorylation of the C-terminal fragment of TP2 was also demonstrated in vivo. Phosphorylation was not stimulated by either protein kinase C activators or cGMP but was inhibited by protein kinase A inhibitor (PKI) peptide, showing the involvement of protein kinase A in the phosphorylation of TP2. Phosphorylation of TP2 greatly reduced its DNA condensation property. TP2 when complexed with DNA was not a good substrate for phosphorylation by PKA. Dephosphorylation of the DNA-TP2 complex by calf intestinal alkaline phosphatase restored the DNA condensation property to a level equivalent to that observed with TP2. The physiological significance of the phosphorylation-dephosphorylation cycle is discussed with reference to the two-domain model of TP2.     

The phosphorylation of eukaryotic ribosomal protein S6 by protein kinase C

Purified Ca2+-dependent and phospholipid-dependent protein kinase (protein kinase C) from bovine brain catalysed the phosphorylation of ribosomal protein S6 when incubated with 40S ribosomal subunits from rat liver or from hamster fibroblasts. The phosphorylation was dependent on Ca2+ and phospholipid, and occurred under ionic conditions similar to those which support protein biosynthesis in vitro. Protein kinase C phosphorylated at least three sites on ribosomal protein S6 when incubated with unphosphorylated ribosomes, and increased the extent of phosphorylation of ribosomes previously phosphorylated predominantly on two sites by cyclic-AMP-dependent protein kinase, converting some molecules to the tetraphosphorylated or pentaphosphorylated form. This indicates that protein kinase C can phosphorylate sites on ribosomal protein S6 other than those phosphorylated by the cyclic-AMP-dependent protein kinase, and this conclusion was confirmed by analysis of tryptic phosphopeptides. These results strengthen the possibility that protein kinase C might be involved in catalysing the multisite phosphorylation of ribosomal protein S6 in certain circumstances in vivo.     

Phosphorylation of cytoplasmic poly (ADP-ribose) polymerase linked to free ribonucleoprotein particles by an associated protein kinase C

Considering the eventuality of an interaction between the two post-translational modifications, phosphorylation and ADP-ribosylation, we investigated the possibility of phosphorylation of the mRNP polyADPR polymerase by a protein kinase C associated to these particles. We demonstrated that cytoplasmic poly (ADP-ribose) polymerase associated with ribonucleoprotein particles containing silent mRNA is phosphorylated by a specifically activated endogenous protein kinase C which in turn induces an inhibition of the polymerase activity. In the absence of protein kinase C activators the mRNP polyADPR-P is also phosphorylated but without changes of its enzymatic activity.     

Phosphorylation of rhodopsin by protein kinase C in vitro

Calium/phospholipid-dependent protein kinase (protein kinase C) was purified from bovine retinae rod outer segments (ROS). In the presence of 0.1-2 microM calcium protein kinase C binds tightly to ROS and phosphorylates rhodopsin in the absence or presence of illumination. This property of protein kinase C contrasts with that of rhodopsin kinase, which in vitro phosphorylates only bleached rhodopsin. Peptide maps of rhodopsin phosphorylated by protein kinase C or rhodopsin kinase were compared using limited Staphylococcus aureus V8 protease digestion or complete tryptic digestion. Phosphorylation sites map to serine and threonine residues on the cytoplasmic carboxylterminal domain of rhodopsin for both kinases. The functional consequence of protein kinase C phosphorylation of rhodopsin was a reduced ability to stimulate the light-dependent rhodopsin activation of [35S]guanosine 5'-O-(thiotriphosphate) binding to transducin, the GTP-binding regulatory protein present in ROS. Properties of the calcium-stimulated interaction of protein kinase C with membranes and in vitro phosphorylation of intrinsic proteins are discussed based upon the findings.     

Phosphorylation of liver gap junction protein by protein kinase C

The 27 kDa protein, a major component of rat liver gap junctions, was shown to be phosphorylated in vitro by protein kinase C. The stoichiometry of the phosphorylation indicated that approx. 0.33 mol phosphate was incorporated per mol 27 kDa protein. Phosphorylation was entirely dependent on the presence of calcium and was virtually specific for serine residues. For comparison, the gap junction protein was also examined for its phosphorylation by cAMP-dependent protein kinase, the extent of phosphorylation being one-tenth that exerted by protein kinase C.     

Phosphorylation of protein phosphatase inhibitor-1 by protein kinase C

Inhibitor-1 becomes a potent inhibitor of protein phosphatase 1 when phosphorylated by cAMP-dependent protein kinase at Thr(35). Moreover, Ser(67) of inhibitor-1 serves as a substrate for cyclin-dependent kinase 5 in the brain. Here, we report that dephosphoinhibitor-1 but not phospho-Ser(67) inhibitor-1 was efficiently phosphorylated by protein kinase C at Ser(65) in vitro. In contrast, Ser(67) phosphorylation by cyclin-dependent kinase 5 was unaffected by phospho-Ser(65). Protein kinase C activation in striatal tissue resulted in the concomitant phosphorylation of inhibitor-1 at Ser(65) and Ser(67), but not Ser(65) alone. Selective pharmacological inhibition of protein phosphatase activity suggested that phospho-Ser(65) inhibitor-1 is dephosphorylated by protein phosphatase 1 in the striatum. In vitro studies confirmed these findings and suggested that phospho-Ser(67) protects phospho-Ser(65) inhibitor-1 from dephosphorylation by protein phosphatase 1 in vivo. Activation of group I metabotropic glutamate receptors resulted in the up-regulation of diphospho-Ser(65)/Ser(67) inhibitor-1 in this tissue. In contrast, the activation of N-methyl-d-aspartate-type ionotropic glutamate receptors opposed increases in striatal diphospho-Ser(65)/Ser(67) inhibitor-1 levels. Phosphomimetic mutation of Ser(65) and/or Ser(67) did not convert inhibitor-1 into a protein phosphatase 1 inhibitor. On the other hand, in vitro and in vivo studies suggested that diphospho-Ser(65)/Ser(67) inhibitor-1 is a poor substrate for cAMP-dependent protein kinase. These observations extend earlier studies regarding the function of phospho-Ser(67) and underscore the possibility that phosphorylation in this region of inhibitor-1 by multiple protein kinases may serve as an integrative signaling mechanism that governs the responsiveness of inhibitor-1 to cAMP-dependent protein kinase activation.     

Phosphorylation of the 27-kDa gap junction protein by protein kinase C in vitro and in rat hepatocytes

We previously demonstrated that the 27-kDa major component protein in rat liver gap junctions was phosphorylated by protein kinase C in vitro (Takeda, A. et al. (1987) FEBS Lett. 210, 169-172). In this study, we examined this further and examined the phosphorylation of the 27-kDa gap junction protein in rat hepatocytes by metabolically labeling cells with [32P]orthophosphate and using a monoclonal antibody to immunoprecipitate the protein. The in vitro phosphorylation was inhibited by monoclonal antibodies recognizing the carboxyl- (C-)terminal domain of the 27-kDa protein. Protease digestion analysis revealed that phosphorylation occurred at the C-terminal domain. In rat hepatocytes, the phorbol esters, 12-O-tetradecanoylphorbol-13-acetate and phorbol-12,13-dibutyrate, stimulated the 27-kDa protein phosphorylation, whereas 4 alpha-phorbol-12,13-didecanoate did not. 1-Oleoyl-2-acetyl-sn-glycerol also stimulated the 27-kDa protein phosphorylation. In addition, norepinephrine stimulated the phosphorylation and pretreatment of hepatocytes with staurosporine, a potent inhibitor of protein kinase C, inhibited this stimulatory effect of norepinephrine. Both in vitro and in hepatocytes, analysis of chemical cleavage of the 27-kDa phosphoprotein revealed that phosphorylation occurred mainly at a 10-kDa fragment which the antibodies recognized. These results indicate that protein kinase C phosphorylates the 27-kDa gap junction protein, not only in vitro but also in hepatocytes, at the C-terminal domain of the protein.     

Phosphorylation of ras oncogene product by protein kinase C

The Harvey (H)-ras oncogene product, p21, can be phosphorylated by protein kinase C in vitro at sites distinct from the site of autophosphorylation of p21. Serine was found to be the main phosphate acceptor. Kinetic studies revealed a high apparent affinity but a much lower turnover for the phosphorylation of p21 as compared with that of the phosphorylation of histone by protein kinase C. Indirect association between protein kinase C and p21 was suggested by the co-immunoprecipitation of both proteins with either anti-protein kinase C or anti-p21 antibodies.     

Ca2+/calmodulin-dependent protein kinase II regulates Tiam1 by reversible protein phosphorylation

A number of guanine nucleotide exchange factors have been identified that activate Rho family GTPases, by promoting the binding of GTP to these proteins. We have recently demonstrated that lysophosphatidic acid and several other agonists stimulate phosphorylation of the Rac1-specific exchange factor Tiam1 in Swiss 3T3 fibroblasts, and that protein kinase C is involved in Tiam1 phosphorylation (Fleming, I. N., Elliott, C. M., Collard, J. G., and Exton, J. H. (1997) J. Biol. Chem. 272, 33105-33110). We now show, through manipulation of intracellular [Ca2+] and the use of protein kinase inhibitors, that both protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II are involved in the phosphorylation of Tiam1 in vivo. Furthermore, we show that Ca2+/calmodulin-dependent protein kinase II phosphorylates Tiam1 in vitro, producing an electrophoretic retardation on SDS-polyacrylamide gel electrophoresis. Significantly, phosphorylation of Tiam1 by Ca2+/calmodulin-dependent protein kinase II, but not by protein kinase C, enhanced its nucleotide exchange activity toward Rac1, by approximately 2-fold. Furthermore, Tiam1 was preferentially dephosphorylated by protein phosphatase 1 in vitro, and treatment with this phosphatase abolished the Ca2+/calmodulin-dependent protein kinase II activation of Tiam1. These data demonstrate that protein kinase Calpha and Ca2+/calmodulin-dependent protein kinase II phosphorylate Tiam1 in vivo, and that the latter kinase plays a key role in regulating the activity of this exchange factor in vitro.     

Purification of two distinct proteins of approximate Mr 80,000 from human epithelial cells and identification as proper substrates for protein kinase C.

A Mr-80,000 acidic phosphoprotein ('80K protein') is a specific substrate for protein kinase C. We attempted to purify the 80K protein from a human squamous-cell carcinoma cell line, Ca9-22, by the sequential use of heat treatment, (NH4)2SO4 precipitation, Mono Q column chromatography, proRPC column chromatography and gel filtration. The 80K protein was assayed by phosphorylation in vitro by using partially purified human type III protein kinase C, and was fractionated into two distinct molecular species with slightly different Mr values, designated 80K-L and 80K-H proteins. Phosphorylation occurred mainly at serine residues of these proteins. Two-dimensional phosphopeptide maps after trypsin digestion and kinetic profiles of phosphorylation were different from each other. Ca2(+)- and phospholipid-dependency of the phosphorylation in vitro confirmed that both 80K-L and 80K-H proteins are true substrates for three subtypes of protein kinase C. The 80K-L protein was a preferential substrate for type III protein kinase C, and the 80K-H protein was phosphorylated more effectively by type I and type II protein kinase C. The possible roles of these two distinct 80K proteins in signal transduction are discussed.     

Effect of core protein phosphorylation by protein kinase C on encapsidation of RNA within core particles of hepatitis B virus.

Phosphorylation of core particles derived either from hepatitis B viruses or from livers of hepatitis B-infected individuals has been long recognized, but the nature and function of the phosphorylating enzyme remained unknown. By immunoblotting with a monoclonal antibody, we have now detected protein kinase C within the liver-derived core particles. To study the significance of the encapsidated protein kinase C for the viral life cycle, we established an in vitro assembly system consisting of Escherichia coli-expressed core protein, protein kinase C, and in vitro-synthesized hepatitis B virus RNA. Phosphorylation of the core protein led to a reduced RNA encapsidation capacity of the core particles. Furthermore, RNA and protein kinase C competed for their target sequence, which is the carboxy-terminal arginine-rich domain of the core protein. This finding implies that phosphorylation of the nucleic acid binding site in the core protein occurs within the particles after encapsidation of protein kinase C, pregenomic RNA, and viral polymerase at a later step during viral genome maturation.