A virion-associated protein kinase induces apoptosis

Apoptosis and inhibition of host gene expression are often associated with virus infections. Many viral polypeptides modulate apoptosis by direct interaction with highly conserved apoptotic pathways. Some viruses induce apoptosis during late stages of the infection cycle, while others inhibit apoptosis to facilitate replication or maintain persistent infection. In previous work, we showed that Chilo iridescent virus (CIV) or CIV virion protein extract induces apoptosis in spruce budworm and cotton boll weevil cell cultures. Here, we characterize the product of a CIV gene (iridovirus serine/threonine kinase; istk) with signature sequences for S/T kinase and ATP binding. ISTK appears to belong to the superfamily, vaccinia-related kinases (VRKs). The istk gene was expressed in Pichia pastoris vectors. Purified ISTK (48 kDa) exhibited S/T kinase activity. Treatment with ISTK induced apoptosis in budworm cells. A 35-kDa cleavage product of ISTK retaining key signature sequences was identified during purification. Pichia-expressed 35-kDa polypeptide, designated iridoptin, induced apoptosis and inhibition of host protein synthesis in budworm and boll weevil cells. A mutation in the ATP-binding site eliminated both kinase and apoptosis activity of iridoptin, suggesting that kinase activity is essential for induction of apoptosis. Analysis with custom antibody confirmed that ISTK is a structural component of CIV particles. This is the first demonstration of a viral kinase inducing apoptosis in any virus-host system and the first identification of a factor inducing apoptosis or host protein shutoff for the family Iridoviridae.     

Human cytomegalovirus virion-associated protein with kinase activity.

Protein kinase activity was detected in immunoprecipitates of human cytomegalovirus virions and infected cells by using a monoclonal antibody directed against an abundant 68,000-dalton virion structural protein. Purification of this protein by electrophoresis confirmed that the kinase activity was associated with this protein. The kinase activity was dependent on divalent cations (Mg2+, Mn2+) and cyclic nucleotide independent and exhibited optimal activity at pH 7 to 8. The kinase phosphorylated threonine and serine but not tyrosine.     

pp54 microtubule-associated protein 2 kinase. A novel serine/threonine protein kinase regulated by phosphorylation and stimulated by poly-L-lysine

An hepatic protein kinase that phosphorylates microtubule-associated protein 2 (MAP-2) on Ser/Thr residues is markedly activated after intraperitoneal injection of cycloheximide in the rat. The enzyme has been purified greater than 10,000-fold to near homogeneity and corresponds to a 54-kDa polypeptide, based on auto-phosphorylation, renaturation of activity from sodium dodecyl sulfate gels, and gel filtration. The protein kinase activity is unaffected by prior autophosphorylation, Ca2+, diacylglycerol and phospholipids, cyclic nucleotides, staurosporine, and protein kinase inhibitor, but can be totally and specifically deactivated by the Ser/Thr protein phosphatase 2A. The enzyme is inhibited completely but reversible by transition metals and p-chloromercuribenzoate, and is strongly stimulated by poly-L-lysine toward most, but not all protein substrates. The activity of the cycloheximide-stimulated MAP-2 kinase (pp54 MAP-2 kinase) toward potential polypeptide substrates was compared to that of an insulin-stimulated MAP-2 kinase (pp42 MAP-2 kinase). Although both MAP-2 kinases exhibited little or no ability to phosphorylate histones and casein, the two kinases had a distinguishable substrate specificity. At comparable MAP-2 phosphorylating activities, pp42 MAP-2 kinase, but not pp54 MAP-2 kinase, phosphorylated and activated the Xenopus S6 protein kinase II. Moreover, pp42 MAP-2 kinase phosphorylated myelin basic protein at 10-12-fold higher rates than did pp54 MAP-2 kinase. Cycloheximide-activated pp54 MAP-2 protein kinase appears to be a previously uncharacterized protein kinase that is itself regulated through Ser/Thr phosphorylation and, perhaps, polypeptide regulators with basic domains. The identity of the upstream regulatory elements and the native substrates remain to be established.     

Expression of a foreign protein by influenza A virus.

In this report we describe the rescue of a transfectant influenza A virus which stably expresses a heterologous protein, bacterial chloramphenicol acetyltransferase (CAT). The foreign sequences encoding CAT are expressed as part of an essential influenza virus segment, that coding for the neuraminidase (NA) protein. The novel way by which this was achieved involved inserting in frame the 16-amino-acid self-cleaving 2A protease of foot-and-mouth disease virus between the CAT and the NA coding sequences. The resultant gene produces a polyprotein which is proteolytically cleaved to release both CAT and NA. The intramolecular cleavage occurs at the C terminus of the 2A sequence between a glycine-proline dipeptide motif such that the released NA protein has an additional N-terminal proline residue. The transfectant virus is stable upon passage in tissue culture. CAT activity is expressed at high levels in cell culture supernatants and in the allantoic fluid of infected eggs. Since the chimeric segment must maintain the heterologous reading frame to retain viability, the virus stability is dependent upon concomitant synthesis of the heterologous protein. This design may be particularly appropriate for utilization of influenza virus as a mammalian expression vector.     

Regulation of arachidonate-mobilizing phospholipase A2 by phosphorylation via protein kinase C in macrophages.

Stimulation of 32P-labeled macrophages with phorbol ester caused an increase in phosphorylation of the intracellular, high molecular weight phospholipase A2. This increase in phosphorylation was accompanied by an increase in enzyme activity, but led to no detectable shift in the concentration dependence for Ca(2+)-induced activation. The phosphorylated phospholipase A2 could be dephosphorylated by treatment with acid phosphatase, and such treatment also reduced its catalytic activity. Together with previous data, these results indicate that the arachidonate-mobilizing phospholipase A2 is dually regulated by Ca2+ (membrane interaction) and by phosphorylation (catalytic activity).     

Inhibition of 2'-phosphodiesterase by cAMP-dependent protein kinase. Involvement of phosphorylation of protein inhibitor

2'-Phosphodiesterase from NIH 3T3 cells was purified about 530-fold. Treatment of the cell lysate with the cAMP-dependent protein kinase causing the 2'-phosphodiesterase inhibition did not result in phosphorylation of the enzyme itself. The kinase was found to phosphorylate a specific 18-kDa protein, the phosphorylated form of this protein being the inhibitor of 2'-phosphodiesterase.     

West Nile virus capsid protein interaction with importin and HDM2 protein is regulated by protein kinase C-mediated phosphorylation

West Nile virus (WNV) capsid (C) protein was shown to enter the nucleus via importin-mediated pathway and induce apoptosis although the precise regulatory mechanisms for such events have remained elusive. In this study, it was shown that WNV C protein was phosphorylated by protein kinase C (PKC). PKC-mediated phosphorylation influenced nuclear trafficking of C protein by modulating the efficiency of C protein–importin-α binding. Combination of bio-informatics, site-directed mutagenesis, co-immunoprecipitation, immuno-fluorescence and mammalian two-hybrid analyses showed that phosphorylation at amino acid residues residing near (Ser83) or within (Ser99 and Thr100) the bipartite nuclear localization motif of WNV C protein was essential for efficient interaction between C protein and importin-α. In addition, phosphorylation of WNV C protein by PKC was shown to enhance its binding to HDM2 and could subsequently induce p53-dependent apoptosis. Collectively, this study highlighted that phosphorylation is an important post-translational modification required to execute the functions of C protein.     

Preferential cleavage of chromatin-bound cohesin after targeted phosphorylation by Polo-like kinase

The final irreversible step in the duplication and dissemination of eukaryotic genomes takes place when sister chromatid pairs split and separate in anaphase. This is triggered by the protease separase that cleaves the Scc1 subunit of 'cohesin', the protein complex responsible for holding sister chromatids together in metaphase. Only part of cellular cohesin is bound to chromosomes in metaphase, and it is unclear whether and how separase specifically targets this fraction for cleavage. We established an assay to compare cleavage of chromatin-bound versus soluble budding yeast cohesin. Scc1 in chromosomal cohesin is significantly preferred by separase over Scc1 in soluble cohesin. The difference is most likely due to preferential phosphorylation of chromatin-bound Scc1 by Polo-like kinase. Site-directed mutagenesis of 10 Polo phosphorylation sites in Scc1 slowed cleavage of chromatin-bound cohesin, and hyperphosphorylation of soluble Scc1 by Polo overexpression accelerated its cleavage to levels of chromosomal cohesin. Polo is bound to chromosomes independently of cohesin's presence, providing a possible explanation for chromosome-specific cohesin modification and targeting of separase cleavage.     

Carboxy-terminal phosphorylation of SIRT1 by protein kinase CK2

Previous analyses of the sirtuin family of histone deacetylases and its most prominent member SIRT1 have focused primarily on the identification of cellular targets exploring the underlying molecular mechanisms of its implicated function in the control of metabolic homeostasis, differentiation, apoptosis and cell survival. So far, little is known about the regulation of SIRT1 itself. In the study presented herein, we assigned the main region of SIRT1 in vivo phosphorylation to amino acids 643-691 of the unique carboxy-terminal domain. Furthermore, we demonstrate that SIRT1 is a substrate for protein kinase CK2 both in vitro and in vivo. Both, deletion construct analyses and serine-to-alanine mutations identified SIRT1 Ser-659 and Ser-661 as major CK2 phosphorylation sites that are phosphorylated in vivo as well.     

The meiosis-specific protein kinase Ime2 directs phosphorylation of replication protein A

In Saccharomyces cerevisiae, the cellular single-stranded DNA-binding protein replication protein A (RPA) becomes phosphorylated during meiosis in two discrete reactions. The primary reaction is first observed shortly after cells enter the meiotic program and leads to phosphorylation of nearly all the detectable RPA. The secondary reaction, which requires the ATM/ATR homologue Mec1, is induced upon initiation of recombination and only modifies a fraction of the total RPA. We now report that correct timing of both RPA phosphorylation reactions requires Ime2, a meiosis-specific protein kinase that is critical for proper initiation of meiotic progression. Expression of Ime2 in vegetative cells leads to an unscheduled RPA phosphorylation reaction that does not require other tested meiosis-specific kinases and is distinct from the RPA phosphorylation reaction that normally occurs during mitotic growth. In addition, immunoprecipitated Ime2 catalyzes phosphorylation of purified RPA. Our data strongly suggest that Ime2 is an RPA kinase in vivo. We propose that Ime2 directly catalyzes RPA phosphorylation in the primary reaction and indirectly promotes the Mec1-dependent secondary reaction by advancing cells through meiotic progression. Our studies have identified a novel meiosis-specific reaction that targets a key protein required for DNA replication, repair, and recombination. This pathway could be important in differentiating mitotic and meiotic DNA metabolism.     

Possible involvement of double-stranded RNA-activated protein kinase in cell death by influenza virus infection.

We reported previously that influenza virus infection induces the apoptotic death of HeLa cells associated with activation of the Fas gene. In this report, we show that transfection with a PKR having a point mutation in the catalytic domain of K at 296 to R suppressed both the augmented expression of Fas and cell death by influenza virus infection. These results suggested the involvement of PKR in influenza virus-induced cell death.     

Human immunodeficiency virus vpr product is a virion-associated regulatory protein.

The vpr product of the human immunodeficiency virus type 1 (HIV-1) acts in trans to accelerate virus replication and cytopathic effect in T cells. Here it is shown that the HIV-1 viral particle contains multiple copies of the vpr protein. The vpr product is the first regulatory protein of HIV-1 to be found in the virus particle. This observation raises the possibility that vpr acts to facilitate the early steps of infection before de novo viral protein synthesis occurs.     

Stimulation of A(2A) adenosine receptor phosphorylation by protein kinase C activation: evidence for regulation by multiple protein kinase C isoforms.

Activation of the A(2A) adenosine receptor (A(2A)AR) contributes to the neuromodulatory and neuroprotective effects of adenosine in the central nervous system. Here we demonstrate that, in rat C6 glioma cells stably expressing an epitope-tagged canine A(2A)AR, receptor phosphorylation on serine and threonine residues can be increased by pretreatment with either the synthetic protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA) or endothelin 1, which increases PKC activity via binding to endogenous endothelin(A) receptors. Under conditions in which PMA was maximally effective, activation of other second messenger-regulated kinases was without effect. While basal and PMA-stimulated phosphorylation were unaffected by the A(2A)AR-selective antagonist ZM241385, they were both blocked by GF109203X (a selective inhibitor of conventional and novel PKC isoforms) and rottlerin (a PKCdelta-selective inhibitor) but not Go6976 (selective for conventional PKC isoforms). However, coexpression of the A(2A)AR with each of the alpha, betaI, and betaII isoforms of PKC increased basal and PMA-stimulated phosphorylation. Mutation of the three consensus PKC phosphorylation sites within the receptor (Thr298, Ser320, and Ser335) to Ala failed to inhibit either basal or PMA-stimulated phosphorylation. In addition, phosphorylation of the receptor was not associated with detectable changes in either its signaling capacity or cell surface expression. These observations suggest that multiple PKC isoforms can stimulate A(2A)AR phosphorylation via activation of one or more downstream kinases which then phosphorylate the receptor directly. In addition, it is likely that phosphorylation controls interactions with regulatory proteins distinct from those involved in the classical cAMP signaling pathway utilized by this receptor.     

Transglutaminase 2 kinase activity facilitates protein kinase A-induced phosphorylation of retinoblastoma protein

Transglutaminase 2 (TG2, tissue transglutaminase) is a multifunctional protein involved in cross-linking a variety of proteins, including retinoblastoma protein (Rb). Here we show that Rb is also a substrate for the recently identified serine/threonine kinase activity of TG2 and that TG2 phosphorylates Rb at the critically important Ser780 residue. Furthermore, phosphorylation of Rb by TG2 destabilizes the Rb.E2F1 complex. TG2 phosphorylation of Rb was abrogated by high Ca2+ concentrations, whereas TG2 transamidating activity was inhibited by ATP. TG2 was itself phosphorylated by protein kinase A (PKA). Phosphorylation of TG2 by PKA attenuated its transamidating activity and enhanced its kinase activity. Activation of PKA in mouse embryonic fibroblasts (MEF) with dibutyryl-cAMP enhanced phosphorylation of both TG2 and Rb by a process that was inhibited by the PKA inhibitor H89. Treatment with dibutyryl-cAMP enhanced Rb phosphorylation in MEFtg2+/+ cells but not in MEFtg2-/- cells. These data indicate that Rb is a substrate for TG2 kinase activity and suggest that phosphorylation of Rb, which results from activation of PKA in fibroblasts, is indirect and requires TG2 kinase activity.     

Deletion of protein kinase A phosphorylation sites in the HERG potassium channel inhibits activation shift by protein kinase A.

We investigated the role of protein kinase A (PKA) in regulation of the human ether-a-go-go-related gene (HERG) potassium channel activation. HERG clones with single mutations destroying one of four consensus PKA phosphorylation sites (S283A, S890A, T895A, S1137A), as well as one clone carrying all mutations with no PKA phosphorylation sites (HERG 4M) were constructed. These clones were expressed heterologously in Xenopus oocytes, and HERG potassium currents were measured with the two microelectrode voltage clamp technique. Application of the cAMP-specific phosphodiesterase (PDE IV) inhibitor Ro-20-1724 (100 microM), which results in an increased cAMP level and PKA stimulation, induced a reduction of HERG wild type outward currents by 19.1% due to a shift in the activation curve of 12.4 mV. When 100 microM Ro-20-1724 was applied to the HERG 4M channel, missing all PKA sites, there was no significant shift in the activation curve, and the current amplitude was not reduced. Furthermore, the adenylate cyclase activator forskolin that leads to PKA activation (400 microM, 60 min), shifted HERG wild type channel activation by 14.1 mV and reduced currents by 39.9%, whereas HERG 4M channels showed only a small shift of 4.3 mV and a weaker current reduction of 22.3%. We conclude that PKA regulates HERG channel activation, and direct phosphorylation of the HERG channel protein has a functional role that may be important in regulation of cardiac repolarization.     

Phosphorylation of histone H2A by protein kinase C and identification of the phosphorylation site.

In regenerating rat liver, nuclear protein histone H2A was shown to be phosphorylated on its amino-terminal serine residue [Sung et al. (1971) J. Biol. Chem. 246, 1358-1364], but the protein kinase which phosphorylates this residue has not been identified. To evaluate the possibility that protein kinase C can phosphorylate this residue, calf thymus histone H2A was 32P-labeled by incubation with [gamma-32P]ATP and highly purified protein kinase C from rat brain in the presence of calcium and phospholipid. About 1 mol of 32P was incorporated per mol of histone H2A and the Km and apparent Vmax of the reaction were calculated to be 2.1 microM and 0.35 mumol/min/mg, respectively. So histone H2A seemed to be a good substrate for protein kinase C. Further, the proteolytic phosphopeptides of 32P-labeled histone H2A were isolated by means of a series of column chromatographies and analyzed for their amino acid compositions. Comparison of the data with the known primary structure of histone H2A revealed their amino acid sequence as 1Ser-Gly-Arg. These data suggest that protein kinase C may be a candidate for the protein kinase which phosphorylates the amino-terminal serine residue of histone H2A during the regeneration of rat liver.     

Differential phosphorylation of SNAP-25 in vivo by protein kinase C and protein kinase A

SNAP-25 is a key protein required for the fusion of synaptic vesicles with the plasma membrane during exocytosis. This study establishes that SNAP-25 is differentially phosphorylated by protein kinase C and protein kinase A in neuroendocrine PC12 cells. Using phosphopeptide mapping and site-directed mutagenesis we identified both Thr138 and Ser187 as the targets of SNAP-25 phosphorylation by protein kinase C and Thr138 as the exclusive site of SNAP-25 phosphorylation by protein kinase A in vivo. Finally, despite published data to the contrary, we demonstrate that stimulation of regulated exocytosis under physiological conditions is independent of a measurable increase in SNAP-25 phosphorylation in PC12 cells.