Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.11.1 (protein kinase)
81,284 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The phosphoprotein (P) and the large protein (L) constitute the RNA-dependent RNA polymerase of vesicular stomatitis virus (VSV). We show that phosphate-free P protein expressed in bacteria is transcriptionally inactive when reconstituted with L protein and viral N-RNA template free of cellular protein kinase. Phosphorylation of P protein by a cellular kinase(s) was essential for transcription as well as for further phosphorylation by an L-associated kinase, the two kinases acting in a sequential (cascade) manner. Phosphate groups introduced by cell kinase were stable, whereas those due to L kinase underwent a turnover which was coupled to ongoing transcription. We present a model for the phosphorylation pathway of P protein and propose that continued phosphorylation and dephosphorylation of P protein may represent a transcriptional regulatory (on-off) switch of nonsegmented negative-strand RNA viruses.
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PMID:Sequential phosphorylation of the phosphoprotein of vesicular stomatitis virus by cellular and viral protein kinases is essential for transcription activation. 130 93

Transcription-competent cores of vesicular stomatitis virus (VSV) contain two tightly bound protein kinase activities capable of phosphorylating the viral P protein (Beckes and Perrault, Virology 184, 383-386, 1991). We examined here the specificity of these kinases for the P protein substrate and their activity during the in vitro transcription process. Conditions favoring the VSVK1 kinase activity resulted in phosphorylation of the P1 species predominantly whereas conditions favoring VSVK2, or transcription conditions, led to an increase in the proportion of the faster migrating P2 and P3 species. A minimum of 2 mol phosphate/mol P protein was incorporated in 1 hr under optimal transcription conditions. Pulse-chase experiments revealed that the VSVK2 activity converted phosphorylated P1 to P2/P3 species. Most or all of the sites modified by VSVK1 (serines only) mapped to the 78 amino acid-long N-terminal fragment of the P protein; additional serine acceptor sites of undetermined location were also phosphorylated under VSVK2 conditions. Pretreatment of virion cores with 5'-p-fluorosulfonylbenzoyl adenosine had little or no effect on P1 phosphorylation but inhibited P1 to P2/P3 conversion nearly completely, with no effect on subsequent transcription. Likewise, the addition of cell extracts had relatively little effect on P1 phosphorylation but strongly inhibited the appearance of P2/P3, without affecting concurrent transcription. We conclude that phosphorylation of the P protein during transcription in vitro is a two-step process carried out by two distinct kinase activities, but only the first step may be essential for viral mRNA synthesis.
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PMID:Stepwise phosphorylation of vesicular stomatitis virus P protein by virion-associated kinases and uncoupling of second step from in vitro transcription. 131 76

We have previously shown that phosphorylation of vesicular stomatitis virus (VSV) phosphoprotein P by cellular protein kinase activity is an essential prerequisite for its transcriptional function. We have now purified this protein kinase by monitoring its ability to phosphorylate bacterially expressed, unphosphorylated P protein. Biochemical studies showed that the kinase is indistinguishable from casein kinase II, a ubiquitous cyclic AMP-independent protein kinase present in a wide variety of eukaryotic cells and tissues. Functional VSV transcription could be reconstituted with viral L protein, N-RNA template, and P protein phosphorylated by either purified cellular protein kinase or purified casein kinase II. The unusual role of casein kinase II in the transcription process of a nonsegmented negative-strand RNA virus would have important implications in host-virus interactions and antiviral therapy.
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PMID:Phosphorylation by cellular casein kinase II is essential for transcriptional activity of vesicular stomatitis virus phosphoprotein P. 132 44

The phosphorylated state of the vesicular stomatitis virus phosphoprotein (P), an essential component of the virion-associated RNA polymerase complex, has been shown to be important for the transcriptional activity of the complex. Recent studies indicate that phosphorylation within the acidic domain of the P protein by cellular casein kinase II is necessary for its activity. In an attempt to identify the exact location of the cell kinase-mediated phosphorylation, we altered specific serine and threonine residues within the acidic domain of the New Jersey serotype of P protein by site-directed mutagenesis. The altered P proteins were then tested to determine what effect these mutations had on the phosphorylated state of the protein in vivo as well as its transcriptional activity in vitro. We report that serine residues 59 and 61 within the acidic domain of the P protein must be phosphorylated for it to be functionally active in a reconstituted transcription assay. These results demonstrate the importance of site-specific phosphorylation in the transcriptional activity of a negative-strand RNA viral phosphoprotein and the crucial role played by a cell protein kinase in this process.
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PMID:Phosphorylation of specific serine residues within the acidic domain of the phosphoprotein of vesicular stomatitis virus regulates transcription in vitro. 132 45

Vesicular stomatitis virus (VSV) fails to replicate in mouse T lymphocytes unless the cells have been mitogenically stimulated with concanavalin A (Con A). We have examined the possibility that the failure of VSV to replicate in unstimulated T lymphocytes can be attributed to a deficiency in a host protein kinase which activates the viral P protein by phosphorylation, thus rendering it transcriptionally competent. Soluble extracts were prepared from purified mouse T lymphocytes, with or without prior treatment with Con A. The ability of these extracts to phosphorylate bacterially synthesized P protein of two VSV serotypes was measured in vitro. Activity of the protein kinase on the P proteins of the Indiana or New Jersey serotypes of VSV increased, on average 2.4- and 2.1-fold respectively, after treatment of the cells with 3 micrograms/ml Con A. Higher concentrations of Con A induced proportional increases (up to 10-fold) in the activity of the host protein kinase. Activities of the kinase phosphorylating the P protein in separate populations of CD4- and CD8-containing murine T lymphocytes increased similarly on mitogenic activation. No biochemical or immunological differences were observed between the T cell protein kinase and the previously characterized protein kinase (casein kinase II) from BHK-21 cells. The activity of the kinase that phosphorylates the P protein did not vary in CV-1 cells on treatment with alpha- or gamma-interferon, both of which inhibited VSV replication. Similarly, casein kinase II activities in Raji and SIRC cells, which do not normally support VSV growth, were the same as in BHK-21 cells. Thus restriction of VSV replication in these cells, in contrast to T lymphocytes, was not associated with a deficiency in the host casein kinase II activity.
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PMID:Restricted replication of vesicular stomatitis virus in T lymphocytes is coincident with a deficiency in a cellular protein kinase required for viral transcription. 133 23

Hepadnaviruses, as well as other pararetroviruses, express their pol (P) gene product unfused to the preceding core gene implying that these retroelements have developed a mechanism for initiating assembly and replication that is principally different from the one used by retroviruses and retrotransposons. We have analysed this mechanism for the human hepatitis B virus by using a newly developed, highly sensitive detection method based upon radiolabelling of the P protein at newly introduced target sites for protein kinase A. The results obtained demonstrate that polymerase encapsidation depends on the concomittant encapsidation of the HBV RNA pregenome and that packaging of the viral RNA, in turn, depends on the presence of P protein. Loss of P protein encapsidation by mutations inactivating the HBV RNA encapsidation signal epsilon could be compensated by trans-complementation with recombinant RNA molecules carrying the epsilon sequence. Thus, in contrast to retroviral replication, the interaction of the hepadnaviral P protein and the RNA genome at its packaging signal appears to be crucial for initiating the formation of replication-competent nucleocapsids. Furthermore, RNA control of P protein packaging stringently limits the number of polymerase molecules that can be encapsidated.
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PMID:Hepadnaviral assembly is initiated by polymerase binding to the encapsidation signal in the viral RNA genome. 138 Apr 55

Sodium dodecyl sulfate-solubilized Sendai virus large (L) protein was highly purified by a one-step procedure, using hydroxylapatite column chromatography. Monoclonal antibodies addressed to the carboxyl-terminal amino acid sequence of the L protein were used for monitoring L protein during purification. By removing sodium dodecyl sulfate from purified L protein, a protein kinase activity was successfully renatured. P and NP proteins served as its substrates. After immunoprecipitation with anti-L antibodies, the immunocomplex already showed protein kinase activity. In the presence of P protein, the NP protein was more highly phosphorylated. The results show that Sendai virus L protein possesses a protein kinase activity phosphorylating the other proteins of the viral nucleocapsid in vitro.
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PMID:Purification, renaturation, and reconstituted protein kinase activity of the Sendai virus large (L) protein: L protein phosphorylates the NP and P proteins in vitro. 216 16

The phosphoprotein P of human respiratory syncytial virus (RSV) was expressed in eukaryotic cells in phosphorylated form. Site-directed mutagenesis of the recombinant protein established Ser232 as the major site of phosphorylation in vivo. Phosphorylation of bacterially made P protein in vitro by purified casein kinase II (CKII) resulted in the phosphorylation of Ser237, whereas mainly Ser232 was phosphorylated by a crude cell extract. The P kinase activity in the cell extract exhibited properties characteristic of CKII. While the Ser232,237 to Ala double mutant was nearly completely defective for phosphorylation and transcription, phosphorylation at Ser232, through the use of appropriate P mutant or kinase, activated P protein. Phosphorylation of Ser237 restored activity only to the extent it facilitated phosphorylation of Ser232. Phosphate groups of P protein in RSV-infected cells were highly stable; inhibitors of protein serine phosphatases had no effect on the intracellular turnover of the phosphates. Highly purified viral polymerase L was transcriptionally active but devoid of P protein kinase activity. Thus, CKII-mediated phosphorylation of Ser232 appears to be the primary regulator of P protein activity while phosphorylation of Ser237 may be involved in a modulatory role under certain conditions.
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PMID:Phosphorylation of Ser232 directly regulates the transcriptional activity of the P protein of human respiratory syncytial virus: phosphorylation of Ser237 may play an accessory role. 749 65

It was previously shown that the phosphoprotein (P) of vesicular stomatitis virus must undergo phosphorylation-dependent multimerization to become transcriptionally active. Phosphorylation at S-60 and/or T-62 by casein kinase II or substitution of these residues by D is required for multimer formation. We now find that substitution of either one of these residues by A prevents phosphorylation by casein kinase II and multimer formation. The binding of multimeric P to the other two transcriptional components of vesicular stomatitis virus (L protein and the N-RNA template) has been characterized by using P immobilized on beads through its poly(His) tag to facilitate recovery of bound complexes. Multimerization of P was absolutely required for binding to both L and template. Multimeric P combined with the polymerase enzyme (L) in a stoichiometric 1:1 complex, which bound to the N-RNA template much more strongly than multimeric P alone. Substitution of S-227 and S-233 by A residues had no effect on multimerization or binding of L to P but prevented binding of both P and L to template and abolished transcriptional activity. In contrast, substitution of these residues with D residues had no effect on template binding or activity. However, substitution at these sites by either D or A largely abolished phosphorylation by L-associated kinases, thus identifying S-227 and S-233 as the major sites targeted by these kinases and confirming that phosphorylation of P protein by L-associated kinases is without transcriptional effect.
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PMID:Cooperative binding of multimeric phosphoprotein (P) of vesicular stomatitis virus to polymerase (L) and template: pathways of assembly. 749 81

The phosphorylation of the P protein of vesicular stomatitis virus by cellular casein kinase II (CKII) is essential for its activity in viral transcription. Recent in vitro studies have demonstrated that CKII converts the inactive unphosphorylated form of P (P0) to an active phosphorylated form P1, after phosphorylation at two serine residues, Ser-59 and Ser-61. To gain insight into the role of CKII-mediated phosphorylation in the structure and function of the P protein, we have carried out circular dichroism (CD) and biochemical analyses of both P0 and P1. The results of CD analyses reveal that phosphorylation of P0 to P1 significantly increases the predicted alpha-helical structure of the P1 protein from 27 to 48%. The phosphorylation defective double serine mutant (P59/61), which is transcriptionally inactive, possesses a secondary structure similar to that of P0. P1, at a protein concentration of 50 micrograms/ml, elutes from a gel filtration column apparently as a dimer, whereas both P0 and the double serine mutant elute as a monomer at the same concentration. Interestingly, unlike wild-type P1 protein, the P mutants in which either Ser-59 or Ser-61 is altered to alanine required a high concentration of CKII for optimal phosphorylation. We demonstrate here that phosphorylation of either Ser-59 or Ser-61 is necessary and sufficient to transactivate L polymerase although alteration of one serine residue significantly decreases its affinity for CKII. We have also shown that P1 binds to the N-RNA template more efficiently than P0 and the formation of P1 is a prerequisite for the subsequent phosphorylation by L protein-associated kinase. In addition, mutant P59/61 acts as a transdominant negative mutant when used in a transcription reconstitution assay in the presence of wild-type P protein.
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PMID:Role of cellular casein kinase II in the function of the phosphoprotein (P) subunit of RNA polymerase of vesicular stomatitis virus. 759 11


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