HIV Nef Increases T Cell ERK MAP Kinase Activity
2002; Elsevier BV; Volume: 277; Issue: 8 Linguagem: Inglês
10.1074/jbc.m107322200
ISSN1083-351X
AutoresJeffrey A. Schrager, Violette Der Minassian, Jon W. Marsh,
Tópico(s)Immune Cell Function and Interaction
ResumoThe human immunodeficiency regulatory protein Nef enhances viral replication and is central to viral pathogenesis. Although Nef has displayed a capacity to associate with a diverse assortment of cellular molecules and to increase T cell activity, the biochemical activity of Nef in T cells remains poorly defined. In this report we examine the bioactivity of Nef in primary CD4 T cells and, in particular, focus on the biochemical pathways known to be central to T cell activity. The extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway was dramatically affected by Nef expression with increases in ERK, MEK, and Elk induction. The capacity of Nef to increase the MAP kinase pathway activity was dependent on T cell receptor stimulation. By increasing ERK MAP kinase activity, Nef is functionally associated with a kinase known to affect T cell activity, viral replication, and viral infectivity. The human immunodeficiency regulatory protein Nef enhances viral replication and is central to viral pathogenesis. Although Nef has displayed a capacity to associate with a diverse assortment of cellular molecules and to increase T cell activity, the biochemical activity of Nef in T cells remains poorly defined. In this report we examine the bioactivity of Nef in primary CD4 T cells and, in particular, focus on the biochemical pathways known to be central to T cell activity. The extracellular signal-regulated kinase (ERK) mitogen-activated protein (MAP) kinase pathway was dramatically affected by Nef expression with increases in ERK, MEK, and Elk induction. The capacity of Nef to increase the MAP kinase pathway activity was dependent on T cell receptor stimulation. By increasing ERK MAP kinase activity, Nef is functionally associated with a kinase known to affect T cell activity, viral replication, and viral infectivity. Most of the viral gene products of HIV 1HIV-1human immunodeficiency virus type 1ERKextracellular signal-regulated kinaseMAPmitogen-activated proteinMEKextracellular signal-regulated kinase kinase 1/2MKKMAP kinase kinaseJNKc-Jun N-terminal kinaseSEK1stress-activated kinase kinase 1IL-2interleukin-2CCDcharge-coupled deviceAMacetoxymethyl ester have established structural or biochemical functions. Nef, which is the predominant early transcript (1Robert-Guroff M. Popovic M. Gartner S. Markham P. Gallo R.C. Reitz M.S. J. Virol. 1990; 64: 3391-3398Crossref PubMed Google Scholar, 2Guatelli J.C. Gingeras T.R. Richman D.D. J. Virol. 1990; 64: 4093-4098Crossref PubMed Google Scholar), is largely defined by cellular and viral phenotypes and by in vivo effects. Nef expression modulates cell surface receptors (3Garcia J.V. Miller A.D. Nature. 1991; 350: 508-511Crossref PubMed Scopus (658) Google Scholar, 4Schwartz O. Marechal V., Le Gall S. Lemonnier F. Heard J.M. Nat. 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The study of CD4 and the major histocompatibility antigen I down-modulation has been the most productive and has resulted in identification of numerous cellular moieties, largely restricted to endocytotic machinery, that can associate with Nef (for reviews, see Refs. 9Piguet V. Schwartz O., Le Gall S. Trono D. Immunol. Rev. 1999; 168: 51-63Crossref PubMed Scopus (179) Google Scholar and10Skowronski J. Greenberg M.E. Lock M. Mariani R. Salghetti S. Swigut T. Iafrate A.J. Cold Spring Harbor Symp. Quant. Biol. 1999; 64: 453-463Crossref PubMed Scopus (24) Google Scholar). From this work, it has been suggested that Nef serves as an adapter for coupling endocytotic molecules to the targeted membrane receptors. human immunodeficiency virus type 1 extracellular signal-regulated kinase mitogen-activated protein extracellular signal-regulated kinase kinase 1/2 MAP kinase kinase c-Jun N-terminal kinase stress-activated kinase kinase 1 interleukin-2 charge-coupled device acetoxymethyl ester The study of Nef-mediated effects on activation pathways has been less conclusive. Studies addressing Nef function in T cells define capacities that both inhibit and enhance T cell activity (for review, see Refs. 10Skowronski J. Greenberg M.E. Lock M. Mariani R. Salghetti S. Swigut T. Iafrate A.J. Cold Spring Harbor Symp. Quant. Biol. 1999; 64: 453-463Crossref PubMed Scopus (24) Google Scholar and 11Marsh J.W. Arch. Biochem. Biophys. 1999; 365: 192-198Crossref PubMed Scopus (34) Google Scholar). From work with a CD8-Nef fusion protein, it was proposed that cellular location defined whether Nef expression resulted in negative or positive effects on T cell activity (12Baur A.S. Sawai E.T. Dazin P. Fantl W.J. Cheng-Mayer C. Peterlin B.M. Immunity. 1994; 1: 373-384Abstract Full Text PDF PubMed Scopus (279) Google Scholar). Recent efforts from our laboratory have demonstrated that the opposing effects of Nef on T cell activation are also mediated by different Nef concentrations (13Liu X. Schrager J.A. Lange G.D. Marsh J.W. J. Biol. Chem. 2001; 276: 32763-32770Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Nef can increase T cell interleukin-2 synthesis in both T cell lines and human primary CD4 T cells (14Rhee S.S. Marsh J.W. J. Immunol. 1994; 152: 5128-5134PubMed Google Scholar, 15Alexander L., Du, Z. Rosenzweig M. Jung J.U. Desrosiers R.C. J. Virol. 1997; 71: 6094-6099Crossref PubMed Google Scholar, 16Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (161) Google Scholar, 17Wang J.K. Kiyokawa E. Verdin E. Trono D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 394-399Crossref PubMed Scopus (251) Google Scholar). Furthermore, Nef has been shown to increase both T cell nuclear factor of activated T cells (17Wang J.K. Kiyokawa E. Verdin E. Trono D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 394-399Crossref PubMed Scopus (251) Google Scholar,18Manninen A. Renkema G.H. Saksela K. J. Biol. Chem. 2000; 275: 16513-16517Abstract Full Text Full Text PDF PubMed Scopus (90) Google Scholar) and NF-κB (17Wang J.K. Kiyokawa E. Verdin E. Trono D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 394-399Crossref PubMed Scopus (251) Google Scholar) reporter activities. Thus, there is evolving support for Nef as a positive T cell factor. Attempts to define the biochemical capacity of Nef are numerous. Nef can bind to a number of cellular signaling moieties. For example, Nef binds to and activates the tyrosine kinase Hck (19Briggs S.D. Sharkey M. Stevenson M. Smithgall T.E. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar, 20Moarefi I. LaFevre-Bernt M. Sicheri F. Huse M. Lee C.H. Kuriyan J. Miller W.T. Nature. 1997; 385: 650-653Crossref PubMed Scopus (541) Google Scholar), and the co-expression of Nef and Hck in Rat-2 fibroblasts resulted in cellular transformation, although Nef expression alone had no effect (19Briggs S.D. Sharkey M. Stevenson M. Smithgall T.E. J. Biol. Chem. 1997; 272: 17899-17902Abstract Full Text Full Text PDF PubMed Scopus (180) Google Scholar). In a macrophage cell line, Nef, through Hck and MAP kinase, induces the transcription factor activating protein-1 (21Biggs T.E. Cooke S.J. Barton C.H. Harris M.P. Saksela K. Mann D.A. J. Mol. Biol. 1999; 290: 21-35Crossref PubMed Scopus (56) Google Scholar). Hck is not expressed in T cells, but with regard to T cell kinases, Nef has been shown to bind and inhibit Lck and MAP kinase activity (22Greenway A. Azad A. Mills J. McPhee D. J. Virol. 1996; 70: 6701-6708Crossref PubMed Google Scholar). Nef expression has also been demonstrated to alter calcium signaling. When expressed in NIH3T3 cells, Nef inhibited inositol trisphosphate-mediated calcium flux (23De S.K. Marsh J.W. J. Biol. Chem. 1994; 269: 6656-6660Abstract Full Text PDF PubMed Google Scholar), an effect similar to that seen in a Jurkat cell expressing a Nef-CD8 fusion protein (12Baur A.S. Sawai E.T. Dazin P. Fantl W.J. Cheng-Mayer C. Peterlin B.M. Immunity. 1994; 1: 373-384Abstract Full Text PDF PubMed Scopus (279) Google Scholar). However, in Nef-expressing transgenic murine thymocytes, T cell stimulation resulted in elevated calcium responses (24Skowronski J. Parks D. Mariani R. EMBO J. 1993; 12: 703-713Crossref PubMed Scopus (256) Google Scholar), a finding more consistent with T cell activation enhancement. Nef also binds to an activated serine kinase, p21-activated kinase or Pak (25Nunn M.F. Marsh J.W. J. Virol. 1996; 70: 6157-6161Crossref PubMed Google Scholar), and this association occurs in HIV-infected primary T cells (26Brown A. Wang X. Sawai E. Cheng-Mayer C. J. Virol. 1999; 73: 9899-9907Crossref PubMed Google Scholar). In a recent exploration of Nef activity, it was demonstrated that the co-expression of Nef with Vav, through Pak, increased c-Jun N-terminal kinase (JNK) activity in NIH3T3 cells (27Fackler O.T. Luo W. Geyer M. Alberts A.S. Peterlin B.M. Mol. Cell. 1999; 3: 729-739Abstract Full Text Full Text PDF PubMed Scopus (205) Google Scholar). However, it is unclear how relevant these studies are to the activity of Nef in peripheral CD4 T cells, the main target of HIV infection. The biochemistry of T cell activation is highly complex, but many of the molecular pathways leading to IL-2 expression have been characterized (for reviews, see Refs. 28van Leeuwen J.E.M. Samelson L.E. Curr. Opin. Immunol. 1999; 11: 242-248Crossref PubMed Scopus (217) Google Scholar and 29Kane L.P. Lin J. Weiss A. Curr. Opin. Immunol. 2000; 12: 242-249Crossref PubMed Scopus (428) Google Scholar). As used here, IL-2 is both a reporter for the metabolic activity of a T cell and the end-product of a highly characterized and dissectible biochemical process of the CD4 T cell. Per se, an increase in IL-2 levels does not significantly contribute to HIV replication (30Kovacs J.A. Vogel S. Albert J.M. Falloon J. Davey R.T., Jr. Walker R.E. Polis M.A. Spooner K. Metcalf J.A. Baseler M. Fyfe G. Lane H.C. N. Engl. J. Med. 1996; 335: 1350-1356Crossref PubMed Scopus (400) Google Scholar). However, with a recent demonstration that Nef, as expressed from HIV infection, increased T cell activity (as defined by IL-2 secretion) and viral production (31Wu Y. Marsh J.W. Science. 2001; 293: 1503-1506Crossref PubMed Scopus (307) Google Scholar), an understanding of the biochemical activity of Nef in these contexts appears relevant. Events mediated by engagement of the T cell receptor and CD28 co-receptor result in activation of the MAP kinases extracellular signal-regulated kinase (ERK), JNK, and p38, in addition to phosphorylation of IκB, elevation of cytosolic calcium, and activation of the kinase Akt. The pathways leading to these cytosolic signaling moieties are briefly outlined in Fig.1. The choice of kinases and signaling molecules is based on the assumption that Nef, as a cytosolic protein, would affect pathways in this cellular compartment. The choice was also to look at late cytosolic events, yielding the greatest opportunity to "capture" Nef effects. We make use of a system that expresses HIV Nef at concentrations similar to those seen in HIV infection (13Liu X. Schrager J.A. Lange G.D. Marsh J.W. J. Biol. Chem. 2001; 276: 32763-32770Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar) in hopes of identifying an indigenous pathway in primary CD4 T cells that would permit relevant molecular dissection. In this report, we demonstrate that Nef expression in primary CD4 T cells specifically increases activity of the ERK MAP kinase cascade. The peripheral lymphocyte fraction from healthy donors was obtained by leukapheresis and countercurrent centrifugal elutriation from the Department of Transfusion Medicine at the National Institutes of Health (32Czerniecki B.J. Carter C. Rivoltini L. Koski G.K. Kim H.I. Weng D.E. Roros J.G. Hijazi Y.M., Xu, S.W. Rosenberg S.A. Cohen P.A. J. Immunol. 1997; 159: 3823-3837PubMed Google Scholar) and purified as previously described (16Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (161) Google Scholar). Cells were grown in complete growth medium (RPMI 1640 supplemented with 10% heat-inactivated fetal calf serum, 25 mm Hepes, 2 g/liter sodium bicarbonate, 1 mmnonessential amino acids, 10 mm sodium pyruvate, 4 μl/liter β-mercaptoethanol, and 50 μg/ml gentamicin, adjusted to pH 7.4). Proliferation of purified CD4 T cells was achieved by the addition of CD3 plus CD28 antibody immobilized on magnetic beads (16Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (161) Google Scholar). Primary CD4 T cells were transduced with the PA-317 retroviral LXSN system (3Garcia J.V. Miller A.D. Nature. 1991; 350: 508-511Crossref PubMed Scopus (658) Google Scholar) expressing either the NL4–3 Nef or the nonmyristylated NL4–3 Nef mutant, which was generated by a glycine to alanine switch at residue position 2 (G2A) (33Guy B. Riviere Y. Dott K. Regnault A. Kieny M.P. Virology. 1990; 176: 413-425Crossref PubMed Scopus (89) Google Scholar). Following selection in G418, Nef was detected by Western analysis (16Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (161) Google Scholar). For T cell functions, the G2A cells were found to be similar to the nontransduced cells, as previously noted (16Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (161) Google Scholar). All transductions were tested and found positive for Nef expression. For cell activation studies, magnetic beads were removed from proliferating cell cultures after gentle pipetting of the cells, followed by re-exposure to a magnetic field. Cells no longer bound to beads were removed. The bead-containing fraction was cycled through this process two or three times. The cells were resuspended at 2 million cells/ml in fresh RPMI with fetal calf serum and then rested overnight. The next day 4 million cells were resuspended in 1 ml of serum-free RPMI 25 mm Hepes, pH 7.4. At time 0, either 10 μg of anti-CD3 (clone HIT3A) or anti-CD3 plus anti-CD28 (clone CD28.2) beads (five beads per cell) was added at 37 °C with mixing. At defined times, cells were centrifuged at 4 °C for 30 s, and the cell pellet was resuspended in 200 μl of lithium dodecyl sulfate sample buffer (Novex), heated for 10 min at 70 °C, and sonicated for 20 s to shear DNA, and similar aliquots were applied to a 10% NuPage gel (Novex). The electrophoresis gel run was then transferred to nitrocellulose and probed with kinase-, substrate-, or phosphospecific antibodies (Cell Signaling Technology or Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Binding of antibodies was assayed by secondary peroxidase conjugate antibody (Kirkegaard and Perry) and developed with West Dura (Pierce) substrate. Measurements of generated light were achieved on an Alpha Innotech ChemiImager with a cooled CCD camera. For reprobing, blots were treated with Restore stripping solution (Pierce). For analysis of ERK phosphorylation of recombinant Elk-1, 4 million cells were suspended in 1 ml of serum-free RPMI, 25 mmHepes, pH 7.4. At time 0, cells were activated by the addition of antibody as described above. At defined times, cells were centrifuged at 4 °C for 30 s, and the cell pellet was resuspended into 1 ml of lysis buffer (20 mm Tris (pH 7.5), 150 mmNaCl, 1 mm EDTA, 1 mm EGTA, 1% Triton X-100, 2.5 mm sodium pyrophosphate, 1 mmβ-glycerolphosphate, 1 mm Na3VO4, 1 μg/ml leupeptin, 1 mm 4-(2-aminoethyl)-benzenesulfonyl fluoride). Lysates were sonicated and centrifuged at 14 K for 10 min, and the supernatant was aliquoted for kinase assays or frozen at −70 °C. Total cellular protein in the lysate was determined by micro-BCA protein assay, and then all samples to be compared were made equivalent in protein concentration. Immobilized phospho-ERK kinase antibody (Thr202/Tyr204) was used to purify active ERK kinase from the cell lysate, followed by an in vitro kinase assay utilizing recombinant Elk-1 protein (as purchased in assay kit form from New England Biolabs). A Western analysis for phospho-Ser383 Elk-1 was then performed as above. For this assay, we followed the protocols of the manufacturer. Free cytosolic calcium was determined by the procedure outlined for fluo-3 acetoxymethyl ester (AM) (34Kao J.P. Harootunian A.T. Tsien R.Y. J. Biol. Chem. 1989; 264: 8179-8184Abstract Full Text PDF PubMed Google Scholar), but using the fluo-4 AM plus Pluronic F-127 reagent from Molecular Probes, Inc. (Eugene, OR) with minor modifications. Cell loading of 4 μm fluo-4 AM was achieved in Hanks' balanced salt buffer with Hepes 25 mm, pH 7.4, 37 °C, 1 h. Loaded and washed cells were aliquoted in a 96-well plate (105 cells). Cells were stimulated through the addition of 2 μg of anti-CD3 antibody, and calcium levels were determined by measurement of emitted fluorescence at 37 °C on a CytoFluor 4000 (Perseptive Biosystems) fluorimeter fitted with a 485-nm excitation filter and a 530-nm emission filter. Calculation of intracellular calcium was achieved following the sequential addition of ionomycin and EGTA, as previously described (35Sharp B.M. Shahabi N.A. Heagy W. McAllen K. Bell M. Huntoon C. McKean D.J. Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 8294-8299Crossref PubMed Scopus (49) Google Scholar, 36Tsien R.Y. Pozzan T. Rink T.J. Nature. 1982; 295: 68-71Crossref PubMed Scopus (776) Google Scholar), but a value of K d = 345 nm was used for fluo-4. For each experiment, cell number and volume were determined on a Coulter Z2 particle analyzer. We transduced purified primary CD4 T cells with either wild type NL4–3 Nef or the nonmyristylated G2A mutant of NL4–3 Nef retroviral expression vectors. The G2A mutant has previously been demonstrated to lack T cell enhancement capacity (16Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (161) Google Scholar, 17Wang J.K. Kiyokawa E. Verdin E. Trono D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 394-399Crossref PubMed Scopus (251) Google Scholar) but serves as a control for the cellular manipulations of transduction and selection. Under the conditions used in this report, the G2A Nef-transduced cell performed identically to a nontransduced cell. Both native and G2A Nef-transduced cells specifically expressed Nef protein (Refs. 13Liu X. Schrager J.A. Lange G.D. Marsh J.W. J. Biol. Chem. 2001; 276: 32763-32770Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar and 16Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (161) Google Scholar; data not shown). Activation of the ERK MAP kinase pathway is essential for IL-2 synthesis (37Owaki H. Varma R. Gillis B. Bruder J.T. Rapp U.R. Davis L.S. Geppert T.D. EMBO J. 1993; 12: 4367-4373Crossref PubMed Scopus (78) Google Scholar, 38Izquierdo M. Bowden S. Cantrell D. J. Exp. Med. 1994; 180: 401-406Crossref PubMed Scopus (57) Google Scholar). ERK is also central to the induction of cellular transcription and translation factors and activation of proliferation machinery (39Whitmarsh A.J. Davis R.J. Nature. 2000; 403: 255-256Crossref PubMed Scopus (111) Google Scholar). In T cells, stimulation of the T cell receptor (CD-28 co-stimulus is not necessary) leads to ERK activity (40Izquierdo M. Leevers S.J. Marshall C.J. Cantrell D. J. Exp. Med. 1993; 178: 1199-1208Crossref PubMed Scopus (153) Google Scholar). T cell activation involves two ERK species (ERK1 and ERK2) of different molecular weights (∼44,000 and 42,000, respectively). Activation of this kinase is achieved by MEK1/2 phosphorylation of a pair of threonine and tyrosine residues of ERK (41Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (842) Google Scholar). This active dual phosphorylated species was quantitated by phosphospecific antibody binding on Western blots developed from electrophoresis gels of cell lysates. Control and Nef-expressing primary CD4 T cells were maintained by stimulation with CD3-CD28 antibodies immobilized on magnetic beads (see "Experimental Procedures"). Prior to activation, the cells were removed from beads and rested overnight. As shown in Fig.2 A, active phosphorylated forms of both ERK1 (p44) and ERK2 (p42) are generated following stimulation with soluble CD3 antibody. The level of activated ERK was dramatically increased in Nef-expressing cells, but not in the nonmyristylated G2A Nef mutant transduced cell. Probing this blot for total ERK protein (activated plus nonactivated) displayed no differences, demonstrating that the Nef-mediated increase in ERK1 and -2 activity was not due to changes in total ERK protein. For comparative purposes, we have plotted the induction of ERK activity with time (Fig. 2 B). The inability of the nonmyristylated G2A mutant Nef to mediate the Nef increase in ERK activity is consistent with its lack of effect on T cell activation enhancement (16Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (161) Google Scholar, 17Wang J.K. Kiyokawa E. Verdin E. Trono D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 394-399Crossref PubMed Scopus (251) Google Scholar). The ERK activity was increased by Nef in cells from three different donors and, in addition, was evident in cells stimulated by CD3-CD28 beads (data not shown). T cells also possess two other inducible MAP kinases, JNK and p38. JNK is induced in T cells by CD3 plus CD28 co-stimulation (42Su B. Jacinto E. Hibi M. Kallunki T. Karin M. Ben-Neriah Y. Cell. 1994; 77: 727-736Abstract Full Text PDF PubMed Scopus (849) Google Scholar). JNK activation in T cells is achieved by SEK1 (also known as MKK4)-mediated phosphorylation of a proximal threonine and tyrosine pair in JNK (43Derijard B. Raingeaud J. Barrett T., Wu, I.H. Han J.H. Ulevitch R.J. Davis R.J. Science. 1995; 267: 682-685Crossref PubMed Scopus (1415) Google Scholar). Rested primary human CD4 T cells were stimulated with beads containing immobilized anti-CD3 and CD28 antibodies. As shown in Fig.3 A, JNK activation was dependent on stimulation. Unlike the activity of MAP kinase ERK, there is no measurable effect on JNK activity in primary CD4 T cells by HIV Nef. Comparison of total JNK protein is also displayed in Fig.3 A. A third MAP kinase, p38, is also activated by threonine-tyrosine phosphorylation (44Han J. Lee J.D. Bibbs L. Ulevitch R.J. Science. 1994; 265: 808-811Crossref PubMed Scopus (2420) Google Scholar, 45Raingeaud J. Gupta S. Rogers J.S. Dickens M. Han J.H. Ulevitch R.J. Davis R.J. J. Biol. Chem. 1995; 270: 7420-7426Abstract Full Text Full Text PDF PubMed Scopus (2046) Google Scholar). Relative to ERK, the phosphorylation of p38 was less intense and required higher exposure. As shown in Fig.3 B, activation of p38 in the absence or presence of Nef was similar. Akt kinase (also known as protein kinase B) is downstream of phosphoinositide 3-kinase activity and is phosphorylated on Thr308 by a phosphatidylinositol trisphosphate-dependent Akt/protein kinase B kinase (46Stephens L. Anderson K. Stokoe D. Erdjument-Bromage H. Painter G.F. Holmes A.B. Gaffney P.R.J. Reese C.B. McCormick F. Tempst P. Coadwell J. Hawkins P.T. Science. 1998; 279: 710-714Crossref PubMed Scopus (916) Google Scholar) and on Ser473 by autophosphorylation (47Toker A. Newton A.C. J. Biol. Chem. 2000; 275: 8271-8274Abstract Full Text Full Text PDF PubMed Scopus (422) Google Scholar). Engagement of the surface receptors resulted in the anticipated phosphorylation of Akt, as shown in Fig. 3 B, and Nef expression appeared to have no effect. The NF-κB transcription factor is inactive when bound to the inhibitory factor IκB (48Baeuerle P.A. Baltimore D. Science. 1988; 242: 540-546Crossref PubMed Scopus (1689) Google Scholar). The phosphorylation of Ser32/Ser36 (49Traenckner E.B.M. Pahl H.L. Henkel T. Schmidt K.N. Wilk S. Baeuerle P.A. EMBO J. 1995; 14: 2876-2883Crossref PubMed Scopus (934) Google Scholar, 50Brown K. Gerstberger S. Carlson L. Franzoso G. Siebenlist U. Science. 1995; 267: 1485-1488Crossref PubMed Scopus (1317) Google Scholar) leads to release and degradation of IκBα, which correlates with NF-κB nuclear localization following T cell activation (51Kalli K. Huntoon C. Bell M. McKean D.J. Mol. Cell. Biol. 1998; 18: 3140-3148Crossref PubMed Google Scholar, 52Khoshnan A. Kempiak S.J. Bennett B.L. Bae D., Xu, W. Manning A.M. June C.H. Nel A.E. J. Immunol. 1999; 163: 5444-5452PubMed Google Scholar). To examine the effect of Nef on IκBα phosphorylation, Nef-expressing and control cells were stimulated with CD3-CD28 beads. Cells were lysed and applied to gel electrophoresis as above, blotted onto cellulose nitrate, and probed by anti-phospho-IκBα antibody (see Fig. 3 C). Stimulation of cells led to rapid phosphorylation of IκBα, and expression of Nef appeared to play no role in enhancing this process. Stimulation of the T cell receptor also results in a rapid elevation of intracellular calcium, a downstream effect of phospholipase Cγ1 activity. Cytosolic calcium was determined by fluorimetric measurement of free calcium binding by fluo-4. Response of control and Nef cells to T cell receptor stimulation resulted in similar cellular calcium elevations as shown in Fig. 3 D. Nef increased the induction of ERK MAP kinase but did not appear to affect other pathways. To further define pathway specificity, we then compared the phosphosphorylation state of MEK1/2 and SEK1 (MKK4), the specific upstream T cell kinases for ERK and JNK, respectively. As with the previous studies, T cells were rested overnight, then activated by antibody engagement of the T cell receptor. Like ERK, MEK1/2 activation-associated phosphorylation was found to be transient, with the highest levels around 5 min (Fig.4 A). Compared with the nontransduced controls and the G2A mutant Nef cells, the Nef-expressing cells displayed an increase in the induction of MEK1/2 activity, as measured by antibody recognition of the dual serine (Ser217/Ser221) phosphorylation by Raf. As with the ERK induction, we did not see Nef-mediated activation in the absence of T cell receptor stimulation. An examination of the induction of the corresponding kinase in the JNK pathway, SEK1 (MEKK4), demonstrated a similar dependence on surface receptor stimulation (Fig.4 B). Consistent with the lack of Nef on JNK activation, SEK1 activity was unchanged by Nef expression. The increased phosphorylation of ERK Thr202/Tyr204 by MEK (Fig. 2) and of MEK Ser217/Ser221 by Raf (Fig. 4) defines the pathway specifically enhanced by Nef expression. These phosphorylation measurements define activities upstream to ERK MAP kinase. Once active, ERK MAP kinase induces numerous downstream transcription factors, including the phosphorylation (Ser383) of Elk-1 (53Gille H. Kortenjann M. Thomae O. Moomaw C. Slaughter C. Cobb M.H. Shaw P.E. EMBO J. 1995; 14: 951-962Crossref PubMed Scopus (590) Google Scholar). We performed Western analysis on the lysate of activated control and Nef-expressing primary CD4 T cells. Increased specific phosphorylation of the Elk-1 Ser383 was evident in the Nef-expressing cells, compared with the control G2A Nef mutant cell (Fig.5 A) or nontransduced cells (data not shown). The increased phosphorylation of Elk was not due to an increase in Elk protein levels in the Nef-positive cell. In addition to MAP kinase ERK, Elk-1 can be phosphorylated by the MAP kinases JNK and p38 (54Janknecht R. Hunter T. EMBO J. 1997; 16: 1620-1627Crossref PubMed Scopus (204) Google Scholar, 55Price M.A. Cruzalegui F.H. Treisman R. EMBO J. 1996; 15: 6552-6563Crossref PubMed Scopus (301) Google Scholar, 56Whitmarsh A.J. Yang S.H., Su, M.S.S. Sharrocks A.D. Davis R.J. Mol. Cell. Biol. 1997; 17: 2360-2371Crossref PubMed Scopus (438) Google Scholar), although activation of JNK or p38 in these studies does not appear to be affected by Nef. We then measured, in an in vitro kinase assay, Elk-1 phosphorylation by ERK kinase activity that was purified (separated from JNK and p38) from lysates of activated cells. As shown in Fig. 5 B, there was an increased induction of ERK MAP kinase activity in Nef-expressing T cells following T cell stimulation, consistent with increased Elk-1 phosphorylation in Nef-expressing cells (Fig. 5 A). We conclude that the ERK MAP kinase activation cascade is enhanced in Nef-expressing primary CD4 T cells. The lack of effect on other pathways emanating from the T cell surface receptor suggests that this activity is responsible for the enhancement in T cell activity. In addition, the mediated increase in ERK MAP kinase activity also connects Nef to previously characterized HIV infection processes. ERK activity has been demonstrated to increase HIV retroviral long terminal repeat expression (57Yang X. Chen Y. Gabuzda D. J. Biol. 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First, the injection of plasmids into NIH3T3 cells would probably result in higher expression levels of Nef and Vav than those seen in the cells examined here. Second, while Pak can induce JNK in numerous cells (65Polverino A. Frost J. Yang P. Hutchison M. Neiman A.M. Cobb M.H. Marcus S. J. Biol. Chem. 1995; 270: 26067-26070Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 66Frost J.A., Xu, S. Hutchison M.R. Marcus S. Cobb M.H. Mol. Cell. Biol. 1996; 16: 3707-3713Crossref PubMed Scopus (242) Google Scholar, 67Brown J.L. Stowers L. Baer M. Trejo J. Coughlin S. Chant J. Curr. Biol. 1996; 6: 598-605Abstract Full Text Full Text PDF PubMed Scopus (228) Google Scholar), Pak activity in T cells, following receptor stimulation, has been shown to be essential for ERK activity but not JNK (68Yablonski D. Kane L.P. Qian D. Weiss A. EMBO J. 1998; 17: 5647-5657Crossref PubMed Scopus (109) Google Scholar). 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Nef has also been reported to bind Raf (73Hodge D.R. Dunn K.J. Pei G.K. Chakrabarty M.K. Heidecker G. Lautenberger J.A. Samuel K.P. J. Biol. Chem. 1998; 273: 15727-15733Abstract Full Text Full Text PDF PubMed Scopus (30) Google Scholar), but the effect of this association on kinase activity is unknown. The positive effect of Nef on T cell IL-2 secretion following stimulation has been documented in murine, simian, and human T cell lines, as well as in primary human T cells (14Rhee S.S. Marsh J.W. J. Immunol. 1994; 152: 5128-5134PubMed Google Scholar, 15Alexander L., Du, Z. Rosenzweig M. Jung J.U. Desrosiers R.C. J. Virol. 1997; 71: 6094-6099Crossref PubMed Google Scholar, 16Schrager J.A. Marsh J.W. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 8167-8172Crossref PubMed Scopus (161) Google Scholar, 17Wang J.K. Kiyokawa E. Verdin E. Trono D. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 394-399Crossref PubMed Scopus (251) Google Scholar). The use of this system to resolve the biochemical activity of Nef in T cells is validated by the recent demonstration that Nef from HIV infection also increases this T cell response (31Wu Y. Marsh J.W. Science. 2001; 293: 1503-1506Crossref PubMed Scopus (307) Google Scholar). Given that Nef increases both murine and human T cell activity, we believe the conclusion that Nef expression results in increased ERK activity in human T cells is corroborated by the transgenic study of Hanna et al. (74Hanna Z. Kay D.G. Rebai N. Guimond A. Jothy S. Jolicoeur P. Cell. 1998; 95: 163-175Abstract Full Text Full Text PDF PubMed Scopus (426) Google Scholar). These authors found that Nef-expressing, CD3-stimulated murine thymocytes display enhanced phosphorylation of several proteins, including the Thr202/Tyr204 phosphorylation of ERK, characteristic of its activation by MEK1 (41Payne D.M. Rossomando A.J. Martino P. Erickson A.K. Her J.H. Shabanowitz J. Hunt D.F. Weber M.J. Sturgill T.W. EMBO J. 1991; 10: 885-892Crossref PubMed Scopus (842) Google Scholar, 75Seger R. Ahn N.G. Posada J. Munar E.S. Jensen A.M. Cooper J.A. Cobb M.H. Krebs E.G. J. Biol. Chem. 1992; 267: 14373-14381Abstract Full Text PDF PubMed Google Scholar). Thus, the findings for Nef bioactivity in primary CD4 T cells presented here appear to be reproducible in other cellular systems. Although the actual pathogenesis may indeed be different, this Nef activity is correlated with a state in these mice that is remarkably similar to the disease seen in humans; thus, further studies of the molecular activity of Nef that affects the MAP kinase pathway is warranted. In conclusion, the specificity for ERK MAP kinase activity over other T cell activation pathways offers an opportunity for biochemical resolution of Nef molecular activity. We thank Thomas Trischmann of the Blood Services Section, Department of Transfusion Medicine, National Institutes of Health, for providing elutriated lymphocytes; Dr. Sundararajan Venkatesan for the Nef expression vectors; and Drs. Kuan-Teh Jeang, Xiaolan Qian, and Yuntao Wu for critical review of the manuscript.
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