Association of ERK2 mitogen-activated protein kinase with human immunodeficiency virus particles.

Here we report the presence of a protein kinase activity associated with human immunodeficiency virus type 1 (HIV-1) particles. We observed phosphorylation of five major proteins by the endogenous protein kinase activity. Phosphoamino acid analysis revealed phosphorylated serine and threonine residues. In addition, we observed autophosphorylation of two proteins in the presence of gamma-ATP in an in-gel phosphorylation assay. These two proteins are not linked by a disulfide bond, suggesting that two different protein kinases are associated with HIV-1 virions. Our results indicate the presence of ERK2 mitogen-activated protein kinase and of a 53,000-molecular-weight protein kinase associated with virions. Moreover, the use of different HIV strains derived from T cells and promonocytic cells, as well as the use of human T-cell leukemia virus type 1 particles, demonstrates that ERK2 is strongly associated with retrovirus particles in a cell-independent manner. Exogenous substrates, such as histone proteins, and a viral substrate, such as Gag protein, are phosphorylated by virus-associated protein kinases.     

Regulation of the mitogen-activated protein kinase signaling pathway by SHP2.

Gab1-SHP2 association is required for Erk mitogen-activated protein kinase activation by several growth factors. Gab1-SHP2 interaction activates SHP2. However, an activated SHP2 still needs to associate with Gab1 to mediate Erk activation. It was unclear whether SHP2 is required to dephosphorylate a negative phosphorylation site on Gab1 or whether SHP2 needs the Gab1 pleckstrin homology (PH) domain to target it to the plasma membrane. We found that expression of a fusion protein consisting of the Gab1 PH domain and an active SHP2 (Gab1PH-SHP2DeltaN) induced constitutive Mek1 and Erk2 activation. Linking the active SHP2DeltaN to the PDK1 PH domain or the FRS2beta myristoylation sequence also induced Mek1 activation. Mek1 activation by Gab1PH-SHP2DeltaN was inhibited by an Src inhibitor and by Csk. Significantly, Gab1PH-SHP2DeltaN induced Src activation. Gab1PH-SHP2DeltaN expression activated Ras, and the Gab1PH-SHP2DeltaN-induced Mek1 activation was blocked by RasN17. These findings suggest that Gab1PH-SHP2DeltaN activated a signaling step upstream of Src and Ras. The SHP2 tyrosine phosphatase activity is essential for the function of the fusion protein. Together, these data show that the Gab1 sequence, besides the PH domain and SHP2 binding sites, is dispensable for Erk activation, suggesting that the primary role of Gab1 association with an activated SHP2 is to target it to the membrane.     

Interaction of the CC-chemokine RANTES with glycosaminoglycans activates a p44/p42 mitogen-activated protein kinase-dependent signaling pathway and enhances human immunodeficiency virus type 1 infectivity

The interaction of the CC-chemokine RANTES with its cell surface receptors transduces multiple intracellular signals: low concentrations of RANTES (1 to 10 nM) stimulate G-protein-coupled receptor (GPCR) activity, and higher concentrations (1 microM) activate a phosphotyrosine kinase (PTK)-dependent pathway. Here, we show that the higher RANTES concentrations induce rapid tyrosine phosphorylation of multiple proteins. Several src-family kinases (Fyn, Hck, Src) are activated, as is the focal adhesion kinase p125 FAK and, eventually, members of the p44/p42 mitogen-activated protein kinase (MAPK) family. This PTK signaling pathway can be activated independently of known seven-transmembrane GPCRs for RANTES because it occurs in cells that lack any such RANTES receptors. Instead, activation of the PTK signaling pathway is dependent on the expression of glycosaminoglycans (GAGs) on the cell surface, in that it could not be activated by RANTES in GAG-deficient cells. We have previously demonstrated that RANTES can both enhance and inhibit infection of cells with human immunodeficiency virus type 1 (HIV-1). Here we show that activation of both PTK and MAPK is involved in the enhancement of HIV-1 infectivity caused by RANTES in cells that lack GPCRs for RANTES but which express GAGs.     

Human immunodeficiency virus type 1 Vpr-dependent cell cycle arrest through a mitogen-activated protein kinase signal transduction pathway

The human immunodeficiency virus type 1 (HIV-1) Vpr protein has important functions in advancing HIV pathogenesis via several effects on the host cell. Vpr mediates nuclear import of the preintegration complex, induces host cell apoptosis, and inhibits cell cycle progression at G(2), which increases HIV gene expression. Some of Vpr's activities have been well described, but some functions, such as cell cycle arrest, are not yet completely characterized, although components of the ATR DNA damage repair pathway and the Cdc25C and Cdc2 cell cycle control mechanisms clearly play important roles. We investigated the mechanisms underlying Vpr-mediated cell cycle arrest by examining global cellular gene expression profiles in cell lines that inducibly express wild-type and mutant Vpr proteins. We found that Vpr expression is associated with the down-regulation of genes in the MEK2-ERK pathway and with decreased phosphorylation of the MEK2 effector protein ERK. Exogenous provision of excess MEK2 reverses the cell cycle arrest associated with Vpr, confirming the involvement of the MEK2-ERK pathway in Vpr-mediated cell cycle arrest. Vpr therefore appears to arrest the cell cycle at G(2)/M through two different mechanisms, the ATR mechanism and a newly described MEK2 mechanism. This redundancy suggests that Vpr-mediated cell cycle arrest is important for HIV replication and pathogenesis. Our findings additionally reinforce the idea that HIV can optimize the host cell environment for viral replication.     

The ERK mitogen-activated protein kinase pathway contributes to Ebola virus glycoprotein-induced cytotoxicity

Ebola virus is a highly lethal pathogen that causes hemorrhagic fever in humans and nonhuman primates. Among the seven known viral gene products, the envelope glycoprotein (GP) alone induces cell rounding and detachment that ultimately leads to cell death. Cellular cytoxicity is not seen with comparable levels of expression of a mutant form of GP lacking a mucin-like domain (GPDeltamuc). GP-induced cell death is nonapoptotic and is preceded by downmodulation of cell surface molecules involved in signaling pathways, including certain integrins and epidermal growth factor receptor. To investigate the mechanism of GP-induced cellular toxicity, we analyzed the activation of several signal transduction pathways involved in cell growth and survival. The active form of extracellular signal-regulated kinases types 1 and 2 (ERK1/2), phospho-ERK1/2, was reduced in cells expressing GP compared to those expressing GPDeltamuc as determined by flow cytometry, in contrast to the case for several other signaling proteins. Subsequent analysis of the activation states and kinase activities of related kinases revealed a more pronounced effect on the ERK2 kinase isoform. Disruption of ERK2 activity by a dominant negative ERK or by small interfering RNA-mediated ERK2 knockdown potentiated the decrease in alphaV integrin expression associated with toxicity. Conversely, activation of the pathway through the expression of a constitutively active form of ERK2 significantly protected against this effect. These results indicate that the ERK signaling cascade mediates GP-mediated cytotoxicity and plays a role in pathogenicity induced by this gene product.     

Catalytic reaction pathway for the mitogen-activated protein kinase ERK2

The structural, functional, and regulatory properties of the mitogen-activated protein kinases (MAP kinases) have long attracted considerable attention owing to the critical role that these enzymes play in signal transduction. While several MAP kinase X-ray crystal structures currently exist, there is by comparison little mechanistic information available to correlate the structural data with the known biochemical properties of these molecules. We have employed steady-state kinetic and solvent viscosometric techniques to characterize the catalytic reaction pathway of the MAP kinase ERK2 with respect to the phosphorylation of a protein substrate, myelin basic protein (MBP), and a synthetic peptide substrate, ERKtide. A minor viscosity effect on k(cat) with respect to the phosphorylation of MBP was observed (k(cat) = 10 +/- 2 s(-1), k(cat)(eta) = 0.18 +/- 0.05), indicating that substrate processing occurs via slow phosphoryl group transfer (12 +/- 4 s(-1)) followed by the faster release of products (56 +/- 4 s(-1)). At an MBP concentration extrapolated to infinity, no significant viscosity effect on k(cat)/K(m(ATP)) was observed (k(cat)/K(m(ATP)) = 0.2 +/- 0.1 microM(-1) s(-1), k(cat)/K(m(ATP))(eta) = -0.08 +/- 0.04), consistent with rapid-equilibrium binding of the nucleotide. In contrast, at saturating ATP, a full viscosity effect on k(cat)/K(m) for MBP was apparent (k(cat)/K(m(MBP)) = 2.4 +/- 1 microM(-1) s(-1), k(cat)/K(m(MBP))(eta) = 1.0 +/- 0.1), while no viscosity effect was observed on k(cat)/K(m) for the phosphorylation of ERKtide (k(cat)/K(m(ERKtide)) = (4 +/- 2) x 10(-3) microM(-1) s(-1), k(cat)/K(m(ERKtide))(eta) = -0.02 +/- 0.02). This is consistent with the diffusion-limited binding of MBP, in contrast to the rapid-equilibrium binding of ERKtide, to form the ternary Michaelis complex. Calculated values for binding constants show that the estimated value for K(d(MBP)) (/= 1.5 mM). The dramatically higher catalytic efficiency of MBP in comparison to that of ERKtide ( approximately 600-fold difference) is largely attributable to the slow dissociation rate of MBP (/=56 s(-1)), from the ERK2 active site.     

Regulation of cartilage formation and maturation by mitogen-activated protein kinase signaling

The majority of bones comprising the adult vertebrate skeleton are generated from hyaline cartilage templates that form during embryonic development. A process known as endochondral ossification is responsible for the conversion of these transient cartilage anlagen into mature, calcified bone. Endochondral ossification is a highly regulated, multistep cell specification program involving the initial differentiation of prechondrogenic mesenchymal cells into hyaline chondrocytes, terminal differentiation of hyaline chondrocytes into hypertrophic chondrocytes, and finally, apoptosis of hypertrophic chondrocytes followed by bone matrix deposition. Recently, extensive research has been carried out describing roles for the three major mitogen-activated protein kinase (MAPK) signaling pathways, the extracellular signal-regulated kinase 1/2 (ERK1/2), p38, and c-jun N-terminal kinase (JNK) pathways, in the successive stages of chondrogenic differentiation. In this review, we survey this research examining the involvement of ERK1/2, p38, and JNK pathway signaling in all aspects of the chondrogenic differentiation program from embryonic through postnatal stages of development. In addition, we summarize evidence from in vitro studies examining MAPK function in immortalized chondrogenic cell lines and adult mesenchymal stem cells. We also provide suggestions for future studies that may help ameliorate existing confusion concerning the specific roles of MAPK signaling at different stages of chondrogenesis.     

Wild-type levels of human immunodeficiency virus type 1 infectivity in the absence of cellular emerin protein

Preintegration complexes (PICs) mediate retroviral integration, and recent results indicate an important role for the inner nuclear membrane protein emerin in orienting human immunodeficiency virus type 1 (HIV-1) PICs to chromatin for integration. Two other host cell proteins, the barrier-to-autointegration factor (BAF) and lamina-associated polypeptide 2alpha (LAP2alpha), seemed to play a similar preintegrative role for Moloney murine leukemia virus (MMLV) in addition to HIV-1. In contrast, we determined efficient HIV-1 and MMLV infection of HeLa-P4 cells following potent down-regulation of emerin, BAF, or LAP2alpha protein by using short interfering RNA. Mouse embryo fibroblasts ablated for emerin protein through gene knockout support the same level of HIV-1 infection as cells derived from wild-type littermate control animals. As the expression of human emerin in mouse knockout cells fails to affect the level of infectivity achieved in its absence, we conclude that HIV-1 efficiently infects cells in the absence of emerin protein and, by extension, that emerin is not a universally important regulator of HIV-1 infectivity.     

Human immunodeficiency virus type 1 Tat induces B7-H1 expression via ERK/MAPK signaling pathway

In HIV-infected subjects, B7-H1 synthesis and expression are up-regulated, and the degree of dysregulation correlates with the severity of disease. HIV-1 Tat protein, the viral transactivating factor, represents a key target for the host immune response. However, the relationship between B7-H1 and Tat protein has not been addressed. Here, we chose human endothelial cells which provide costimulatory signals sufficiently to influence T cells. We used recombinant pcDNA3.1(+)–Tat plasmid to transfect human endothelial cells ECV304 to establish stable Tat-expressed cell strain, and found that HIV-1 Tat was able to induce B7-H1 expression in ECV304 cells by Real-time PCR and flow cytometry analysis, and inhibited lymphocyte proliferation in co-culture system. Moreover, by using pharmacological inhibitor of ERK pathway, HIV-1 Tat induces B7-H1 expression via ERK/MAPK signaling pathway was corroborated. In summary, our results indicate that HIV-1 Tat could induce B7-H1 synthesis in ECV304 cells through ERK/MAPK signaling pathway.     

Regulation of cellular gene expression and function by the human immunodeficiency virus type 1 tat protein

The human immunodeficiency virus type 1 Tat protein is a potent activator of viral gene expression and replication. Tat can also affect the expression of cellular genes including cytokines, extracellular matrix proteins, enzymes degrading the basement membrane and cell cycle-related proteins, and can regulate cellular functions such as growth, migration and angiogenesis. In addition, under certain circumstances, Tat may have tumorigenic effects. These activities of Tat appear to be mediated by different mechanisms such as the transactivation of cellular gene expression or the interaction of extracellular Tat with the cell membrane through both receptor-mediated and nonreceptor-mediated interactions. Deregulation of cellular gene expression and function by Tat cause abnormalities which may participate in AIDS pathogenesis and in the development of AIDS-associated disorders.     

Target cell cyclophilin A modulates human immunodeficiency virus type 1 infectivity

The peptidyl-prolyl isomerase cyclophilin A (CypA) increases the kinetics by which human immunodeficiency virus type 1 (HIV-1) spreads in tissue culture. This was conclusively demonstrated by gene targeting in human CD4(+) T cells, but the role of CypA in HIV-1 replication remains unknown. Though CypA binds to mature HIV-1 capsid protein (CA), it is also incorporated into nascent HIV-1 virions via interaction with the CA domain of the Gag polyprotein. These findings raised the possibility that CypA might act at multiple steps of the retroviral life cycle. Disruption of the CA-CypA interaction, either by the competitive inhibitor cyclosporine (CsA) or by mutation of CA residue G89 or P90, suggested that producer cell CypA was required for full virion infectivity. However, recent studies indicate that CypA within the target cell regulates HIV-1 infectivity by modulating Ref1- or Lv1-mediated restriction. To examine the relative contribution to HIV-1 replication of producer cell CypA and target cell CypA, we exploited multiple tools that disrupt the HIV-1 CA-CypA interaction. These tools included the drugs CsA, MeIle(4)-CsA, and Sanglifehrin; CA mutants exhibiting decreased affinity for CypA or altered CypA dependence; HeLa cells with CypA knockdown by RNA interference; and Jurkat T cells homozygous for a deletion of the gene encoding CypA. Our results clearly demonstrate that target cell CypA, and not producer cell CypA, is important for HIV-1 CA-mediated function. Inhibition of HIV-1 infectivity resulting from virion production in the presence of CsA occurs independently of the CA-CypA interaction or even of CypA.     

Human immunodeficiency virus type 1 Nef binds directly to Lck and mitogen-activated protein kinase, inhibiting kinase activity.

It is now well established that human immunodeficiency virus type I (HIV-1) Nef contributes substantially to disease pathogenesis by augmenting virus replication and markedly perturbing T-cell function. The effect of Nef on host cell activation could be explained in part by its interaction with specific cellular proteins involved in signal transduction, including at least a member of the src family kinase, Lck, and the serine/threonine kinase, mitogen-activated protein kinase (MAPK). Recombinant Nef directly interacted with purified Lck and MAPK in coprecipitation experiments and binding assays. A proline-rich repeat sequence [(Pxx)4] in Nef occurring between amino acid residues 69 to 78 is highly conserved and bears strong resemblance to a defined consensus sequence identified as an SH3 binding domain present in several proteins which can interact with the SH3 domain of various signalling and cytoskeletal proteins. Binding and coprecipitation assays with short synthetic peptides corresponding to the proline-rich repeat sequence [(Pxx)4] of Nef and the SH2, SH3, or SH2 and SH3 domains of Lck revealed that the interaction between these two proteins is at least in part mediated by the proline repeat sequence of Nef and the SH3 domain of Lck. In addition to direct binding to full-length Nef, MAPK was also shown to bind the same proline repeat motif. Nef protein significantly decreased the in vitro kinase activity of Lck and MAPK. Inhibition of key members of signalling cascades, including those emanating from the T-cell receptor, by the HIV-1 Nef protein undoubtedly alters the ability of the infected T cell to respond to antigens or cytokines, facilitating HIV-1 replication and contributing to HIV-1-induced disease pathogenesis.     

Nef enhances human immunodeficiency virus type 1 infectivity in the absence of matrix

Nef enhances the serine phosphorylation of the human immunodeficiency virus type 1 matrix (MA) protein, which suggests that MA may be a functional target of Nef. Using mutants that remain infectious despite the absence of most or all of MA, we show in the present study that the ability of Nef to enhance virus infectivity is not compromised even if MA is entirely replaced by a heterologous lipid anchor.     

Nef can enhance the infectivity of receptor-pseudotyped human immunodeficiency virus type 1 particles

Nef is an accessory protein of human immunodeficiency virus type 1 (HIV-1) that enhances the infectivity of progeny virions when expressed in virus-producing cells. The requirement for Nef for optimal infectivity is, at least in part, determined by the envelope (Env) glycoprotein, because it can be eliminated by pseudotyping HIV-1 particles with pH-dependent Env proteins. To investigate the role of Env in the function of Nef, we have examined the effect of Nef on the infectivity of Env-deficient HIV-1 particles pseudotyped with viral receptors for cells expressing cognate Env proteins. We found that Nef significantly enhances the infectivity of CD4-chemokine receptor pseudotypes for cells expressing HIV-1 Env. Nef also increased the infectivity of HIV-1 particles pseudotyped with Tva, the receptor for subgroup A Rous sarcoma virus (RSV-A), even though Nef had no effect if the pH-dependent Env protein of RSV-A was used for pseudotyping. However, Nef does not always enhance viral infectivity if the normal orientation of the Env-receptor interaction is reversed, because the entry of Env-deficient HIV-1 into cells expressing the vesicular stomatitis virus G protein was unaffected by Nef. Together, our results demonstrate that the presence of a viral Env protein during virus production is not required for the ability of Nef to increase viral infectivity. Furthermore, since the infectivity of Tva pseudotypes was blocked by inhibitors of endosomal acidification, we conclude that low-pH-dependent entry does not always bypass the requirement for Nef.     

Regulation of the Epstein-Barr virus C promoter by AUF1 and the cyclic AMP/protein kinase A signaling pathway

EBNA2 is an Epstein-Barr virus (EBV)-encoded protein that regulates the expression of viral and cellular genes required for EBV-driven B-cell immortalization. Elucidating the mechanisms by which EBNA2 regulates viral and cellular gene expression is necessary to understand EBV-induced B-cell immortalization and viral latency in humans. EBNA2 targets to the latency C promoter (Cp) through an interaction with the cellular DNA binding protein CBF1 (RBPJk). The EBNA2 enhancer in Cp also binds another cellular factor, C promoter binding factor 2 (CBF2), whose protein product(s) has not yet been identified. Within the EBNA2 enhancer in Cp, we have previously identified the DNA sequence required for CBF2 binding and also determined that this element is required for efficient activation of Cp by EBNA2. In this study, the CBF2 activity was biochemically purified and microsequenced. The peptides sequenced were identical to the hnRNP protein AUF1. Antibodies against AUF1 but not antibodies to related hnRNP proteins reacted with CBF2 in gel mobility shift assays. In addition, stimulation of the cellular cyclic AMP (cAMP)/protein kinase A (PKA) signal transduction pathway results in an increase in detectable CBF2/AUF1 binding activity extracted from stimulated cells. Furthermore, the CBF2 binding site was able to confer EBNA2 responsiveness to a heterologous promoter when transfected cells were treated with compounds that activate PKA or by cotransfection of plasmids expressing a constitutively active catalytic subunit of PKA. EBNA2-mediated stimulation of the latency Cp is also increased in similar cotransfection assays. These results further support an important role for CBF2 in mediating EBNA2 transactivation; they identify the hnRNP protein AUF1 as a major component of CBF2 and are also the first evidence of a cis-acting sequence other than a CBF1 binding element that is able to confer responsiveness to EBNA2.