Herpes simplex virus 1 protein kinase Us3 phosphorylates viral envelope glycoprotein B and regulates its expression on the cell surface

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). As reported here, we attempted to identify the previously unreported physiological substrate of Us3 in HSV-1-infected cells. Our results were as follows. (i) Bioinformatics analysis predicted two putative Us3 phosphorylation sites in the viral envelope glycoprotein B (gB) at codons 557 to 562 (RRVSAR) and codons 884 to 889 (RRNTNY). (ii) In in vitro kinase assays, the threonine residue at position 887 (Thr-887) in the gB domain was specifically phosphorylated by Us3, while the serine residue at position 560 was not. (iii) The phosphorylation of gB Thr-887 in Vero cells infected with wild-type HSV-1 was specifically detected using an antibody that recognized phosphorylated serine or threonine residues with arginine at the −3 and −2 positions. (iv) The phosphorylation of gB Thr-887 in infected cells was dependent on the kinase activity of Us3. (v) The replacement of Thr-887 with alanine markedly upregulated the cell surface expression of gB in infected cells, whereas replacement with aspartic acid, which sometimes mimics constitutive phosphorylation, restored the wild-type phenotype. The upregulation of gB expression on the cell surface also was observed in cells infected with a recombinant HSV-1 encoding catalytically inactive Us3. These results supported the hypothesis that Us3 phosphorylates gB and downregulates the cell surface expression of gB in HSV-1-infected cells.     

Herpes Simplex Virus 1 Protein Kinase Us3 Phosphorylates Viral dUTPase and Regulates Its Catalytic Activity in Infected Cells

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). In this study, a large-scale phosphoproteomic analysis of titanium dioxide affinity chromatography-enriched phosphopeptides from HSV-1-infected cells using high-accuracy mass spectrometry (MS) and subsequent analyses showed that Us3 phosphorylated HSV-1-encoded dUTPase (vdUTPase) at serine 187 (Ser-187) in HSV-1-infected cells. Thus, the following observations were made. (i) In in vitro kinase assays, Ser-187 in the vdUTPase domain was specifically phosphorylated by Us3. (ii) Phosphorylation of vdUTPase Ser-187 in HSV-1-infected cells was detected by phosphate-affinity polyacrylamide gel electrophoresis analyses and was dependent on the kinase activity of Us3. (iii) Replacement of Ser-187 with alanine (S187A) in vdUTPase and an amino acid substitution in Us3 that inactivated its kinase activity significantly downregulated the enzymatic activity of vdUTPase in HSV-1-infected cells, whereas a phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type enzymatic activity of vdUTPase. (iv) The vdUTPase S187A mutation as well as the kinase-dead mutation in Us3 significantly reduced HSV-1 replication in human neuroblastoma SK-N-SH cells at a multiplicity of infection (MOI) of 5 but not at an MOI of 0.01, whereas the phosphomimetic substitution at vdUTPase Ser-187 restored the wild-type viral replication at an MOI of 5. In contrast, these mutations had no effect on HSV-1 replication in Vero and HEp-2 cells. Collectively, our results suggested that Us3 phosphorylation of vdUTPase Ser-187 promoted HSV-1 replication in a manner dependent on cell types and MOIs by regulating optimal enzymatic activity of vdUTPase.     

Herpes simplex virus 1 protein kinase Us3 and major tegument protein UL47 reciprocally regulate their subcellular localization in infected cells

Us3 is a serine-threonine protein kinase encoded by herpes simplex virus 1 (HSV-1). We have identified UL47, a major virion protein, as a novel physiological substrate of Us3. In vitro kinase assays and systematic analysis of mutations at putative Us3 phosphorylation sites near the nuclear localization signal of UL47 showed that serine at residue 77 (Ser-77) was required for Us3 phosphorylation of UL47. Replacement of UL47 Ser-77 by alanine produced aberrant accumulation of UL47 at the nuclear rim and impaired the nuclear localization of UL47 in a significant fraction of infected cells. The same defect in UL47 localization was produced by an amino acid substitution in Us3 that inactivated its protein kinase activity. In contrast, a phosphomimetic mutation at UL47 Ser-77 restored wild-type nuclear localization. The UL47 S77A mutation also reduced viral replication in the mouse cornea and the development of herpes stromal keratitis in mice. In addition, UL47 formed a stable complex with Us3 in infected cells, and nuclear localization of Us3 was significantly impaired in the absence of UL47. These results suggested that Us3 phosphorylation of UL47 Ser-77 promoted the nuclear localization of UL47 in cell cultures and played a critical role in viral replication and pathogenesis in vivo. Furthermore, UL47 appeared to be required for efficient nuclear localization of Us3 in infected cells. Therefore, Us3 protein kinase and its substrate UL47 demonstrated a unique regulatory feature in that they reciprocally regulated their subcellular localization in infected cells.     

Conserved protein kinases encoded by herpesviruses and cellular protein kinase cdc2 target the same phosphorylation site in eukaryotic elongation factor 1delta

Earlier studies have shown that translation elongation factor 1delta (EF-1delta) is hyperphosphorylated in various mammalian cells infected with representative alpha-, beta-, and gammaherpesviruses and that the modification is mediated by conserved viral protein kinases encoded by herpesviruses, including UL13 of herpes simplex virus type 1 (HSV-1), UL97 of human cytomegalovirus, and BGLF4 of Epstein-Barr virus (EBV). In the present study, we attempted to identify the site in EF-1delta associated with the hyperphosphorylation by the herpesvirus protein kinases. Our results are as follows: (i) not only in infected cells but also in uninfected cells, replacement of the serine residue at position 133 (Ser-133) of EF-1delta by alanine precluded the posttranslational processing of EF-1delta, which corresponds to the hyperphosphorylation. (ii) A purified chimeric protein consisting of maltose binding protein (MBP) fused to a domain of EF-1delta containing Ser-133 (MBP-EFWt) is specifically phosphorylated in in vitro kinase assays by purified recombinant UL13 fused to glutathione S-transferase (GST) expressed in the baculovirus system. In contrast, the level of phosphorylation by the recombinant UL13 of MBP-EFWt carrying an alanine replacement of Ser-133 (MBP-EFS133A) was greatly impaired. (iii) MBP-EFWt is also specifically phosphorylated in vitro by purified recombinant BGLF4 fused to GST expressed in the baculovirus system, and the level of phosphorylation of MBP-EFS133A by the recombinant BGLF4 was greatly reduced. (iv) The sequence flanking Ser-133 of EF-1delta completely matches the consensus phosphorylation site for a cellular protein kinase, cdc2, and in vitro kinase assays revealed that purified cdc2 phosphorylates Ser-133 of EF-1delta. (v) As observed with EF-1delta, the casein kinase II beta subunit (CKIIbeta) was specifically phosphorylated by UL13 in vitro, while the level of phosphorylation of CKIIbeta by UL13 was greatly diminished when a serine residue at position 209, which has been reported to be phosphorylated by cdc2, was replaced with alanine. These results indicate that the conserved protein kinases encoded by herpesviruses and a cellular protein kinase, cdc2, have the ability to target the same amino acid residues for phosphorylation. Our results raise the possibility that the viral protein kinases mimic cdc2 in infected cells.     

Differences in the Regulatory and Functional Effects of the Us3 Protein Kinase Activities of Herpes Simplex Virus 1 and 2

Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) are serine/threonine protein kinases and play critical roles in viral replication and pathogenicity in vivo. In the present study, we investigated differences in the biological properties of HSV-1 and HSV-2 Us3 protein kinases and demonstrated that HSV-2 Us3 did not have some of the HSV-1 Us3 kinase functions, including control of nuclear egress of nucleocapsids, localization of UL31 and UL34, and cell surface expression of viral envelope glycoprotein B. In agreement with the observations that HSV-2 Us3 was less important for these functions, the effect of HSV-2 Us3 kinase activity on virulence in mice following intracerebral inoculation was much lower than that of HSV-1 Us3. Furthermore, we showed that alanine substitution in HSV-2 Us3 at a site (aspartic acid at position 147) corresponding to one that can be autophosphorylated in HSV-1 Us3 abolished HSV-2 Us3 kinase activity. Thus, the regulatory and functional effects of Us3 kinase activity are different between HSV-1 and HSV-2.Us3 protein kinases encoded by herpes simplex virus 1 (HSV-1) and 2 (HSV-2) are serine/threonine protein kinases with amino acid sequences that are conserved in the subfamily Alphaherpesvirinae (6, 24, 36). Based on studies showing that recombinant Us3 mutants of HSV-1 and HSV-2 have significantly impaired viral replication and virulence in mice models, it has been concluded that both HSV-1 and HSV-2 Us3 protein kinases play important roles in viral replication and pathogenicity in vivo (25, 33, 41). In contrast, HSV-1 and HSV-2 Us3 protein kinases are not essential for growth in tissue culture cells (33, 36). Thus, recombinant Us3 mutants grow as well as wild-type viruses in Vero cells, and the mutants exhibit modestly impaired replication in HEp-2 cells (33, 36, 39, 40). The possible functions of Us3 have been extensively studied and gradually elucidated for HSV-1 Us3, but much less is known about HSV-2 Us3. These functions include (i) blocking apoptosis (1, 22, 30, 31, 35); (ii) promoting nuclear egress of progeny nucleocapsids through the nuclear membrane (39, 40, 45); (iii) redistributing and phosphorylating nuclear membrane-associated viral nuclear egress factors UL31 and UL34 (14, 37, 38) and cellular proteins, including lamin A/C and emerin (21, 27, 28); (iv) controlling infected cell morphology (13, 31, 32); and (v) downregulating cell surface expression of viral envelope glycoprotein B (gB) (12).To determine the molecular mechanisms for a viral protein kinase''s effects in infected cells, the kinase''s physiological substrates and its phosphorylation sites must be identified. This can involve studies showing that the altered phenotypes observed in cells infected with a mutant virus lacking the protein kinase activity is also detected in cells infected with a mutant virus in which the substrate''s phosphorylation sites have been modified by mutations. Although more than 15 potential HSV Us3 substrates have been reported, HSV-1 Us3 phosphorylation of only three substrates (Us3 itself, gB, and UL31) has been demonstrated to be linked directly with Us3 functions in infected cells (12, 13, 29, 41) as follows. (i) Us3 has been reported to autophosphorylate serine at position 147 (Ser-147), and this phosphorylation augments Us3''s kinase activity in infected cells (13, 41). Even though only a small fraction of Us3 is autophosphorylated at Ser-147 in infected cells, alanine replacement of Ser-147 in Us3 significantly reduced HSV-1 replication in the mouse cornea and pathogenic manifestations of herpes stroma keratitis and periocular skin disease in mice (41). These results indicated that Us3 kinase activity was, in part, regulated by autophosphorylation of Ser-147, and regulation of Us3 activity by autophosphorylation played a critical role in viral replication in vivo and HSV-1 pathogenesis. (ii) It has been reported that HSV-1 Us3 phosphorylates Thr-887 in the cytoplasmic tail of gB, and this phosphorylation downregulates the cell surface expression of gB (12). Us3 phosphorylation of gB at Thr-887 also has been proposed to be involved in the regulation of fusion of the nascent progeny virion envelope with the cell''s outer nuclear membrane, based on the observation that virions accumulated aberrantly in the perinuclear space in cells infected with mutant viruses carrying the amino acid substitution mutation T887A in gB and lacking the capacity to produce gH (45). The Us3 phosphorylation of gB at Thr-887 appeared to be critical for HSV-1 replication and pathogenesis in vivo, based on studies showing that the T887A substitution in the phosphorylation site in gB significantly reduced viral replication in the mouse cornea and pathogenic manifestations of herpes stroma keratitis and periocular skin disease in mice (Takahiko Imai, Ken Sagou, and Yasushi Kawaguchi, unpublished observations). (iii) It has been shown that Us3 phosphorylated some or all of the six serines in the UL31 N-terminal region, and this phosphorylation regulated the proper localization of UL31 and UL34 at the nuclear membrane and nuclear egress of nucleocapsids (29). Thus, the molecular basis of HSV-1 Us3 effects in infected cells have been gradually elucidated.However, the Us3 phosphorylation sites in Us3 itself and in gB are not conserved between HSV serotypes (12, 13). The amino acid residues in HSV-2 Us3 and gB corresponding to HSV-1 Us3 Ser-147 and gB Thr-887 are aspartic acid (Asp-147) and alanine (Ala-887), respectively. These results suggest that some HSV-1 Us3 functions, such as regulation of its own catalytic activity and control of gB expression on the cell surface, are not regulated by HSV-2 Us3 or are regulated in a manner(s) different from HSV-1 Us3. In agreement with this suggestion, there is a marked difference between HSV-1 and HSV-2 virulence in mice following intracerebral infection, with the HSV-1 Us3 null mutant being >10⁴-fold less virulent than the parent wild-type virus (25), while the HSV-2 Us3 null mutant was only ∼10-fold less virulent (33). Although these results were from different reports and the mouse strains used in the studies were different, they indicate that some HSV-1 Us3 functions are different from those of HSV-2 Us3.Therefore, we investigated differences in the biological properties of HSV-1 and HSV-2 Us3 protein kinases. It was of particular interest to examine whether Asp-147 in HSV-2 Us3 is required for its own kinase activity, since it is well established that acidic amino acids such as Asp or glutamic acid sometimes mimic the negative charges produced by phosphorylation (29, 46). In the present study, using a genetic manipulation system of HSV-2 with our newly constructed HSV-2 bacterial artificial chromosome (BAC) clone, we have shown that HSV-2 Us3 exhibited marked differences from HSV-1 Us3 in its catalytic functions, including the regulation of UL31/UL34 localization, nuclear egress of nucleocapsids, cell surface expression of gB, and virulence in mice. We also found that Asp-147 in HSV-2 Us3 was critical for its kinase activity, raising a possibility that the activity of Us3 kinases was regulated differently in HSV-1 and HSV-2.     

Phosphorylation and activation of phospholipase D1 by protein kinase C in vivo: determination of multiple phosphorylation sites.

Protein kinase C (PKC) is an important regulator of phospholipase D1 (PLD1). Currently there is some controversy about a phosphorylation-dependent or -independent mechanism of the activation of PLD1 by PKC. To solve this problem, we examined whether PLD1 is phosphorylated by PKC in vivo. For the first time, we have now identified multiple basal phophopeptides and multiple phorbol myristate acetate (PMA) induced phosphopeptides of endogenous PLD1 in 3Y1 cells as well as of transiently expressed PLD1 in COS-7 cells. Down regulation or inhibition of PKC greatly attenuated the PMA-induced phosphorylation as well as the activation of PLD1. In the presence of PMA, purified PLD1 from rat brain was also found to be phosphorylated by PKCalpha in vitro at multiple sites generating seven distinct tryptic phosphopeptides. Four phosphopeptides generated in vivo and in vitro correlated well with each other, suggesting direct phosphorylation of PLD1 by PKCalpha in the cells. Serine 2, threonine 147, and serine 561 were identified as phosphorylation sites, and by mutation of these residues to alanine these residues were proven to be specific phosphorylation sites in vivo. Interestingly, threonine 147 is located in the PX domain and serine 561 is in the negative regulatory "loop" region of PLD1. Mutation of serine 2, threonine 147, or serine 561 significantly reduced PMA-induced PLD1 activity. These results strongly suggest that phosphorylation plays a pivotal role in PLD1 regulation in vivo.     

Phosphorylation of amphiphysin I by minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase, a kinase implicated in Down syndrome

Minibrain kinase/dual-specificity tyrosine phosphorylation-regulated kinase (Mnb/Dyrk1A) is a proline-directed serine/threonine kinase encoded in the Down syndrome critical region of human chromosome 21. This kinase has been shown to phosphorylate dynamin 1 and synaptojanin 1. Here we report that amphiphysin I (Amph I) is also a Mnb/Dyrk1A substrate. This kinase phosphorylated native Amph I in rodent brains and recombinant human Amph I expressed in Escherichia coli. Serine 293 (Ser-293) was identified as the major site, whereas serine 295 and threonine 310 were found as minor kinase sites. In cultured cells, recombinant Amph I was phosphorylated at Ser-293 by endogenous kinase(s). Because mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) has been suggested to phosphorylate Amph I at Ser-293, our efforts addressed whether Ser-293 is phosphorylated in vivo by MAPK/ERK or by Mnb/Dyrk1A. Overnight serum-withdrawal inactivated MAPK/ERK; nonetheless, Ser-293 was phosphorylated in Chinese hamster ovary and SY5Y cells. Epigallocatechin-3-gallate, a potent Mnb/Dyrk1A inhibitor in vitro, apparently reduced the phosphorylation at Ser-293, whereas PD98059, a potent MAPK/ERK inhibitor, did not. High frequency stimulation of mouse hippocampal slices reduced the phosphorylation at Ser-293, albeit in the midst of MAPK/ERK activation. The endophilin binding in vitro was inhibited by phosphorylating Amph I with Mnb/Dyrk1A. However, phosphorylation at Ser-293 did not appear to alter cellular distribution patterns of the protein. Our results suggest that Mnb/Dyrk1A, not MAPK/ERK, is responsible for in vivo phosphorylation of Amph I at Ser-293 and that phosphorylation changes the recruitment of endophilin at the endocytic sites.     

Dual specificity protein kinase activity of testis-specific protein kinase 1 and its regulation by autophosphorylation of serine-215 within the activation loop

TESK1 (testis-specific protein kinase 1) is a protein kinase with a structure composed of an N-terminal protein kinase domain and a C-terminal proline-rich domain. Whereas the 3.6-kilobase TESK1 mRNA is expressed predominantly in the testis, a faint 2.5-kilobase TESK1 mRNA is expressed ubiquitously. The kinase domain of TESK1 contains in the catalytic loop in subdomain VIB an unusual DLTSKN sequence, which is not related to the consensus sequence of either serine/threonine kinases or tyrosine kinases. In this study, we show that TESK1 has kinase activity with dual specificity on both serine/threonine and tyrosine residues. In an in vitro kinase reaction, the kinase domain of TESK1 underwent autophosphorylation on serine and tyrosine residues and catalyzed phosphorylation of histone H3 and myelin basic protein on serine, threonine, and tyrosine residues. Site-directed mutagenesis analyses revealed that Ser-215 within the "activation loop" of the kinase domain is the site of serine autophosphorylation of TESK1. Replacement of Ser-215 by alanine almost completely abolished serine autophosphorylation and histone H3 kinase activities. In contrast, replacement of Ser-215 by glutamic acid abolished serine autophosphorylation activity but retained histone H3 kinase activity. These results suggest that autophosphorylation of Ser-215 is an important step to positively regulate the kinase activity of TESK1.     

The Us3 protein kinase of herpes simplex virus 1 blocks apoptosis and induces phosporylation of the Bcl-2 family member Bad

Viruses have evolved different strategies to interfere with apoptotic pathways in order to halt cellular responses to infection. The herpes simplex virus 1 (HSV-1) Us3 open-reading frame encodes a serine/threonine protein kinase that participates in the inhibition of apoptosis induced by virus infection and other stress agents. Previous studies have shown that Us3 counteracts the virus-induced activation of caspase-3 by acting at a premitochondrial stage. Using stable transfectants that express Us3 under the control of constitutive or inducible promoters we demonstrate that apoptosis induced by treatment with anti-Fas antibody and sorbitol is blocked when Us3 is expressed at levels comparable to those achieved during virus infection. Expression of Us3 correlated with phosphorylation of Bad, a BH3-only proapoptotic Bcl-2 family member that is also a target for growth factor-induced cellular kinases. Bad was phosphorylated by Us3 in in vitro kination assays. These results point to a strategy for viral inhibition of apoptosis based on functional inactivation of a critical component of the cellular death machinery.     

Phosphorylation of mammalian eukaryotic translation initiation factor 6 and its Saccharomyces cerevisiae homologue Tif6p: evidence that phosphorylation of Tif6p regulates its nucleocytoplasmic distribution and is required for yeast cell growth

The synthesis of 60S ribosomal subunits in Saccharomyces cerevisiae requires Tif6p, the yeast homologue of mammalian eukaryotic translation initiation factor 6 (eIF6). In the present work, we have isolated a protein kinase from rabbit reticulocyte lysates on the basis of its ability to phosphorylate recombinant human eIF6. Mass spectrometric analysis as well as antigenic properties of the purified kinase identified it as casein kinase I. The site of in vitro phosphorylation, which is highly conserved from yeast to mammals, was identified as the serine residues at positions 174 (major site) and 175 (minor site). The homologous yeast protein Tif6p was also phosphorylated in vivo in yeast cells. Mutation of Tif6p at serine-174 to alanine reduced phosphorylation drastically and caused loss of cell growth and viability. When both Ser-174 and Ser-175 were mutated to alanine, phosphorylation of Tif6p was completely abolished. Furthermore, while wild-type Tif6p was distributed both in nuclei and the cytoplasm of yeast cells, the mutant Tif6p (with Ser174Ala and Ser175Ala) became a constitutively nuclear protein. These results suggest that phosphorylatable Ser-174 and Ser-175 play a critical role in the nuclear export of Tif6p.     

Effects of Phosphorylation of Herpes Simplex Virus 1 Envelope Glycoprotein B by Us3 Kinase In Vivo and In Vitro

We recently reported that the herpes simplex virus 1 (HSV-1) Us3 protein kinase phosphorylates threonine at position 887 (Thr-887) in the cytoplasmic tail of envelope glycoprotein B (gB) (A. Kato, J. Arii, I. Shiratori, H. Akashi, H. Arase, and Y. Kawaguchi, J. Virol. 83:250-261, 2009; T. Wisner, C. C. Wright, A. Kato, Y. Kawaguchi, F. Mou, J. D. Baines, R. J. Roller and D. C. Johnson, J. Virol. 83:3115-3126, 2009). In the studies reported here, we examined the effect(s) of this phosphorylation on viral replication and pathogenesis in vivo and present data showing that replacement of gB Thr-887 by alanine significantly reduced viral replication in the mouse cornea and development of herpes stroma keratitis and periocular skin disease in mice. The same effects have been reported for mice infected with a recombinant HSV-1 carrying a kinase-inactive mutant of Us3. These observations suggested that Us3 phosphorylation of gB Thr-887 played a critical role in viral replication in vivo and in HSV-1 pathogenesis. In addition, we generated a monoclonal antibody that specifically reacted with phosphorylated gB Thr-887 and used this antibody to show that Us3 phosphorylation of gB Thr-887 regulated subcellular localization of gB, particularly on the cell surface of infected cells.The herpes simplex virus 1 (HSV-1) Us3 gene encodes a serine/threonine protein kinase with an amino acid sequence that is conserved in the subfamily Alphaherpesvirinae (9, 20, 29). The Us3 kinase phosphorylation target site has been reported to be similar to that of protein kinase A (PKA), a cellular cyclic AMP-dependent protein kinase (3, 12). Us3 catalytic activity plays important roles in viral replication and pathogenesis in vivo, based on studies showing that recombinant Us3 null mutant viruses and recombinant viruses encoding catalytically inactive Us3 have significantly reduced virulence, pathogenicity, and replication in mouse models (21, 34). In contrast, Us3 is not essential for growth in tissue culture cells (29). Thus, recombinant Us3 mutants grow as well as wild-type virus in Vero cells and have modestly impaired growth in a specific cell line such as HEp-2 cells (32, 33). The catalytic activity of Us3 is, in part, regulated by autophosphorylation of its serine at position 147 (Ser-147), and regulation of Us3 activity by autophosphorylation of Ser-147 appears to play a critical role in HSV-1 replication in vivo and in HSV-1 pathogenesis (34). Numerous studies have elucidated the potential downstream effects of Us3, including blocking apoptosis (18, 26-28), promoting nuclear egress of progeny nucleocapsids through the nuclear membrane (24, 32, 33), redistributing and phosphorylating nuclear membrane-associated viral nuclear egress factors UL31 and UL34 (13, 24, 30, 31) and cellular proteins including lamin A/C and emerin (16, 22, 23), controlling infected cell morphology (12, 27), and downregulating cell surface expression of viral envelope glycoprotein B (gB) (11).Two substrates that mediate some of the Us3 functions described above have been identified. First, it has been shown that Us3 phosphorylates Thr-887 in the cytoplasmic tail of gB, which appears to downregulate cell surface expression of gB (11). This conclusion is based on the observation that a T887A mutation in gB (gB-T887A) markedly upregulated cell surface expression of gB in infected cells: this upregulation was also observed with a recombinant virus encoding a Us3 kinase-inactive mutant, whereas a phosphomimetic substitution for gB Thr-887 restored wild-type cell surface expression of gB (11). Us3 phosphorylation of gB Thr-887 has also been proposed to be involved in regulation of fusion of the nascent progeny virion envelope with the cell''s outer nuclear membrane, based on the observation that virions accumulated aberrantly in the perinuclear space in cells infected with a mutant virus carrying the gB-T887A substitution mutation and lacking the capacity to produce gH (42). Second, it has been shown that Us3 may phosphorylate some or all of the six serines in the UL31 N-terminal region (24). Such phosphorylation might regulate proper localization of UL31 and UL34 at the nuclear membrane, nuclear egress of nucleocapsids, and viral growth in cell cultures since the Us3 kinase-inactive mutant phenotype for nuclear egress (i.e., mislocalization of UL31 and UL34 at the nuclear membrane, aberrant accumulation of virions within herniations of the nuclear membrane, and decreased viral growth in cell cultures) is also produced by replacement of the six serines in the UL31 N-terminal region with alanines while phosphomimetic substitutions of the six serines restored the wild-type phenotype (24).Thus, the molecular mechanisms for some of the downstream effects of Us3 phosphorylation have been gradually elucidated. However, it remains to be shown whether the Us3 functions reported to date are in fact involved in viral replication and pathogenicity in vivo. In the present study, we focused on Us3 phosphorylation of gB Thr-887 and examined the effect(s) of this phosphorylation on viral replication and pathogenesis in vivo. These studies have shown that replacement of gB Thr-887 by alanine significantly reduced viral replication in the mouse cornea and development of herpes stroma keratitis (HSK) and periocular skin disease in mice, as reported for infection of mice with a recombinant virus carrying a Us3 kinase-inactive mutant (34). These observations suggested that Us3 phosphorylation of gB Thr-887 played a critical role in viral replication in vivo and in HSV-1 pathogenesis. In addition, we generated a monoclonal antibody that specifically recognized phosphorylated gB Thr-887 and used this antibody to directly study the functional consequences of Us3 phosphorylation of gB Thr-887 in infected cells. We also present data showing that Us3 phosphorylation of gB Thr-887 regulated subcellular localization of gB, particularly gB localization on the cell surface of infected cells.     

TAK1 mitogen-activated protein kinase kinase kinase is activated by autophosphorylation within its activation loop

TAK1, a member of the mitogen-activated kinase kinase kinase family, is activated in vivo by various cytokines, including interleukin-1 (IL-1), or when ectopically expressed together with the TAK1-binding protein TAB1. However, this molecular mechanism of activation is not yet understood. We show here that endogenous TAK1 is constitutively associated with TAB1 and phosphorylated following IL-1 stimulation. Furthermore, TAK1 is constitutively phosphorylated when ectopically overexpressed with TAB1. In both cases, dephosphorylation of TAK1 renders it inactive, but it can be reactivated by preincubation with ATP. A mutant of TAK1 that lacks kinase activity is not phosphorylated either following IL-1 treatment or when coexpressed with TAB1, indicating that TAK1 phosphorylation is due to autophosphorylation. Furthermore, mutation to alanine of a conserved serine residue (Ser-192) in the activation loop between kinase domains VII and VIII abolishes both phosphorylation and activation of TAK1. These results suggest that IL-1 and ectopic expression of TAB1 both activate TAK1 via autophosphorylation of Ser-192.     

Regulation of human cytidine triphosphate synthetase 1 by glycogen synthase kinase 3

Cytidine triphosphate synthetase (CTPS) catalyzes the rate-limiting step in the de novo synthesis of CTP, and both the yeast and human enzymes have been reported to be regulated by protein kinase A or protein kinase C phosphorylation. Here, we provide evidence that stimulation or inhibition of protein kinase A and protein kinase C does not alter the phosphorylation of endogenous human CTPS1 in human embryonic kidney 293 cells under the conditions tested. Unexpectedly, we found that low serum conditions increased phosphorylation of endogenous CTPS1 and this phosphorylation was inhibited by the glycogen synthase kinase 3 (GSK3) inhibitor indirubin-3'-monoxime and GSK3beta short interfering RNAs, demonstrating the involvement of GSK3 in phosphorylation of endogenous human CTPS1. Separating tryptic peptides from [(32)P]orthophosphate-labeled cells and analyzing the phosphopeptides by mass spectrometry identified Ser-574 and Ser-575 as phosphorylated residues. Mutation of Ser-571 demonstrated that Ser-571 was the major site phosphorylated by GSK3 in intact human embryonic kidney 293 cells by GSK3 in vitro. Furthermore, mutation of Ser-575 prevented the phosphorylation of Ser-571, suggesting that phosphorylation of Ser-575 was necessary for priming the GSK3 phosphorylation of Ser-571. Low serum was found to decrease CTPS1 activity, and incubation with the GSK3 inhibitor indirubin-3'-monoxime protected against this decrease in activity. Incubation with an alkaline phosphatase increased CTPS1 activity in a time-dependent manner, demonstrating that phosphorylation inhibits CTPS1 activity. This is the first study to investigate the phosphorylation and regulation of human CTPS1 in human cells and suggests that GSK3 is a novel regulator of CTPS activity.     

The alphaherpesvirus serine/threonine kinase us3 disrupts promyelocytic leukemia protein nuclear bodies

Us3, a serine/threonine kinase encoded by all alphaherpesviruses, plays diverse roles during virus infection, including preventing virus-induced apoptosis, facilitating nuclear egress of capsids, stimulating mRNA translation and promoting cell-to-cell spread of virus infection. Given this diversity, the full spectrum of Us3 function may not yet be recognized. We noted, in transiently transfected cells, that herpes simplex virus type 2 (HSV-2) Us3 disrupted promyelocytic leukemia protein nuclear bodies (PML-NBs). However, PML-NB disruption was not observed in cells expressing catalytically inactive HSV-2 Us3. Analysis of PML-NBs in Vero cells transfected with pseudorabies virus (PRV) Us3 and those in Vero cells infected with Us3-null or -repaired PRV strains indicated that PRV Us3 expression also leads to the disruption of PML-NBs. While loss of PML-NBs in response to Us3 expression was prevented by the proteasome inhibitor MG132, Us3-mediated degradation of PML was not observed in infected cells or in transfected cells expressing enhanced green fluorescent protein (EGFP)-tagged PML isoform IV. These findings demonstrate that Us3 orthologues derived from distantly related alphaherpesviruses cause a disruption of PML-NBs in a kinase- and proteasome-dependent manner but, unlike the alphaherpesvirus ICP0 orthologues, do not target PML for degradation.     

Isoform specific phosphorylation of protein phosphatase 2C expressed in COS7 cells

Of the six distinct isoforms of mouse protein phosphatase 2C (PP2C) (α, β-1, β-2, β-3, β-4 and β-5), PP2Cα was specifically phosphorylated on the serine residue(s) when expressed in COS7 cells. Analysis of phosphorylation sites using site-directed mutagenesis demonstrated that Ser-375 and/or Ser-377 were phosphorylated in vivo. These serine residues were the sites of phosphorylation by casein kinase II in vitro. Phosphorylation of PP2Cα was enhanced two-fold by the addition of okadaic acid to the culture medium, but addition of cyclosporin A had no such effect. These results suggest that the expressed PP2Cα is phosphorylated by a casein kinase II-like protein kinase and dephosphorylated by PP1 and/or PP2A in COS7 cells.     

Cofilin phosphorylation by protein kinase testicular protein kinase 1 and its role in integrin-mediated actin reorganization and focal adhesion formation

Testicular protein kinase 1 (TESK1) is a serine/threonine kinase with a structure composed of a kinase domain related to those of LIM-kinases and a unique C-terminal proline-rich domain. Like LIM-kinases, TESK1 phosphorylated cofilin specifically at Ser-3, both in vitro and in vivo. When expressed in HeLa cells, TESK1 stimulated the formation of actin stress fibers and focal adhesions. In contrast to LIM-kinases, the kinase activity of TESK1 was not enhanced by Rho-associated kinase (ROCK) or p21-activated kinase, indicating that TESK1 is not their downstream effector. Both the kinase activity of TESK1 and the level of cofilin phosphorylation increased by plating cells on fibronectin. Y-27632, a specific inhibitor of ROCK, inhibited LIM-kinase-induced cofilin phosphorylation but did not affect fibronectin-induced or TESK1-induced cofilin phosphorylation in HeLa cells. Expression of a kinase-negative TESK1 suppressed cofilin phosphorylation and formation of stress fibers and focal adhesions induced in cells plated on fibronectin. These results suggest that TESK1 functions downstream of integrins and plays a key role in integrin-mediated actin reorganization, presumably through phosphorylating and inactivating cofilin. We propose that TESK1 and LIM-kinases commonly phosphorylate cofilin but are regulated in different ways and play distinct roles in actin reorganization in living cells.     

Interaction of PLD1b with actin in antigen-stimulated mast cells

Phosphatidic acid, the product of phospholipase D catalysed phosphatidylcholine hydrolysis is an important signalling molecule that has been implicated in regulation of actin cytoskeleton remodelling and secretion from mast cells. We show that human PLD1b (hPLD1b) is an actin-binding protein and the N-terminus is predominantly involved in this interaction. Protein kinase C (PKC) is a major upstream regulator of PLD activity and PKC phosphorylation sites have been identified within the N-terminus of PLD1b at serine 2 and threonine 147. Over-expression of wild type hPLD1b in mast cells showed that antigen stimulation significantly enhanced co-localisation of PLD1b with actin structures. Mutation of serine 2 to alanine abolished antigen-induced co-localisation whereas mutation of threonine 147 had less dramatic effects on co-localisation. The absence of co-localisation of PLD1b (S2A) with actin coincides with a significant decrease in PLD activity in cells expressing the PLD1b (S2A) mutant. In resting RBL-2H3 cells, mutation of serine 2 to aspartate resulted in constitutive co-localisation of PLD with the actin cytoskeleton, coincident with restored PLD activity. These results reveal that serine 2 is an important regulatory site involved in controlling PLD enzyme activity and the interaction between PLD and actin.