The herpesvirus protein kinase: a new departure in protein phosphorylation?
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Phosphorylation↗
Biomedical subjects
D P Leader
Publications and source records attributed to D P Leader.
Viral aspects of protein phosphorylation.
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Bacteriophages↗
Activation of a ribosomal protein S6 kinase in mouse fibroblasts during infection with herpesvirus.
If confluent fibroblasts are infected with the swine alpha-herpes virus, pseudorabies virus, ribosomal protein S6 becomes phosphorylated after a lag of approximately 2 h. When cell-free extracts were prepared from such cells in the presence of glycerol 2-phosphate and EGTA, a ribosomal protein S6 kinase activity was found to appear at approximately the same time as the phosphorylation in vivo. This protein kinase was similar to that activated in the same cells by replenishing the nutrient medium, and in other quiescent cells by the action of growth factors and mitogens. It was distinct from the previously described pseudorabies virus protein kinase, which is unique to infected cells. When medium from cells infected with pseudorabies virus was freed of virus and added to confluent fibroblasts, rapid activation of the ribosomal protein S6 kinase activity occurred. A similar, although more limited, effect could be seen when the pH of the medium was increased. These results suggest that the phosphorylation of ribosomal protein S6 in cells infected with herpes virus is a consequence of the production of a factor which initiates the metabolic programme for cellular growth. The possible function of this effect in the infective strategy of herpes viruses is discussed in relation to requirements for the replication of viral DNA.
Animals↗
Complete purification of the pseudorabies virus protein kinase.
The recently described pseudorabies virus protein kinase has been purified from infected hamster fibroblasts by a combination of anion-exchange, hydrophobic-interaction and affinity chromatography. The purification resulted in enzyme with a specific activity in excess of 1,000 nmol phosphate mg-1 min-1 in relatively high yield. Gel electrophoresis of the purified enzyme under denaturing conditions revealed a single stained band at a position of migration corresponding to a Mr 38,000. Incubation of the purified enzyme with [gamma-32P]ATP in the absence of added substrate resulted in incorporation of 32P into this protein band, consistent with the 38-kDa protein being a protein kinase with a capacity for autophosphorylation. The phosphorylated form of the protein has an isoelectric point of approximately 4.9. Gel permeation chromatography of the purified enzyme indicated a native Mr 70,000, suggesting that the protein kinase has a homodimeric structure.
Animals↗
Molecular analysis of elements inserted into mouse gamma-actin processed pseudogenes.
DNA from ten mouse genomic clones, each containing distinct gamma-actin processed pseudogenes, was subjected to electron microscopic heteroduplex analysis, and in three cases (lambda mA36, lambda mA118 and lambda mA119) the heteroduplex formed with the DNA of a reference clone was found to be interrupted by a single-stranded loop. The genomic regions corresponding to these loops were subjected to structural analysis and they were found to represent different elements (IEs) inserted into the pseudogenes in a manner that gave rise to short target-site direct repeats. IE 36 (500 base-pairs in length) was found to be an intercisternal A-particle solo long terminal repeat (LTR), a 46 nucleotide region of which had undergone five-fold tandem amplification and subsequent mutation. IE 119 (501 base-pairs in length) was also a solo LTR, bearing similarity to the recently-described GLN-3 class of murine retroviral-like elements. IE 118 (865 base-pairs in length) is repeated 1000-2000 times in the mouse genome. It is not related to any known class of mobile elements, but does possess some sequence motifs that suggest it may be an LTR of a hitherto unrecognized family of retroviral-like elements. It also possesses a 26 out of 27 nucleotide identity to a region of the flanking pseudogene, suggesting that it may have suffered gene conversion.
Actins↗
Induction, partial purification and characterization of a hamster fibroblast protein kinase activity that phosphorylates ribosomal protein S6.
When BHK cells were grown to confluence and the growth medium replenished, there was a large and rapid increase in the phosphorylation of ribosomal protein S6. In postribosomal extracts of these cells, prepared in the presence of glycerol 2-phosphate and EGTA, a ribosomal protein S6 kinase was detected. The increase in activity of this protein kinase broadly reflected the increase in phosphorylation of ribosomal protein S6 observed in vivo. This ribosomal protein S6 kinase activity was substantially purified by a combination of phosphocellulose, DEAE-cellulose, Mono Q and heparin-Sepharose chromatography, and some of its characteristics were examined. When the products of phosphorylation of 40S ribosomal subunits by purified enzyme in vitro were analysed using two-dimensional gel electrophoresis, monophosphorylated and diphosphorylated forms of ribosomal protein S6 were observed to be the predominant radioactively labelled species.
Animals↗
Identification of the herpes simplex virus protein kinase as the product of viral gene US3.
Previous work has shown that a novel protein kinase is induced after infection of cultured cells with herpes simplex virus type 1 (HSV-1). Separately, it has been reported that the protein encoded by HSV-1 gene US3 shows similarity in its amino acid sequence to members of the protein kinase family of eukaryotes. We have investigated the possibility that these two observations are connected by preparing an antiserum to a synthetic oligopeptide corresponding to the carboxy-terminal eight amino acids of the US3 protein. This antiserum reacted on immunoblots with a polypeptide of apparent molecular weight 68,000 from extracts of cells which had been infected with HSV-1. The antiserum also reacted strongly with a 68,000 molecular weight species from a preparation of the novel HSV-1 protein kinase which had been extensively purified and resolved from other protein kinases. In addition, the purified preparation phosphorylated a protein species, also of 68,000 apparent molecular weight, when incubated with [gamma-32P]ATP. These data are consistent with gene US3 encoding the novel protein kinase induced after infection of cells with HSV-1.
Genes, Viral↗
A major phosphoprotein of cells infected with pseudorabies virus is phosphorylated by cellular casein kinase II.
Endogenous protein phosphorylation was studied in extracts of hamster fibroblasts infected with pseudorabies virus. The major phosphorylation was detected quite late in infection and involved an acidic protein of Mr 62,000. It was catalysed by an enzyme activity with the properties of cellular casein kinase II. Two-dimensional gel analysis was used to demonstrate that this same protein was also phosphorylated in vivo. The phosphoprotein was detected in mature virions and is most likely viral in origin.
Casein Kinases↗
Herpes simplex virus 1 protein kinase is encoded by open reading frame US3 which is not essential for virus growth in cell culture.
Earlier reports have described a novel protein kinase in cells infected with herpes simplex or pseudorabies viruses. These novel enzymes were characterized by their acceptance of protamine as a substrate and by their differential chromatographic behavior in anion-exchange chromatography. We report that this activity was not present in extracts of uninfected cells or of cells infected with a mutant constructed so as to contain a deletion in the US3 open reading frame mapping in the small component of herpes simplex virus 1 DNA. The activity was present in extracts of cells infected with wild-type virus and with a recombinant in which the US3 open reading frame had been rescued. Our results are consistent with the observation reported earlier that the coding sequences predict an amino acid motif common to protein kinases and lead to the conclusion that the US3 open reading frame encodes a virus-specific protein kinase that is not required for virus growth in cells in culture.
Amino Acid Sequence↗
The substrate specificity of the protein kinase induced in cells infected with herpesviruses: studies with synthetic substrates [corrected] indicate structural requirements distinct from other protein kinases.
Synthetic peptides have been used to investigate the site specificity of highly purified virus induced protein kinase, a recently discovered protein kinase isolated from cells infected with alpha-herpesviruses. The enzyme from cells infected with pseudorabies virus can catalyse the phosphorylation of both seryl and threonyl residues in peptides that contain several arginyl residues on the amino-terminal side of the target residue. At least two arginyl residues are required, and the best substrates examined contain four to six such residues. Virus induced protein kinase differs in site specificity from protein kinase C in being unable to phosphorylate peptides in which multiple arginyl residues are on the carboxyl-terminal side of the target residue, or to phosphorylate peptides in which the arginyl residues are replaced by ornithyl residues. Virus induced protein kinase from cells infected with herpes simplex virus type I had similar substrate preferences to virus induced protein kinase from cells infected with pseudorabies virus. Although virus induced protein kinase and the cyclic AMP-dependent protein kinase have several peptide substrates in common, their relative preferences for these (as indicated by Km values) were found to be very different.
Animals↗
The phosphorylation of ribosomal protein S6 by protein kinases from cells infected with pseudorabies virus.
We examined the ability of protein kinase activities from BHK (baby-hamster kidney) cells infected with pseudorabies virus to catalyse the phosphorylation of ribosomal protein S6 in vitro. When the cytosol from infected cells was fractionated on DEAE-cellulose, 40S ribosomal protein kinase activity was found associated with the two isoforms of the cyclic AMP-dependent protein kinase, protein kinase C and a protein kinase (ViPK, virus-induced protein kinase) only detected in infected cells. The phosphorylation of ribosomal protein by ViPK was of particular interest because the appearance of the protein kinase and the increase in the phosphorylation of protein S6 in infected cells shared a similar time course. At moderate concentrations of KCl the major ribosomal substrate for ViPK was ribosomal protein S7, a protein not found to be phosphorylated in vivo. However, at 600 mM-KCl, or in the presence of 5-10 mM-spermine at 60-150 mM-KCl, the phosphorylation of ribosomal protein S7 was suppressed and ribosomal protein S6 became the major substrate. The maximum stoichiometry of phosphorylation obtained under the latter conditions was 1-2 mol of phosphate/mol of S6, and only mono- and di-phosphorylated forms of S6 were detected on two-dimensional gel electrophoresis. As the infection of BHK cells by pseudorabies virus results in the appearance of phosphorylated species of S6 containing up to 5 mol of phosphate/mol of S6 protein, it appears unlikely that ViPK alone can be responsible for the multiple phosphorylation seen in vivo. Nevertheless, tryptic phosphopeptide analysis did indicate that in vitro ViPK catalysed the phosphorylation of at least one of the sites on ribosomal protein S6 phosphorylated in vivo, so that a contributory role for the enzyme in the phosphorylation in vivo cannot be excluded.
Cells, Cultured↗
The structure of a cDNA clone corresponding to mouse cardiac muscle actin mRNA.
A cDNA library was constructed from mouse cardiac muscle mRNA, and a clone corresponding to part of the mRNA for the cardiac muscle isoform of actin was isolated from this library. The nucleotide sequence of the cloned insert was determined and was found to contain almost the complete amino acid coding region for actin (only codons for the first two amino acids, absent from the mature protein, were lacking) and a substantial portion derived from the 3' untranslated region of the mRNA. Comparison of the latter with the corresponding region in cardiac actin mRNA from man and rat showed that this 3' untranslated region has been subject to conservational pressure during evolution. However a comparison with the corresponding region in skeletal muscle actin mRNAs indicated that the pattern of conservation is quite different in the two striated muscle actin isoforms.
Actins↗
Characterization of a human orphon 28 S ribosomal DNA.
We have isolated clones in which two regions of the human genome are represented, each containing an orphon: a dispersed copy of 28S rDNA. Nucleotide (nt) sequence analysis established that one of these, H28S-O1, corresponds to nt 3627-4105 of human 28S rDNA, but in a mutated form. The orphon was flanked on one side by a portion of the L1Hs long interspersed repeat family of the human genome. Although H25S-O1 is not flanked by the terminal direct repeats characteristic of transposed DNA, it is possible that it is a processed pseudogene.
Base Sequence↗
Isolation and characterization of cDNA clones from mouse skeletal muscle actin mRNA.
The sequence corresponding to approximately 98% of mouse skeletal muscle actin mRNA was determined from cDNA clones isolated from a library of recombinants in pBR322. One of these clones contains DNA corresponding to the complete amino acid coding region and a large part of the 5' and 3' untranslated regions of the mRNA. Comparison of the mouse coding region (conserved at the amino acid level) and noncoding regions with the corresponding regions of the rat skeletal muscle actin gene indicates that the noncoding regions have also been under selective pressure during evolution.
Actins↗
Characteristics of the induction of a new protein kinase in cells infected with herpesviruses.
The appearance of a recently described protein kinase activity (virus-induced protein kinase, ViPK) has been studied during infection of hamster fibroblasts with pseudorabies virus or with herpes simplex virus type 1 (HSV-1). An enzyme activity with comparable catalytic properties was induced in both cases, and had broadly similar kinetics of appearance to that of the viral DNA polymerase. The amount of active ViPK detected depended on the multiplicity of infection, and no ViPK was induced after the viruses had been subjected to irradiation with u.v. light. When cells were infected with the tsK mutant of HSV-1, ViPK was induced at the permissive but not at the restrictive temperature. The ViPK preparations obtained from cells infected with each virus differed in chromatographic properties on anion-exchange and gel-permeation resins. These results indicate that expression of the viral genome is required for induction of ViPK. They suggest that the enzyme may be encoded by the viral genome, but do not provide proof of this.
Animals↗
Partial purification and characterization of a new phosphoprotein kinase from cells infected with pseudorabies virus.
Cytoplasmic fractions from normal baby hamster kidney fibroblasts and from fibroblasts infected with pseudorabies virus were fractionated by DEAE-cellulose chromatography and fractions assayed for protein kinase activity. In preparations from uninfected and infected cells protein kinase activities identified as casein kinase I and II, the two isoforms of the cyclic-AMP-dependent protein kinase, protein kinase C, and a presumed proteolytic fragment of protein kinase C were present in comparable amounts. However in infected cells a new protein kinase activity was detected, appearing about 4 h after infection and increasing during the following 6 h at least. This new protein kinase was purified 100-fold by high-performance gel-permeation and ion-exchange chromatography, and characterized. It has an apparent relative molecular mass of 68 000 on the basis of gel-permeation chromatography, and a sedimentation coefficient of 4.3 S. It catalysed the phosphorylation of serine residues of basic proteins in vitro, with protamine a better substrate than mixed histones; and used ATP (apparent Km = 60 microM), but not GTP, as phosphoryl donor. Molecules that can serve as effectors for other protein kinases (cyclic AMP, cyclic GMP, Ca2+ + calmodulin, Ca2+ + phospholipid, double-stranded RNA, and heparin) did not significantly alter the activity of this enzyme. A distinguishing characteristic of the protein kinase was a high KCl concentration optimum with the persistence of activity up to 800 mM KCl, at least.
Animals↗
The myosin alkali light chains of mouse ventricular and slow skeletal muscle are indistinguishable and are encoded by the same gene.
We have isolated a cDNA recombinant plasmid (pA29) identified as encoding part of the ventricular muscle myosin light chain MLC1v. This cDNA contains a 300-base pair fragment which under conditions of moderate stringency shows specific hybridization to MLC1v mRNA with no detectable cross-hybridization with the mRNAs encoding the fast skeletal muscle isoforms MLC1F and MLC3F, or the atrial muscle isoform MLC1A. Under these conditions hybridization is seen with an abundant mRNA present in slow skeletal muscle (soleus) which is indistinguishable from ventricular MLC1V mRNA on the basis of size and of thermal stability of hybrids formed with plasmid pA29. The mouse MLC1V and MLC1S proteins are found to co-migrate on two-dimensional gels. We therefore conclude that these isoforms are the same and are encoded by the same mRNA. Analysis of mouse DNA has identified a single region of the genome which hybridizes to this same fragment of pA29. This region has been isolated in a recombinant phage and has been shown to contain a single gene showing homology with MLC1V mRNA by R-loop analysis. We therefore conclude that MLC1V and MLC1S are encoded by a single gene. The pattern of segregation of a restriction fragment length polymorphism identified for this gene between Mus musculus and Mus spretus has been followed in an F1 backcross between these two mouse species. The results show the MLC1V/MLC1S gene to be closely linked to a marker at the distal end of mouse chromosome 9.
Animals↗
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.
Animals↗