hiv glycoprotein gp120 inhibits tcr–cd3- mediated activation of

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International Immunology, Vol. 9, No. 1, pp. 53–64 © 1997 Oxford University Press

HIV glycoprotein gp120 inhibits TCR–CD3-mediated activation of fyn and lckTomohiro Morio, Talal Chatila and Raif S. Geha

Division of Immunology, Children’s Hospital, and Department of Pediatrics, Harvard Medical School,Boston, MA 02115, USA

Keywords: CD4, Fyn, gp120, HIV, Lck

Abstract

HIV major glycoprotein gp120 interacts with CD4 molecules and perturbs signaling through theTCR–CD3 complex. We examined the effects of gp120 on TCR–CD3-induced phosphorylation andactivation of the src- type protein tyrosine kinases (PTK), fyn and lck . gp120 caused minimalchanges in lck phosphorylation or lck enzymatic activity, but preincubation of Jurkat cells withgp120 for 20 min strongly inhibited TCR–CD3-mediated phosphorylation and activation of lck andfyn , as well as phosphorylation of CD3 ζ. Inhibition of TCR–CD3 signaling in T cells preincubatedwith gp120 was paralleled by inhibition of T cell proliferation to the antigen tetanus toxoid. Neithersurface CD4 expression nor CD4– lck association was affected by gp120. Furthermore, gp120inhibited lck phosphorylation induced by cross-linking of TCR–CD3 and CD4 suggesting that theinhibition of lck phosphorylation could not be simply accounted for by sequestration of CD4molecules. gp120 selectively enhanced the phosphorylation of the lck peptide containing theautoinhibitory tyrosine residue Tyr505 relative to the lck peptide containing the positive regulatoryresidue Tyr394, suggesting that a qualitative alteration in lck may underlie the inhibition ofTCR–CD3 signaling by gp120.

Introduction

Infection by HIV-1 results in progressive immunodeficiency specific proliferation and IL-2 production (12). More detailedbiochemical analysis revealed that gp120 inhibited TCR–associated with impaired T cell function followed by a severe

decline in the number of CD41 T cells. Primary association CD3-mediated phosphoinositide (PI) turnover, translocationof protein kinase C (PKC) and Ca21 influx (6,13).of HIV-1 with T cells involves high-affinity interaction of its

envelope glycoprotein gp120 with surface CD4 molecules Activation of src-type protein tyrosine kinases (PTK) is oneof the earliest and most critical events in T cell signaling(1). Engagement of CD4 by viral gp120 has been implicated

in both the defective T cell function and the T cell depletion (14,15). TCR–CD3 cross-linking results in the activation of thesrc-type PTK, lck (16) and fyn (17), which then phosphorylateseen in HIV-1 infection (1–13).

Cross-linking of gp120 on CD41 T cells followed by sig- multiple substrates including themselves and CD3ζ. Phos-phorylated CD3ζ serves as docking site for the PTK ZAP-70naling through TCR–CD3 molecules was shown to result

in activation-dependent programmed cell death (apoptosis) (18), which then undergoes tyrosine phosphorylation andactivation (19). TCR–CD3-coupled PTK up-regulate the(10). This has led to the hypothesis that the gp120–anti-gp120

complexes detected on the surface of lymphocytes from activity of several signaling pathways, most notably thatmediated by phospholipase Cγ1 (PLCγ1) (reviewed in 20).patients infected with HIV-1 disease play an important role in

T cell depletion (4,5,10). The phosphorylation of PLCγ1 by PTK leads to its activation.This results in enhanced hydrolysis of inositol phospholipids,The mechanism of impairment of T cell function by gp120

is not entirely clear. Binding of gp120 on surface CD4 and the release of the second messengers diacylglyceroland inositol phosphates, which act to activate PKC and tomolecules on normal blood T cells has been shown to

inhibit subsequent TCR–CD3-dependent T cell signaling and induce Ca21 influx respectively. TCR–CD3-coupled PTK arealso involved in the activation of pathways regulated by p21rasproliferation as well as antigen-independent T–B cell

interaction (6,12). Similarly, preincubation of a human antigen- and by phosphatidylinositol-39-hydroxyl kinase (20). Theactivation by PTK of intracellular signaling pathways leads tospecific T cell clone with gp120 resulted in altered antigen-

Correspondence to: R. S. Geha, Division of Immunology, Children’s Hospital, 300 Longwood Avenue, Boston, MA 02115, USA

Transmitting editor: E. Unanue Received 31 May 1996, accepted 18 September 1996

54 gp120 inhibition of CD3-mediated activation of fyn and lck

Fig. 1. Effect of gp120 on phosphorylation and activation of lck. (A) gp120 has minimal effects on lck phosphorylation. Human leukemic JurkatT cells were depleted of phosphate by incubation in phosphate-free MEM supplemented with dialyzed FCS. The cells were washed andstimulated with gp120 (2.5 µg/53106 cells) for 30 s, 5 min and 20 min, and phosphorylation of lck was examined as described in Methods.Arrows indicate the position of lck. A 50 kDa radiolabeled band migrating ahead of the lck band was frequently observed and was determinedto be derived from the heavy chain IgG band of the immunoprecipitating antibody (not shown). The experiment shown is representative ofthree separate experiments. (B) Incubation with gp120 does not enhance the enzymatic activity of lck. Jurkat cells (53106 cells/point) werewashed and incubated with gp120 for the indicated time periods. lck immunoprecipitates were subjected to in vitro kinase reaction andresolved by SDS–PAGE as described in Methods. The experiment shown is representative of three separate experiments. Arrow indicates theposition of enolase (E). (C and D) Immunoblotting of lck protein following incubation with gp120. Immunoblotting was carried out as described.Equal volume of eluates from lck precipitates were loaded in each lane. Arrows indicate the position of lck. (E and F) Combined results ofthree separate experiments. The mean relative phosphorylation index for lck and enolase was calculated. This index is the ratio of thedensitometric values of the lck and enolase bands in gp120-stimulated versus unstimulated cells (time 0). Error bars represent 2 SE.

a cascade of biochemical events that culminates in T cell rylation by gp120 could not be simply accounted for bysequestration of CD4 molecules. gp120 caused increasedproliferation and cytokine secretion.

Recent work has shown altered levels of fyn and lck and phosphorylation of the lck peptide containing the auto-inhibitory tyrosine residue Tyr505 relative to the lck peptidedefective tyrosine phosphorylation in T cells of HIV-infected

patients (21). Goldman et al. (22) reported that cross-linking containing the positive regulatory residue Tyr394, suggestingthat a qualitative alteration in lck may underlie the inhibitionof gp120 led to p56lck activation, with no detectable CD3ζ

tyrosine phosphorylation, and resulted in TCR desensitization. of TCR–CD3 signaling by gp120.There is little data on the effect of gp120 on TCR–CD3-mediated activation of fyn and lck, and on the potential

Methodsmechanism of gp120 inhibition of the early biological eventsthat follow engagement of TCR–CD3.

Cells and antibodiesIn this paper, we have examined the effect of gp120 bindingto CD4 on T cell signaling. Engagement of CD4 by gp120 Anti-fyn and anti-lck antisera were obtained by immunizing

rabbits with peptides corresponding to amino acids 11–30strongly inhibited TCR–CD3-mediated phosphorylation andactivation of both lck and fyn as well as phosphorylation of and 39–58 of fyn and lck respectively. OKT3, OKT4 and OKT8

mAb were obtained from the ATCC (Rockville, MD). and werethe key substrate, CD3ζ. This was paralleled by inhibition ofT cell proliferation to the antigen tetanus toxoid (TT). gp120 purified with a Protein A-affinity column (BioRad, Hercules,

CA). Rabbit anti-CD3ζ antibody (N.40) was kindly provided byinhibited lck phosphorylation induced by cross-linking TCR–CD3 and CD4 suggesting that the inhibition of lck phospho- Dr C. Terhorst (Beth-Israel Hospital, Boston, MA). Biotinylated

gp120 inhibition of CD3-mediated activation of fyn and lck 55

Fig. 2. gp120 inhibits anti-CD3-mediated phosphorylation of fyn and lck in Jurkat T cells. (A and B) In situ phosphorylation assay. Jurkat Tcells were depleted of phosphate by incubation in phosphate-free medium supplemented with dialyzed FCS and further incubated in thesame medium in the presence or absence of gp120 (2.5 µg/53106 cells/0.5 ml) for 20 min. After washing, the cells were stimulated with OKT3mAb at 5 µg/53106 cells/0.5 ml for the indicated time periods. Phosphorylation of fyn (A) and lck (B) was examined as described in Methods.Arrows indicate the positions of fyn and lck. The experiment shown is representative of eight independent experiments. (C and D) Immunoblottingof fyn and lck protein carried out in parallel with the in situ phosphorylation assay. (E and F) Pooled results from eight separate experiments.Error bars represent 2 SE. Closed circles represent relative phosphorylation index of fyn or lck in T cells without pretreatment with gp120.Closed squares represent relative phosphorylation index of fyn and lck from T cells preincubated with gp120 for 20 min.

OKT3 and OKT4 were prepared using NHS-LC-biotin (Pierce, Cell surface staining and flow cytometric analysisRockford, IL). The human leukemic T cell line Jurkat (JE6.1) Jurkat cells were stained with biotinylated OKT4 followed bywas obtained from ATCC and maintained in RPMI 1640 phycoerythrin-conjugated streptavidin (Sigma, St Louis, MO).supplemented with 10% heat-inactivated FCS. A subline of Flow cytometric analysis was carried out using a FACScanJE6.1, FACS sorted for high CD4 expression, was obtained (Becton Dickinson, Mountain View, CA) as previouslyfrom Dr C. Terhorst. Human peripheral blood T cells were described (24).prepared as previously described (23). Partially purified CD41

cells were obtained by depleting CD81 T cells with OKT8 mAb Proliferative response of peripheral blood T cells to TT antigenand goat anti-mouse IgG-coated magnetic beads (Advanced

Purified CD41 peripheral blood T cells were preincubatedMagnetics, Cambridge, MA) as previously described (24).either with or without gp120 for 20 min at the dose of 5 µg/ml. Then, 13105 cells were mixed with 23105 non-E rosetteReagentsforming cells and incubated with TT (20 µg/ml) for 120 h in aTetanus toxoid (TT) was obtained from Massachusetts Bio-96-well flat-bottomed plate and were pulsed with [3H]thy-logical Laboratory (Jamaica Plain, MA). Ovalbumin wasmidine (1 µCi) for the last 18 h. Incorporation of the radio-obtained from Sigma (St Louis, MO). HIV major glycoproteinlabeled thymidine was determined as described (23).gp120 and soluble CD4 were obtained through National

Institute of Allergy and Infectious Diseases, AIDS ResearchDetection of phosphorylation in α-lysophosphatidylcholineand Reference Reagent Program. Another preparation of(α-LPC) permeabilized cellsgp120 was obtained from American Biotechnologies

(Cambridge, MA). Both preparations of gp120 were demon- The extent of phosphorylation of fyn, lck and CD3ζ chain wasmonitored in α-LPC permeabilized cells as described beforestrated to bind CD4 with high affinity as evidenced by their

inhibition of CD41 T cell staining with the anti-CD4 mAb Leu3a (25). Briefly, cells were first depleted of phosphate by incuba-tion in phosphate-free MEM supplemented with 10% dialyzed(data not shown).


FCS for 1 h, washed and preincubated with or without 2.5µg/ml gp120 at 53106 cells/ml for 20 min. Cells were washedand stimulated with OKT3 (10 µg/13107 cells) for the indicatedtime periods at 37°C, centrifuged and permeabilized with α-LPC containing permeabilization buffer for 1 min on ice. Thekinase reaction was started by adding 100 µCi/point [γ-32P]ATP and was allowed to proceed for 15 min on ice. Thecells were rapidly pelleted, then lysed in 1% NP-40 containinglysis buffer. Cell lysates were precleared with Protein G–agarose beads preadsorbed with normal rabbit serum (NRS),and the supernatants were collected and immunoprecipitatedwith various antibodies as indicated. Immunoprecipitateswere subjected to SDS–PAGE on a 5–16% gradient gel.Autoradiography and densitometry were carried out as previ-ously described (25).

In vitro immune complex (IC) kinase assay

Cells were stimulated for the indicated time periods andsolubilized as described above. Supernatants from 53106

cells were precleared and then subjected to immunoprecipi-Fig. 3. Soluble CD4 (sCD4) abrogates the inhibitory effect of gp120 tation with antibody preadsorbed with Protein G–agaroseon anti-CD3-mediated lck phosphorylation. Jurkat cells were beads. After washing the immunoprecipitates, IC kinase assaypreincubated either with gp120 alone (1 µg/ml) or with gp120 1 was carried out using rabbit muscle enolase (Sigma) as ansoluble CD4 (5 µg/ml) for 20 min and then stimulated with OKT3 mAb

exogenous substrate as described (25).for 30 s. Phosphorylation of lck was examined as described in Fig.1. The results from three separate experiments were pooled. Closed

Immunoblottingcircles represent relative phosphorylation index of lck in T cellswithout pretreatment with gp120. Closed squares represent relative fyn, lck or CD3ζ immunoprecipitates were resolved by SDS–phosphorylation index of lck in T cells preincubated with gp120 for

PAGE and electrophoretically transferred to PVDF mem-20 min. Open circles represent relative phosphorylation index of lckbranes. Immunoblotting was carried out as previouslyin T cells preincubated with gp120 for 20 min in the presence of

soluble CD4. described (25).

Tryptic phosphopeptide mapping

Tryptic phosphopeptide mapping was carried out accordingto the method described by Hurley and Sefton (26). Jurkatcells were first depleted of phosphate by incubation inphosphate-free MEM supplemented with 10% dialyzed FCSfor 1 h and then loaded with 32P (2 mCi/13107 cells) for 2 h.The cells were washed with medium and incubated with orwithout gp120 for the indicated time periods. The cells werelysed in 1% NP-40-containing lysis buffer, the supernatantwas collected, precleared with NRS-preadsorbed beads andthen immunoprecipitated with anti-lck antisera. The immuno-precipitates were subjected to SDS–PAGE and the lck proteinswere electrophoretically transferred to PVDF membranes.Following autoradiography, bands were cut, washed brieflyand incubated in 0.5% PVP-360 in 100 mM acetic acid for30 min. Peptides were eluted by incubating the membranes in10 µg of N-tosyl-L-phenylalanine chloromethyl ketone (TPCK)-treated trypsin in 200 µl of 0.05 M NH4HCO3 at 37°C overnight.Peptides were lyophilized and analyzed by electrophoresisin 1% NH4HCO3 (pH 8.9) for 27 min at 1.0 kV followed byascending chromatography. Each spot was visualized byautoradiography and identified by comparison with the knownFig. 4. Effect of gp120 on CD3-mediated lck phosphorylation in

peripheral blood CD41 T cells. (A) In situ phosphorylation assay. peptide map of lck (26). The extent of phosphorylation wasPeripheral blood CD41 T cells were prepared by treating E rosette measured using the PhosphorImager (Molecular Dynamics,forming cells with anti-CD8 mAb (OKT8) and goat anti-mouse Ig-

CA). For phosphopeptide mapping of CD4-associated lck,coupled magnetic beads. Purified CD4 T cells were incubated in the32P-labeled cells were lysed in 1% NP-40 containing lysispresence or absence of gp120 for 20 min. The cells were stimulated

with biotinylated OKT3 cross-linked with avidin. Phosphorylation of buffer, precleared with normal mouse serum precoated beads,lck was examined as described in Fig. 1. (B) Immunoblotting of lck then precipitated with the beads preadsorbed with OKT4.protein carried out in parallel with the in situ phosphorylation assay. The OKT4 immunoprecipitates and control lck precipitate were


Fig. 5. Preincubation with gp120 abrogates the increased catalytic activity of fyn and lck in anti-CD3 stimulated Jurkat cells. (A and B) A totalof 53106 Jurkat cells (in the presence or absence of preincubation with 5 µg/ml of gp120) were stimulated with OKT3 (5 µg/53106 cells) asindicated. The cell lysates were precleared, and were subjected to in vitro IC kinase assay, and analyzed by SDS–PAGE and autoradiographyas described in Methods. Arrows indicate the position of enolase (E) phosphorylated either by fyn immunoprecipitates (A) or by lckimmunoprecipitates (B). (C and D) Immunoblots of fyn and lck were carried out in parallel with the in vitro IC kinase assay. (E and F) Pooledresults from six independent experiments. Error bars represent 2 SE. Upper curve depicts relative phosphorylation index of fyn and lck in Tcells without pretreatment with gp120. Lower curve depicts relative phosphorylation index of lck from T cells preincubated with gp120 for 20 min.

subjected to SDS–PAGE and the proteins were transferred to that short-term incubation with gp120 had no discernible effecton the phosphorylation status of lck or on its enzymatic activity.PVDF membranes. The bands corresponding to lck were cut

Engagement of TCR–CD3 by anti-CD3 mAb results inand subjected to phosphopeptide mapping.phosphorylation of fyn and lck. The effect of 20 min preincuba-tion with gp120 on these events was examined. Stimulation

Results of Jurkat cells by anti-CD3 mAb resulted in a rapid andmarked increase in the phosphorylation of both fyn and lck.gp120 inhibits TCR–CD3-mediated phosphorylation of fynPreincubation with gp120 abrogated the capacity of anti-CD3and lckmAb to enhance phosphorylation of both fyn and lck (Fig. 2A

Because lck associates with CD4, the receptor for gp120, and B). Western blot analysis of immunoprecipitates showedand also plays a critical role in TCR–CD3 signaling, we first no change in the amount of fyn and lck (Fig. 2C and D),examined the effect of a short-term incubation with gp120 on indicating that the changes observed were not merely due tolck phosphorylation and activation. Preincubation with gp120 changes in the amount of precipitated kinases nor due tofor up to 20 min did not result in a statistically significant unequal loading. Figure 2(E and F) shows the pooled resultsalteration in lck phosphorylation status (Fig. 1A). Similarly, from eight experiments. Stimulation of T cells with anti-CD3gp120 did not cause any alteration in lck enzymatic activity, mAb for 30 s resulted in a mean 4.3- and 4.5-fold increasesas measured by phosphorylation of the substrate enolase in fyn and lck phosphorylation, which were almost totally(Fig. 1B). Figure 1(C and D) shows that equivalent amounts abrogated by pretreatment with gp120. This effect of gp120of lck protein were loaded in each lane in both experiments. was dose dependent over a range of 0.1–10 µg/ml. InhibitionThe pooled results from three independent experiments are of anti-CD3-mediated phosphorylation of fyn and lck wasshown in Fig. 1(E and F). Twenty minutes following incubation observed with concentrations of gp120 as little as 0.5 µg/mlwith gp120, the average lck phosphorylation index was 1.33 and plateaued at 5 µg/ml (data not shown).6 0.18 (mean 6 SE), while the average phosphorylation index The inhibitory effect of gp120 on lck phosphorylation was

mediated by binding to CD4. Figure 3 demonstrates that addi-of enolase by lck was 0.90 6 0.14. The above results indicated


tion of soluble CD4 (sCD4) to gp120 neutralized the inhibitoryeffect of gp120 on TCR–CD3-mediated lck phosphorylation.Furthermore, inhibition of anti-CD3-mediated phosphorylationof lck by gp120 was not restricted to Jurkat T cells but was alsoobserved with peripheral blood CD41 T cells as shown in Fig.4. In four experiments, stimulation of peripheral blood T cellswith anti-CD3 mAb resulted in a mean increase of 3.2-, 3.4- and4.0-fold increase in lck phosphorylation at 30 s, 5 min and 15min respectively, whereas pretreatment with gp120 inhibitedanti-CD3-mediated lck phosphorylation with a mean of 1.1-,1.5- and 1.4-fold at the 30 s, 5 min and 15 min time points.

gp120 inhibits TCR–CD3-mediated activation of fyn and lck

Previous studies have documented that engagement of TCR–CD3 results in increased enzymatic activity of both fyn (17) andlck (16). These results are reproduced in Fig. 5(A and B), whichshows that treatment of Jurkat T cells with the anti-CD3 mAbOKT3 resulted in enhanced enzymatic activity of fyn and lckrespectively. Importantly, preincubation with gp120 inhibitedanti-CD3-mediated induction of both fyn and lck enzymaticactivity (Fig. 5A and 5B). Western blot analysis confirmed thatequivalent amounts of fyn and lck were present in each lane(Fig. 5C and 5D). The pooled results from six different experi-ments are shown in Fig. 5(E and F). gp120 strongly inhibitedanti-CD3 induction of fyn and lck activity, and exerted no signi-ficant effect on the baseline activity of these enzymes.

Preincubation of Jurkat T cells with gp120 inhibits CD3-medi-ated phosphorylation of CD3ζ

Fig. 6. gp120 inhibits TCR–CD3-mediated CD3ζ phosphorylation. (A)Activation of PTK through TCR–CD3 leads to the phosphoryla- Jurkat cells were preincubated with gp120 (2.5 µg/53106 cells) fortion of CD3ζ. Phospho-CD3ζ plays an important role as an 20 min and stimulated with OKT3 mAb for the indicated time periods.

Phosphorylation of CD3ζ protein was assessed by in situadaptor molecule which, by means of the phosphotyrosinephosphorylation assay. Arrow indicates the position of CD3ζ. (B)residues of its Reth motifs (27), bridges the TCR–CD3 complexImmunoblotting of CD3ζ following stimulation with OKT3 mAb

to intracellular signal transducing molecules such as ZAP-70 performed in parallel with in situ phosphorylation assay. (C) Pooled(18). The effect of gp120 on anti-CD3-mediated phosphoryla- results from six independent experiments. Error bar represents 2 SE.

Closed circles represent relative phosphorylation index of CD3ζ in Ttion of CD3ζ is depicted in Fig. 6(A). Anti-CD3 mAb enhancedcells without pretreatment with gp120. Closed squares representthe phosphorylation of CD3ζ with a peak at 30 s. This wasrelative phosphorylation index of CD3ζ from T cells preincubatedinhibited by gp120. Western blotting showed that equal with gp120 for 20 min.

amounts of protein were recovered and loaded (Fig. 6B). In sixexperiments, the peak phosphorylation of CD3ζ was 7.4-foldand was reduced to 2.1-fold by preincubation with gp120(Fig. 6C). in a TT-specific T cell clone preincubated for 20 min with gp120

(13). The results also indicate that the inhibitory effect of gp120Preincubation of peripheral blood T cells with gp120 results in on T cell proliferation is not restricted to gp120 from a particulara decreased proliferative response to TT HIV-1 strain.We next examined whether inhibition of TCR–CD3 signaling by

Short exposure to gp120 does not down-regulate CD4 or causepreincubation with gp120 was accompanied by inhibition ofdissociation of CD4 and lcklate events triggered by engagement of the TCR. Purified CD41

T cells were incubated with or without gp120 (5 µg/107 cells) It has been shown that surface CD4 expression is down-modu-lated following long-term incubation with gp120 (28). However,for 20 min, washed, mixed at 1:2 ratio with E– cells and incub-

ated with TT for 120 h. Antigen-driven proliferation was CD4 expression in Jurkat cells, as measured by percent ofcells stained with anti-CD4 mAb and by the mean fluorescenceassessed by monitoring [3H]thymidine incorporation. Table 1

demonstrates that preincubation with gp120 but not with the intensity of staining, was unchanged by a 20 min incubationwith gp120 (Fig. 7A and C). Furthermore. gp120 did not alterunrelated protein ovalbumin, which by itself caused no T cell

proliferation (data not shown), inhibited TT-driven T cell prolif- CD4 expression in a subline of JE6.1 cells which was FACS-sorted for high expression of CD4 (Fig. 7B and D).eration. The mean 6 SE of inhibition of proliferative response

to TT for cells treated with gp120 was 28 6 3.2% for gp120 lck is physically associated with CD4 (29,30). It has beenreported that exposure for 4 h or more to gp120 causes dissoci-from the SF2 strain and 30 6 5.3% for gp120 from the IIIB strain

(P , 0.05). These resultswere concordantwithprevious reports ation of lck from CD4 (9). We examined whether short-termincubation with gp120 caused a change in the associationwhich showed ~30% inhibition of TT-driven T cell proliferation


Fig. 8. The association between CD4 antigen and lck is unchangedby short-term incubation with gp120. Jurkat cells were incubatedwith gp120 (2.5 µg/53106 cells) for 20 min and then lysed in 1% NP-40 lysis buffer. Cell lysates were immunoprecipitated with Protein G–agarose beads preadsorbed with OKT4 mAb which recognizes adifferent epitope from gp120. The immunoprecipitates were resolved

Fig. 7. Expression of CD4 in Jurkat T cells after short-term incubation by SDS–PAGE, transferred to a PVDF membrane and immunoblottedwith gp120 is unchanged. Jurkat cells were incubated either in the with anti-lck antisera. The membrane was reacted with [125I]Proteinpresence (C and D) or absence of gp120 (1 µg/106 cells) (A and B) G and bands were visualized by autoradiography. Positions and mol.for 20 min, washed and stained with biotinylated OKT4 mAb followed wt (kDa) of radiolabeled protein markers are indicated. Arrowsby streptavidin–phycoerythrin. (A and C) The effect of gp120 on CD4 indicate the position of lck.expression in JE6.1 Jurkat cells. (B and D) The effect of gp120 onJE6.1 Jurkat cells FACS-sorted for high CD4 expression. Solid linesrepresent staining with isotype-matched control antibody. Dottedlines represent expression of CD4.

led to a modest increase in lck phosphorylation (1.8 6 0.46-fold) which was also inhibited by preincubation with gp120(Fig. 10B). Cross-linking of CD3 and CD4 with the biotin–avidinsystem induced a sustained lck phosphorylation whichbetween lckandCD4asassessedbyprobingCD4 immunopre-

cipitates for the presence of lck by Western blotting. Figure 8 exceeded that induced by cross-linking of CD3 (7.6 6 0.74-fold) as shown in Fig. 10(C). This was inhibited by preincubationshows that the association of CD4 with lck was not affected by

pretreatment of the T cells with gp120 for 20 min. The fraction of with gp120. These results suggested that gp120 does notsimply exert its effect on TCR–CD3 signaling by sequestrationlck that we recovered in anti-CD4 immunoprecipitates equaled

~20% of total cellular lck. of CD4.We next investigated the effect of gp120 on the activation of

gp120 differentially affects the phosphorylation of lck peptideslck molecules associated with CD4. Figure 9(A) shows that theactivity of CD4-associated lck was rapidly increased upon the Phosphorylation of two key tyrosine residues, Tyr505 and

Tyr394, plays a critical role in regulating lck activity.engagement of TCR–CD3. This was completely inhibited bypreincubation of Jurkat cells with gp120. Neither anti-CD3 nor Phosphorylation of Tyr505 negatively regulates lck catalytic

activity, while phosphorylation of Tyr394 up-regulates itsgp120 caused a change in the recovery of lck from CD4immunoprecipitates (Fig. 9B). The pooled results from three enzymatic capacity (32,33). We considered the possibility

that engagement of CD4 by gp120 inactivated lck byexperiments are shown in Fig. 9(C).affecting the phosphorylation of its key tyrosine residues.

InhibitionofTCR–CD3signalingbygp120 isnotdue tosequest- T cells were pulse labeled with [32P]orthophosphate andration of CD4 stimulated for 20 min with either gp120 or medium, and

lck immunoprecipitates were derived and resolved by SDS–Sequestration of CD4 from TCR–CD3 by anti-CD4 results ininhibition of TCR–CD3 signaling (31). This raised the possibility PAGE. Two bands of mol. wt 56 and 60 kDa were specifically

immunoprecipitated. The latter probably represents a fractionthat gp120 binding to CD4 prevents CD4 from associating withTCR–CD3, rendering lck unavailable to participate in signal of lck present in quiescent Jurkat cells that has undergone

post-translational modification, such as serine phosphoryla-transduction following engagement of the TCR–CD3 complex.We tested this hypothesis by examining whether approximation tion (34). The Lck proteins were transferred to a PVDF

membrane and subjected to tryptic phosphopeptide map-of CD4 and TCR–CD3 by biotin-conjugated mAb against each,followed by cross-linking with avidin overcomes gp120-medi- ping. Figure 11(A and B) demonstrates a representative

experiment showing that incubation of T cells with gp120ated inhibition of TCR–CD3 signaling in Jurkat cells. The pooledresults of four independent experiments are shown in Fig. 10. for 20 min results in enhanced phosphorylation of the

Tyr505-containing peptides, whereas the phosphorylation ofFigure 10(A) demonstrates that cross-linking of CD3 withbiotinylated anti-CD3 mAb and avidin induced a strong and the Tyr394-containing peptide remained unchanged. In

four experiments the average increase (mean 6 SE) insustained lck phosphorylation (4.7 6 0.60-fold, mean 6 SE)which was inhibited by preincubation with gp120. Cross-linking phosphorylation of the Tyr505-containing peptides was 46

6 17% 20 min after incubation with gp120, while theof CD4 by biotinylated OKT4 mAb and avidin on Jurkat cells


Discussion

In this paper, we demonstrate that gp120 inhibits TCR–CD3-mediated phosphorylation and activation of the src-type PTK,fyn and lck, as well as TCR–CD3-mediated phosphorylationof CD3ζ. In parallel, gp120 caused increased phosphorylationof the lck peptide containing the autoinhibitory tyrosine residueTyr505, relative to the lck peptide containing the positiveregulatory residue Tyr394, suggesting that a qualitative altera-tion in the phosphorylation and autoregulation of lck mayunderlie the inhibition of TCR–CD3 signaling by gp120.

Incubation of Jurkat cells with gp120 for 20 min did notcause a significant change in baseline lck phosphorylationor activity (Fig. 1). There is conflicting data on the capacityof gp120 to phosphorylate and activate lck. Hivroz et al. (35)reported that a gp120-derived peptide activates lck in theHUT78 cell line. Soula et al. (36) reported the activation oflck by gp160, and speculated that this could be due tocross-linking of CD4 by gp120 and ‘two-fingered’ gp41. Inagreement with our findings, Horak et al. (37) demonstratedthat no PTK activation was observed by gp120 in peripheralblood T cells. These differences may be attributed to thedifferent cell lines utilized and possibly to the source ofgp120 used.

A strong body of evidence implicates lck in TCR–CD3-mediated signaling. Peripheral blood T cells from mice inwhich the lck gene was disrupted by gene targeting showdecreased T cell number and severely impaired T cell sig-Fig. 9. The effect of gp120 on the activity of CD4-associated lck. (A)naling via the TCR–CD3 complex (38). A lck-deficient mutantJurkat cells were preincubated with or without gp120 and stimulatedT cell line also shows impaired T cell signaling through theby OKT3 mAb for the indicated time periods. The cells were lysed in

1% NP-40 lysis buffer, precleared with NMS–Protein G beads and TCR–CD3 complex (39). lck is also required for TCR–CD3-then immunoprecipitated with Protein G coupled with OKT4 mAb. mediated signal transduction in CD4– T cells (40). Further-The OKT4 immunoprecipitate was incubated with 100 mM glycine,

more, ZAP-70 and syk play an important role in recruitingpH 2.5, to dissociate the protein. The pH of the eluates was adjustedCD4–lck to the activated TCR–CD3ζ complex (41). Preincuba-with Tris–HCl and the eluates were diluted with 1% NP-40 containing

lysis buffer. The extracted proteins were then reimmunoprecipitated tion with gp120 strongly inhibited CD3-mediated phosphory-with Protein G preadsorbed with anti-lck antiserum. The kinase lation (Figs 2 and 4) as well as activation of total lck (Fig. 5)reaction using enolase as substrate was carried out as described and of CD4 associated lck (Fig 9). This is in agreement within Methods. (B) Immunoblotting of CD4-associated lck following

a recent study by Hubert et al. which showed that CD3-stimulation with OKT3 mAb performed in parallel with the IC kinaseassay. (C) Combined results of three separate experiments. Error induced lck activation was inhibited by a 30 min preincubationbars represent 2 SE. Closed squares represent relative with gp120 (28).phosphorylation index for enolase without pretreatment with gp120. The src PTK fyn, which associates with CD3 (42), has alsoClosed circles represent relative enolase phosphorylation index from

been shown to participate in TCR signaling. Thymocytes ofT cells pretreated with gp120.mice deficient in fyn expression show defective T cell sig-naling, although the peripheral T cells of these mice exhibita normal response to TCR–CD3 (43,44). It is possible thatinhibition of lck activation plays a role in the inhibition bychange in the phosphorylation of the Tyr394 containinggp120 of anti-CD3-mediated phosphorylation and activationpeptides was –2.7 6 2.4%. The average ratio of phosphoryla-of fyn (Figs 2 and 5). This would be consistent with thetion of Tyr505:Tyr394-containing peptides increased by 1.5observation that lck-deficient lymphocytes exhibit defective6 0.15-fold following incubation with gp120.activation of TCR–CD3-coupled PTK (39,45).The effect of gp120 on the phosphorylation of lck peptides

Our demonstration that gp120 inhibits the phosphorylationwas more pronounced when the fraction of lck associatedand activation of lck and fyn kinases may explain the inhibitorywith CD4 was examined (Fig. 11C and D). In threeeffect of gp120 on CD3ζ phosphorylation (Fig. 6). Inhibitionexperiments the mean 6 SE change in phosphorylationof CD3ζ phosphorylation on tyrosine residues would interferewas 83 6 23% for the Tyr505-containing peptide and –42with its recruitment of ZAP-70 and subsequent phosphory-6 19% for the Tyr394-containing peptide. The average ratiolation and activation of PLCγ triggering downstream eventsof phosphorylation of Tyr505:Tyr394-containing peptidessuch as the generation of inositol triphosphate and Ca21 fluxincreased by 2.8 6 0.74-fold following incubation withboth of which are known to be inhibited by gp120 (6,8,13,28).gp120. These results suggest that gp120 induces changes

In contrast to the almost complete inhibition of early sig-in the phosphorylation of lck which may play a key role inregulating its enzymatic activity. naling events induced via TCR–CD3, preincubation with


Fig. 10. Preincubation with gp120 inhibits lck phosphorylation induced by cross-linking of CD3 and CD4. Jurkat cells were incubated in thepresence or absence of gp120 for 20 min. The cells were stimulated with either biotinylated OKT3 with avidin (A), biotinylated OKT4 withavidin (B) or with biotinylated OKT3 and biotinylated OKT4 cross-linked with avidin (C). Phosphorylation of lck was assessed by in situphosphorylation assay. Pooled results from four independent experiments are shown. Error bars represent 2 SE. Closed circles representrelative phosphorylation index of lck in T cells without pretreatment with gp120. Closed squares represent relative phosphorylation index oflck from T cells preincubated with gp120 for 20 min.

Fig. 11. Short-term incubation of Jurkat cells with gp120 results in hyper-phosphorylation of a tryptic lck peptide containing Tyr505. (A) 32P-labeled lck before and after incubation with gp120. Jurkat cells were first loaded with [32P]orthophosphate and either left unstimulated orstimulated with gp120 for 20 min. lck immunoprecipitates from cell lysates were resolved by SDS–PAGE, transferred to PVDF membranes andlck bands were visualized by autoradiography. A 2 h exposure is shown. (B) Tryptic phosphopeptide mapping of lck. lck proteins were elutedfrom the membranes and digested with TPCK-trypsin. Digests were then resolved by electrophoresis in 1% NH4HCO3 at pH 8.9 in thehorizontal direction with the positive electrode at the left, followed by ascending chromatography in the second dimension. The film wasexposed for 24 h. Spot 1 corresponds to the lck peptide containing Tyr505. Spot 2 corresponds to the lck peptide containing Tyr394. Theexperiment shown is a representative of four independent experiments. (C) 32P-labeled lck associated with CD4 before and after incubationwith gp120. Total lck was recovered by immunoprecipitation with anti-lck antisera and is shown in the left lane. A 15 h exposure is shown. (D)Tryptic phosphopeptide mapping of lck recovered from CD4 immunoprecipitates. The film was exposed for 7 days. Spot 1 corresponds tothe lck peptide containing Tyr505. Spot 2, which corresponds to the lck peptide containing Tyr394, did not reproduce well in the photographbut was clearly visualized on the autoradiograph and was readily detected by the PhosphorImager.


Table 1. Preincubation of T cells with gp120 inhibits TT-driven T cell proliferation

[3H]Thymidine incorporation (c.p.m.)

Experiment 1 Experiment 2

— TT — TT

Medium 502 6 187 6068 6 257 577 6 159 8072 6 444gp120 (SF2) 613 6 129 4618 6 256 (23.9a) 592 6 119 5358 6 529 (33.6a)gp120 (IIIB) 550 6 218 4825 6 199 (20.5a) 585 6 89 5433 6 191 (32.7a)Ovalbumin 646 6 171 6438 6 310 (–6.1) 622 6 128 7346 6 388 (–5.4)

TT-driven T cell proliferation was inhibited by pretreatment with gp120 for 20 min. Purified CD41 T cells (13106 cells) were preincubatedwith gp120 (5 µg/ml) or with ovalbumin (10 µg/ml) for 20 min, washed and mixed with E rosette negative cells at a 1:2 ratio. The cells wereincubated with TT (20 µg/ml) for 120 h and pulsed with [3H]thymidine for the last 18 h. The mean 6 SD of [3H]thymidine incorporation isindicated. Numbers shown in parentheses represent percentage inhibition over control proliferative response.

aP , 0.05.

gp120 for 20 min resulted in only a modest inhibition of the surprising that no significant increase in total lck phosphory-lation could be detected in gp120-treated cells.T cell proliferative response to the antigen TT (Table 1). This

inhibition is in the same range as that previously reported Although hyper-phosphorylation of the lck peptide con-taining the autoinhibitory Tyr505 residue was modest, it maywith a TT-specific T cell clone (13). The modest degree of

inhibition seen in both studies is not totally surprising, because well be biologically important. A modest (~50%) change inphosphorylation of Tyr527 in pp60c-src, which corresponds toCD4 molecules synthesized during the 5 day culture period

would be available to participate, in the absence of gp120 Tyr505 of lck, results in a .4-fold change in its enzymaticactivity (33,52,53). The mechanism of hyper-phosphorylationengagement, in the TCR-mediated response to antigen which

persists on antigen-presenting cells during the culture. of the Tyr505 by gp120-containing peptide by gp120 is atpresent unknown. It is possible that gp120 activates csk, aWe ruled out several potential mechanisms for the inhibitory

effect of gp120 on TCR–CD3 signaling. In contrast to the kinase which negatively regulates the activity of src familyPTK (54) including lck (32) by phosphorylating the regulatoryeffect of long-term incubation with gp120 which induces

down-modulation of surface CD4 expression and dissociation C-terminal tyrosines. Alternatively, gp120 may render CD4-associated lck more available to csk phosphorylation. Anotherof lck from CD4 molecules (7,10,28), a short-term incubation

with gp120 did not alter surface CD4 expression (Fig. 7) nor mechanism may involve the sequestration by gp120 of thephosphatase CD45, which regulates the phosphorylation ofCD4 association with lck (Fig. 8). Thus, it is unlikely that the

inhibition of TCR–CD3 signaling by gp120 is due to down- C-terminal tyrosine residues of several src kinases, includinglck Tyr505 and the homologous fyn residue Tyr531 (reviewedmodulation of CD4 surface expression or to dissociation of

lck from CD4. The CD4–lck complex associates with TCR– in 55).Hyper-phosphorylation of Tyr505 may not be the only mech-CD3 and this association is important for optimal T cell

activation (31,46–49). Our experiments demonstrated that anism of gp120-mediated inactivation of lck. Although cross-linking of CD3 and CD4 molecules did not overcome theapproximation of CD4 and TCR–CD3 by biotin-conjugated

mAb and avidin did not overcome gp120 inhibition of TCR– inhibitory effect of gp120 on lck phosphorylation, it is stillpossible that gp120 interferes qualitatively with CD3–CD4CD3 signaling (Fig. 10), implying that gp120 does not inhibit

TCR–CD3 signaling by simply sequestering CD4 molecules. interaction or with CD4–lck interaction.Recently, Goldman et al. (22) demonstrated that CD4src-type PTK have highly conserved structural features.

They have a short C-terminal regulatory domain, which con- ligation by gp120 and anti-gp120 antibodies led to lck activa-tion and defective CD3-mediated signaling as assayed bytains a conserved negative regulatory tyrosine residue (50).

Phosphorylation of this C-terminal Tyr505 in lck allows it to Ca21 influx and tyrosine phosphorylation. Their data suggestthat gp120 induces uncoupling of TCR from the early signalingbind to its SH2 domain, thus masking the catalytic site and

resulting in inhibition of its kinase activity. Dephosphorylation events. Our results differ in that in the absence of cross-linking, gp120 caused no activation of lck, yet it inhibitedof Tyr505 by the transmembrane phosphatase CD45 releases

this inhibition which is critical for T cell activation (51). src-type CD3 signaling. This suggests that gp120 by itself interruptsCD4–lck signaling, which may be required for CD3 activation,PTK also contain a positive regulatory Tyr autophosphorylation

site in their kinase domain. In this regard, the activated form possibly by causing hyper-phosphorylation of the autoinhibi-tory residue on CD4-associated lck. Thus the mechanism ofof lck contains autophosphorylated Tyr394 in its catalytic

domain (33). Tryptic peptide mapping of total lck and of CD4- inhibition of CD3 signaling by gp120 and gp120 cross-linkingmay well differ.associated lck in T cells simulated with gp120 revealed

that phosphorylation of the Tyr505-containing peptide was Caution should be exercised in drawing conclusions aboutHIV infection, which is a chronic progressive disease, fromincreased while that of the Tyr394-containing peptide was

unchanged or decreased (Fig. 11). Given the modest hyper- results of short-term, in vitro assays such as the one presentedin this report. Our data is consistent with and helps providephosphorylation of the Tyr505-containing peptide, it is not


and p56lck together with impairment of T cell activation.mechanistic explanation for the previously noted defects inJ. Immunol. 149:285.TCR–CD3 signaling following exposure of T cells to gp120

8 Diamond, D. C., Sleckman, B. R. Gregory, T., Lasky, L. A.,in vitro (6,11,13). In vivo, defective signaling via TCR–CD3 Grenstein, J. L. and Burakoff, S. J. 1988. Inhibition of CD41 Thas been noted in lymphocytes of patients with intermediate cell function by HIV envelop protein gp120. J. Immunol. 141:3715.

9 Juszczak, R. J., Turchin, H., Truneh, A., Culp, J. and Kasis, S. 1991.(21) and advanced (56) stages of HIV infection but not inEffect of human immunodeficiency virus gp 120 glycoprotein onthose with early disease (57). Cayota et al. (21) reported thatthe association of the protein tyrosine kinase p56lck with CD4 inCD41 T cells of patients with intermediate stage HIV infectionhuman T lymphocytes. J. Biol. Chem. 266:11176.

exhibit a decreased cellular level of p56lck and an increased 10 Groux, H., Torpier, G., Monte, D., Mouton, Y., Capron, A. andexpression of p59fyn, and demonstrated that anti-CD3-medi- Ameisen, J. C. 1992. Activation-induced death by apoptosis

in CD41 T cells from human immunodeficiency virus-infectedated stimulation is followed by defective tyrosine phosphoryla-asymptomatic individuals. J. Exp. Med. 175:331.tion of cellular proteins. It can be speculated that a role for

11 Mittler, R. S. and Hoffmann, M. K. 1989. Synergism between HIVgp120 in inhibiting TCR–CD3 signaling may become relevantgp120 and gp120-specific antibody in blocking human T cell

in vivo as the viral load increases with advancing HIV disease. activation. Science 245:1380.gp120 inhibition of TCR–CD3 signaling may also be relevant 12 Oyaizu, N., Chirmule, N., Kalyanaraman, V. S., Hall, W. W.,

Pahwa, R., Shuster, M. and Pahwa, S. 1990. Humanin anatomic sites where HIV replicates at high levels such asimmunodeficiency virus type 1 envelop glycoprotein gp 120lymph nodes. Further studies will be required to explore theproduces immune defects in CD41 T lymphocytes by inhibitingrelationship between the in vivo gp120 load, disease stageinterleukin 2 mRNA. Proc. Natl Acad. Sci. USA 87:2379.

and the occurrence of signaling abnormalities. 13 Chirmule, N., Kalyanaraman, V. S., Oyaizu, N., Slade, H. B. andPahwa, S. 1990. Inhibition of functional properties of tetanusantigen-specific T-cell clones by envelop glycoprotein gp120 ofhuman immunodeficiency virus. Blood 75:152.Acknowledgements

14 Klausner, R. and Samelson, L. 1991. T cell antigen receptorThis work was supported by NIH grant AI-29906, and by grants from activation pathways: the tyrosine kinase connection. Cell 44:875.Baxter Healthcare, Alpha Therapeutics and Quantum Corporations. 15 Weiss, A. and Littman, D. R. 1994. Signal transduction byT. M. was a recipient of a postdoctoral fellowship from the Uehara lymphocyte antigen receptors. Cell 76:263.Memorial Foundation and was a Lucille L. Markey Trust Awardee. 16 Danielian, S., Alcover, A., Polissard, L., Stefanescu, M., Acuto, O.,

Fischer, S. and Fagard, R. 1992. Both T cell receptor (TCR)–CD3complex and CD2 increase the tyrosine kinase activity of p56lck:CD2 can mediate TCR-independent and CD45-dependentAbbreviationsactivation of p56lck. Eur. J. Immunol. 22:2915.

α-LPC α-lysophosphatidylcholine 17 Tsygankov, A. Y., Broker, B. M., Fargnolli, J., Ledbetter, J. A. andIC immune complex Bolen, J. A. 1992. Activation of tyrosine kinase p60fyn following TNRS normal rabbit serum cell receptor cross-linking. J. Biol. Chem. 267:18259.PKC protein kinase C 18 Chan, A. C., Irving, B. A., Fraser, J. D. and Weiss, A. 1991. ThePLCγ1 phospholipase Cγ1 ζ-chain is associated with a tyrosine kinase and upon T cell antigenPTK protein tyrosine kinases receptor stimulation associates with ZAP-70, a 70 kilodaltonTPCK N-tosyl-L-phenylalanine chloromethyl ketone phosphoprotein. Proc. Natl Acad. Sci. USA 88:9166.TT tetanus toxoid 19 Iwashima, M., Irving, B. A., Van Oers, N. S. C., Chan, A. C. and

Weiss, A. 1994. Sequential interactions of the TCR with two distincttyrosine kinases. Science 263:1136.

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hiv glycoprotein gp120 inhibits tcr–cd3- mediated activation of

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