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(Journal of Leukocyte Biology. 2003;73:682-688.)
© 2003 by Society for Leukocyte Biology

The lectin jacalin induces phosphorylation of ERK and JNK in CD4+ T cells

Seetha M. Lakshmi Tamma*, V. S. Kalyanaraman{dagger}, Savita Pahwa{ddagger}, Paul Dominguez* and Ron R. Modesto*

* Department of Biomedical Sciences, C. W. Post Campus, Long Island University, Brookville, New York;
{dagger} Department of Cell Biology, Advanced Biosciences Laboratories, Inc., Kensington, Maryland; and
{ddagger} North Shore-LIJ Research Institute, Manhasset, New York

Correspondence: Seetha M. Lakshmi Tamma, Ph.D., Associate Professor, Department of Biomedical Sciences, C. W. Post Campus, Long Island University, 720 Northern Blvd., Life Science 338, Brookville, NY 11548. E-mail: stamma{at}liu.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The CD4 molecule plays an essential role in mediating the transduction of intracellular signals by functioning as a coreceptor for the complex T cell receptor/CD3 and also acts as the primary receptor for human immunodeficiency virus (HIV). Several authors have shown evidence that jacalin, a plant lectin, binds to CD4 and inhibits in vitro HIV infection. We analyzed jacalin-induced intracellular signaling events in CD4+ T cells and have shown that cell activation resulted in tyrosine phosphorylation of intracellular substrates p56lck, p59fyn, ZAP-70, p95 vav, phospholipase C-{gamma}1, and ras activation, as assessed by conversion of ras guanosine 5'-diphosphate to ras guanosine 5'-triphosphate. We further examined extracellular regulated kinase (ERK) and c-jun NH2-terminal kinase (JNK) phosphorylation following stimulation with jacalin. The data indicate that the kinetics of JNK phosphorylation is delayed. Optimum phosphorylation of ERK2 was observed by 10 min, and that of JNK was observed by 30 min. Pretreatment with gp120 followed by stimulation with jacalin resulted in marked inhibition of all of the aforementioned intracellular events. The data presented here provide insight into the intracellular signaling events associated with the CD4 molecule–jacalin–gp120 interactions and HIV-induced CD4+ T cell anergy. Jacalin may be used as a possible tool for the study of CD4-mediated signal transduction and HIV-impaired CD4+ T cell activation.

Key Words: PLC-{gamma}1 • vav • RAS


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The T cell receptor (TCR)/CD3 receptor complex and the coreceptor molecules CD4 or CD8 act in synergy to initiate the T cell activation cascade [1 2 3 ]. Ligation of antigen/major histocompatibility complex with the TCR complex results in the activation of protein tyrosine kinases (PTKs), including p56lck [4 ], which mediate tyrosine phosphorylation of several substrates such as phospholipase C-{gamma}1 (PLC-{gamma}1), phosphatidylinositol 3-kinase (PI-3K), p95vav, and p21 ras [5 6 7 8 ]. PLC-{gamma}1 plays a central role in PTK- and protein kinase C (PKC)-mediated signaling, calcium mobilization, and interleukin (IL)-2 secretion. Several molecules, including Crk/Cbl/C3G [9 ] and vav [6 ], have been implicated in ras activation in T cells. The rate-limiting step in ras activation is the exchange of bound guanosine 5'-diphosphate (GDP) to guanosine 5'-triphosphate (GTP), which leads to the formation of an active ras-GTP complex [10 , 11 ].

This cascade proceeds through activation of Raf-1, which is recruited to the plasma membrane by interaction with ras, and in turn, activates mitogen-activated protein kinases (MAPK), which phosphorylate the extracellular signal-regulated kinases (ERKs), a group of proteins belonging to the MAPK family [12 , 13 ]. This stimulation is a result of dual-specificity protein kinase, MAPK kinase (MEK) [14 , 15 ], which phosphorylates ERK1 and ERK2. Signaling events downstream of ras activation have also been shown to involve multiple pathways leading to activation of MAPK c-jun NH2-terminal kinase (JNK)/stress-activated protein kinases [16 , 17 ]. Second messengers induced upon TCR ligation regulate nuclear-binding proteins. Examples of these include PKC and [Ca2+]ifor the regulation of nuclear factor (NF)-{kappa}B and NF of activated T cells (NF-AT) and PTK/PKC for the regulation of activated protein-1 (AP-1) via the activation of IL-2 gene.

The JNK can be activated in T cells by the combination of TCR and CD28 costimulation or by a variety of stress-related stimuli, including UV light, H202, and hyperosmolar sorbitol solutions [18 ], and has been implicated in regulation of several transcriptional factors including AP-1 [19 ]. It has been shown [20 ] that MEK/ERK kinase kinase 1 (MEKK 1) induces the site-specific phosphorylation of I{kappa}B{alpha} in vivo and in vitro (I{kappa}B{alpha} is involved in the activation of NF-{kappa}B) and concluded that MEKK 1 is a critical component in the c-jun and NF-{kappa}B stress-response pathway. Rincon et al. [21 ] reported that during the development of T cells in the thymus, the ERK pathway is required for differentiation of CD4-CD8- cells into CD4+CD8+ [double-positive (DP)] thymocytes, positive selection of DP cells, and their maturation into CD4+ T cells. Furthermore, these authors concluded that the JNK pathway contributes to the deletion of DP thymocytes by apoptosis. Data from mouse studies indicate that JNK is required for helper T cell differentiation into effector T cells and their cytokine production [22 ]. Others [23 ] have shown that the activation pathways of ERK, JNK, and Ca2+ mobilization are differentially regulated YxxL segments of an immunoreceptor tyrosine-based activation motif.

CD4, which is the primary receptor for human immunodeficiency virus (HIV) envelope glycoprotein, binds HIV envelope glycoprotein-gp120 with strong affinity. Several investigators have shown that the interaction with HIV-gp120 in vitro significantly influences CD4+ T cells, rendering them unresponsive to subsequent TCR activation. Other reported effects of CD4 signaling include activation of p56lck [24 25 26 ], tyrosine phosphorylation of PI-3K and PI-4K [27 ], and activation of Raf-1 [28 ].

Lectins such as phytohemagglutinin (PHA) and concanavalin A are mitogenic to all T cells, whereas jacalin, a purified lectin from jackfruit, is mitogenic only for CD4+ T cells [29 , 30 ]. Jacalin is a plant lectin that induces mitogenic responses selectively in CD4+ T lymphocytes and has been shown to block infection by HIV-1 in a T lymphoid cell line [31 ].

We have reported previously that HIV-1 gp120 inhibits jacalin-induced, proliferative responses and IL-2 secretion in peripheral blood mononuclear cells (PBMC) and in jacalin–T cell blasts. Jacalin-induced proliferative responses of PBMC from HIV-infected patients reflect not only the absolute number of CD4+ T cells within PBMC but also functional responses of CD4+ T cells to jacalin [32 ]. It is possible that the jacalin-induced proliferation observed in HIV+ patients may be representing responses of those functional CD4+ T cells in which the CD4 molecules are not occupied in vivo by gp120/anti-gp120 complexes. Thus, in this respect, jacalin-stimulated responses appear to provide an alternate, surrogate measure of CD4 cell numbers as well as CD4 T cell function.

Based on the effect of jacalin on CD4+ T cells and the effect of interaction of CD4, gp120, and jacalin on CD4+ T cells, we investigated the ability of jacalin to transmit intracellular signals associated with T cell activation. We analyzed early signaling events such as p56lck, p59fyn, ZAP-70, p95vav, PLC-{gamma}1, and ras activation. We further analyzed the downstream events of ras such as phosphorylation of MAPK ERK2 and JNK. As CD4 molecules are the primary receptors for HIV-1 envelope glycoprotein gp120, in this study, we examined the inhibitory effects of HIV-gp120 on jacalin-induced T cell signaling. Our results show that jacalin induces tyrosine phosphorylation of several intracellular substrates, and these events are blocked by pretreatment with gp120.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents
HIV-1-derived gp120 was purified from culture supernatants of a clone of HIV-infected HUT-78 cells, 6D4451, as described earlier [33 ]. The following reagents were used: mouse anti-human CD3 antibody from BD PharMingen (San Diego, CA); antiphosphotyrosine (4G10), anti-p56lck, anti-p59fyn, anti-ZAP-70, anti-p95vav, anti-PLC-{gamma}1, phospho-ERK2, and phospho-JNK antibodies (Upstate Biotechnology, Lake Placid, NY); anti-ras antibody Y13-259, rabbit anti-immunoglobulin (Ig) antibody, and protein A agarose (Oncogene Science, Uniondale, NY); polyethylenimine (PEI)-cellulose plates from Sigma Chemical Co. (St. Louis, MO); 32P-orthophosphate (NEN, Dupont, Boston, MA); sodium dodecyl sulfate (SDS) gradient gels and polyvinylidene difluoride (PVDF) membrane (Biorad, Hercules, CA); jacalin (E-Y Laboratories, San Mateo, CA); PHA and phorbol 12-myristate 13-acetate (PMA; Sigma Chemical Co.); and ionomycin (Calbiochem, La Jolla, CA).

Isolation and purification of cells
PBMC were isolated from healthy volunteers by Ficoll-Hypaque (Lymphoprep, Nycomed, Birmingham, UK) density-gradient centrifugation. T cells were enriched by double-rosetting with neuraminidase-treated sheep red blood cells. Adherent cells were removed by incubation in the petri dishes for 2 h at 37°C. CD4+ T cells were purified by negative selection with anti-CD8 monoclonal antibody (mAb)-coated magnetic beads (Dynal, Great Neck, NY), and CD8+ T cells were purified by negative selection with anti-CD4 mAb-coated magnetic beads.

Cell activation and lysis
For signal transduction studies, CD4+ T cells were cultured with PMA (50 ng/106 cells) + ionomycin (400 ng/106cells) and jacalin (200 µg/106 cells) for 10 min to analyze early phosphorylation events based on kinetic studies. Cells were also stimulated with plate-bound anti-CD3 mAb (1 µg/106 cells) as a positive control for T cell activation and tyrosine phosphorylation of various kinases. For blocking studies, purified CD4+ T cells were incubated with HIV-gp120 (5 µg/mlx106 cells) overnight at 4°C on a rotator. Cells were washed and incubated at 37°C for 10 min or incubated at 37°C for 10 min and then stimulated with jacalin (200 µg/106 cells) for 10 min. Reactions were terminated by washing cells in ice-cold phosphate-buffered saline (PBS) + EDTA + sodium orthovanadate and were lysed in lysis buffer (0.04 mol/L Tris-HCl, 0.276 mol/L NaCl, 20% glycerol, 2% Nonidet P-40, 0.002 mol/L sodium orthovanadate, 0.02 mol/L NaF, 10 µg/ml aprotinin, 10 µg/ml leupeptin, 0.004 mol/L EDTA, and 1 mmol/L phenylmethylsulfonyl fluoride).

Immunoprecipitation and immunoblotting
Cell lysates were precleared with Protein A-agarose and were incubated overnight at 4°C with appropriate antibodies as indicated (see Fig. 2 ) on a rotator, mixed with second antibody-coated Protein A-agarose beads, and allowed to precipitate at 4°C on a rotator overnight. The immunoprecipitates were washed extensively with lysis buffer, and pellets were boiled with sample buffer. Samples were analyzed by SDS-polyacrylamide gel electrophoresis (PAGE) using 4–20% gradient gels (Biorad), followed by immunoblotting with specific antibody or with antiphosphotyrosine antibody (4G10), followed by a peroxidase-conjugated secondary antibody, and developed by an enhanced chemiluminescence (ECL) system.



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  		Figure 2. Jacalin induces tyrosine phosphorylation of p56lck, p59fyn, ZAP-70, p95vav, and PLC-{gamma}1 in CD4+ T cells. To examine the tyrosine phosphorylation of individual cellular substrates, cell lysates were precleared with protein A agarose, immunoprecipitated with appropriate antibodies, and immunoblotted with antiphosphotyrosine (anti-PT) antibody (4G10). Data are representative of three independent experiments.

 
Biosynthetic labeling of cells with [32P]orthophosphate to examine ras activation following stimulation with jacalin
Purified CD4+ T cells (1x108 cells) were labeled with [32P]orthophosphate (400 µCi/ml/1x108 cells) in phosphate-free medium containing dialyzed fetal calf serum for 4 h at 37°C. Following labeling, cells were cultured with anti-CD3 antibody, with PMA + ionomycin, or with jacalin, with or without gp120 as described above. Ras proteins from cell lysates were immunoprecipitated with antibody Y13-259 precoupled to protein A-agarose via rabbit anti-Ig. Immunoprecipitates were washed extensively with lysis buffer and mixed with elution buffer (2 mM EDTA, 2 mM dithiothreitol, and 0.2% SDS) at 68°C for 20 min for elution of GDP and GTP nucleotides. Separation of eluted nucleotides was performed on PEI-cellulose thin-layer chromatography (TLC) plates using the solvent consisting of 1.2 M ammonium formate plus 0.8 M HCl. Labeled nucleotides were separated by TLC, analyzed by autoradiography, and were quantitated by densitometry.

Jacalin-induced kinetics of phosphorylation of ERK2 and JNK
At various time points, cells were lysed and examined for ERK and JNK phosphorylation. Cell extracts were analyzed by SDS-PAGE on gradient (4–20%) gels (Biorad). Protein was electrotransferred to PVDF membrane (Biorad), blocked with a solution of PBS containing 5% milk and 0.1% Tween-20, and probed with a phospho-specific antibody against ERK or JNK (Upstate Biotechnology), followed by a peroxidase-conjugated, secondary antibody and developed by an ECL system.

Enzyme-linked immunosorbent assay
Cells treated as described above were cultured in the presence of accessory cells for 48 h, and culture supernatants were harvested and analyzed for IL-2 using a commercial kit from Biosource (Camarillo, CA), according to the manufacturer’s instructions.

Lymphoproliferation assay
Cells stimulated with anti-CD3 antibody, PMA + ionomycin, PHA (10 µg/ml), and jacalin, with or without gp120 pretreatment, were cultured for 3 days with accessory cells, labeled with [14C]thymidine for 18 h, harvested, and analyzed by scintillation counting. Results were expressed as counts per minute (cpm).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Jacalin induces tyrosine phosphorylation of various cellular substrates
As proliferation and IL-2 secretion were only observed in jacalin-stimulated CD4+T cells [32 ], we analyzed the ability of jacalin to induce protein phosphorylation in CD4+ T cells. CD4+ T cells were cultured in the presence of PMA + ionomycin, plate-bound anti-CD3 antibody, and jacalin for 10 min at 37°C. The cells were washed in wash buffer and lysed with lysis buffer as described in Materials and Methods. Total tyrosine phosphorylation was analyzed by Western blotting using antiphosphotyrosine antibody (4G10). As shown in Figure 1 , jacalin induced tyrosine phosphorylation of several proteins, and the data are comparable with that induced by PMA + ionomycin and anti-CD3 antibody stimulation. However, phosphorylation of several proteins was absent when CD8+ T cells were similarly stimulated with jacalin (Fig. 1) . Although jacalin binds to CD8 molecules, it does not appear to bind to a signal-initiating or transmitting epitope. Herbimycin and staurosporin inhibited these phosphorylation events (data not shown). However, when cells were incubated with gp120 before stimulation with jacalin, tyrosine phosphorylation of several proteins was greatly inhibited. As jacalin binds to several cell-surface molecules, it seems that jacalin cross-links CD4 and CD3 in inducing enhanced phosphorylation. Failure of induction of phosphorylation events in CD4-preligated and jacalin-stimulated cells appears to be a result of dissociation of CD4- from CD3-mediated signals [34 ].



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  		Figure 1. Jacalin induces tyrosine phosphorylation of various cellular substrates in CD4+ T cells. CD4+ T lymphocytes were stimulated as described in Materials and Methods, and total tyrosine phosphorylation was analyzed as described in Materials and Methods. Data are representative of three independent experiments.

 
CD4+ T cells were similarly stimulated with anti-CD3 antibody, PMA + ionomycin, and jacalin; cells were washed and lysed in lysis buffer. Tyrosine phosphorylation of individual substrates was evaluated by immunoprecipitation with specific antibodies followed by immunoblotting with specific antibody or with antiphosphotyrosine antibody. Substrates, including p56lck, p59fyn, ZAP-70, p95vav, and PLC-{gamma}1, were tyrosine-phosphorylated following stimulation with jacalin (Fig. 2 ). Pretreatment with gp120 inhibited jacalin-induced tyrosine phosphorylation of p59fyn, p95vav, ZAP-70, and PLC-{gamma}1. Premixing of native gp120 with soluble CD4 completely abrogated its inhibitory effects on jacalin-induced responses (data not shown).

Jacalin induces ras activation
To elucidate the involvement of ras in T cell signaling, we examined the GDP/GTP exchange of ras protein following stimulation with jacalin. As shown in Figure 3 , treatment with jacalin resulted in conversion of ras GDP to ras GTP. The mean-percent conversion of rasGDP to rasGTP was 33% ± 4 in resting CD4+ T cells, 78% ± 6 in anti-CD3 antibody stimulated, 68% ± 8 in jacalin stimulated, and 35% ± 5 in gp120 treated. Treatment with gp120 followed by stimulation with jacalin resulted in 39% ± 4. Therefore, prior treatment with gp120 resulted in a marked reduction in jacalin-induced ras activation compared with that in untreated cells.



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  		Figure 3. Jacalin induces p21ras activation in CD4+ T cells as measured by conversion of rasGDP to rasGTP. [32P]Orthophosphate labeling, pretreatment, cell stimulation, cell lysing, and immunoprecipitation were performed as described in Materials and Methods. Ras-bound GDP and GTP were eluted and analyzed by TLC, visualized by autoradiography, and quantitated by densitometry. Conversion of rasGDP to rasGTP was calculated as a ratio of percent rasGTP/GDP:GTP. Data are representative of two independent experiments.

 
Jacalin induces ERK2 and JNK phosphorylation
We analyzed the kinetics of phosphorylation of the MAPK ERK2 and JNK downstream to Ras and Raf-1. Maximal ERK2 phosphorylation was detected by 10 min following stimulation with jacalin, and by 90 min, it was reduced by 80%; 1 min, 650 ± 20; 5 min, 1100 ± 35; 10 min, 4030 ± 60; 15 min, 3760 ± 50; 60 min, 2850 ± 65; and 90 min, 760 ± 40 (Fig. 4 ). ERK2 phosphorylation at 10 min is significantly higher (P<0.05) compared with 1 min or by 60 min. As shown in Figure 5 , the kinetics of JNK phosphorylation is delayed when compared with ERK2 phosphorylation or with CD3-mediated phosphorylation of JNK (data not shown). Maximal JNK phosphorylation was observed by 30 min (500±30) following stimulation with jacalin (Fig. 5A) . However, when cells were stimulated with jacalin in the presence of IL-2 (BD PharMingen; 100 ng per 1x106 cells), optimum phosphorylation of JNK was observed by 10 min (1945±35), and it was 40% higher when compared with phosphorylation of JNK induced by jacalin alone (Fig. 5B) . Herbimycin and staurosporin inhibited these phosphorylation events (data not shown). However, when cells were incubated with gp120 before stimulation with jacalin or jacalin + IL-2, phosphorylation of ERK2 and JNK was significantly inhibited (P<0.05; Fig. 6 ).



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  		Figure 4. Kinetics of jacalin-induced phosphorylation of ERK2 in CD4+ T cells. CD4+ T cells were stimulated with jacalin as described in Materials and Methods, harvested, and lysed at various time points as indicated. Lysates were analyzed by SDS-PAGE, transblotted, and probed with phospho-ERK2 antibody. Densitometric readings of scanned immunoblots were as follows: 1 min (lane 1) = 680; 5 min (lane 2) = 1050; 10 min (lane 3) = 4000; 15 min (lane 4) = 3810; 60 min (lane 5) = 2940; and 90 min (lane 6) = 751. Data are representative of three independent experiments.

 


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  		Figure 5. Kinetics of jacalin-induced phosphorylation of JNK in CD4+T cells. CD4+ T cells were stimulated with jacalin alone (A) or jacalin plus IL-2 (B), harvested, and lysed at various time points as indicated. Lysates were analyzed by SDS-PAGE, transblotted, and probed with phospho-JNK antibody. Data are representative of three independent experiments. Densitometric readings of scanned immunoblots were as follows: (A; jacalin) 0 min (lane 1) = 150; 20 min (lane 2) = 280; 30 min (lane 3) = 510; 45 min (lane 4) = 480; 60 min (lane 5) = 390; 90 min (lane 6) = 380; 2 h (lane 7) = 200. (B; jacalin+IL-2): 5 min (lane 1) = 340; 10 min (lane 2) = 1590; 30 min (lane 3) = 1960; 60 min (lane 4) = 1720; 90 min (lane 5) = 1690; 3 h (lane 6) = 380.

 


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  		Figure 6. gp120 pretreatment inhibits jacalin-induced phosphorylation of ERK2 and JNK in CD4+ T cells. CD4+ T cells were stimulated with jacalin alone (top), harvested, and lysed. Lysates were analyzed by SDS-PAGE, transblotted, and probed with phospho-ERK2 antibody. Data are representative of three independent experiments. Densitometric readings of scanned immunoblots for phospho-ERK2 (top) were as follows: lane 1 = 370; lane 2 = 3010; lane 3 = 2960; lane 4 = 2880; lane 5 = 2910; lane 6 = 280. CD4+ T cells were stimulated with jacalin alone (A) or jacalin plus IL-2 (B), harvested, and lysed. Lysates were analyzed by SDS-PAGE, transblotted, and probed with phospho-JNK antibody. Data are representative of three independent experiments. Densitometric readings of scanned immunoblots were for phospho-JNK (middle; B) as follows: lane 1 = 180; lane 2 = 1361; lane 3 = 1400; lane 4 = 1380; lane 5 = 1410; lane 6 = 210; (bottom; C): lane 1 = 210; lane 2 = 2640; lane 3 = 2810; lane 4 = 2841; lane 5 = 3120; lane 6 = 310.

 
Pretreatment of CD4+ T cells with gp120 inhibits jacalin-mediated IL-2 secretion and cell proliferation
Stimulation of CD4+ T cells with jacalin in the presence of accessory cells resulted in proliferation and IL-2 secretion, although the level of IL-2 secretion was much lower than that induced by other stimulants (Table 1 ). No proliferation or IL-2 secretion was observed in CD4+ T cell-depleted PBMC cultured with jacalin (data not shown). These results are in agreement with previous findings [29 , 30 ], confirming that jacalin is a selective CD4+ T cell mitogen. Pretreatment of CD4+T cells in the presence of accessory cells with gp120 followed by jacalin treatment resulted in inhibition of IL-2 secretion and lymphoproliferative responses (Table 1) . Pretreatment with gp120 was also found to result in profound inhibition of anti-CD3 mAb-induced, proliferative responses and IL-2 secretion, without any inhibitory effects on PMA + ionomycin-induced responses (data not shown).


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  		Table 1. Pretreatment with HIV-1 gp120 Inhibits Jacalin-Induced IL-2 Secretion and Lymphoproliferationa

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
CD4 molecules serve as coreceptors for TCR and act synergistically with TCR in T cell activation. [1 ]. It has been previously shown that pretreatment of T cells with gp120 results in inhibition of TCR/CD3-mediated T cell activation but not of responses resulting from activation of CD2 or CD28 nor of those resulting from bypass activation of T cells with PMA plus ionomycin [35 36 37 38 39 ]. Occupancy of the CD4 molecule with gp120/gp160 itself results in transmission of signals, which leads to a moderate activation of ras [40 ] but does not induce PLC-{gamma}1 activation. Signals elicited from CD4 molecules are often referred to as negative signaling, as they render the cells unresponsive to subsequent stimuli, which normally elicit full TCR activation [40 41 42 43 ]. As reported previously [38 ], CD4 preligation selectively inhibits TCR/CD3-mediated signaling and impairs IL-2 secretion [41 ].

In addition to the CD4 molecule, jacalin binds to several other cell-surface molecules; however, jacalin is stimulatory only to CD4+ T cells. We investigated the ability of jacalin to transmit intracellular signals associated with T cell activation. We show that stimulation with jacalin induces tyrosine phosphorylation of early signaling events such as p59fyn, ZAP-70, p95vav, PLC-{gamma}1, and ras activation. Our data also show that jacalin induces phosphorylation of ERK2 and JNK, as assessed by antiphospho-ERK2 and antiphospho-JNK antibodies, respectively. Pretreatment with gp120 followed by stimulation with jacalin inhibited several of the intracellular signaling events.

Several MAPK signal-transduction pathways have been defined in mammalian cells [44 , 45 ]. The ERK family has been associated with the proliferation and differentiation of a number of different cell types, and the JNK and p38 families have been associated with apoptotic death in several systems [44 , 45 ]. JNK activation has been reported to require a second signal provided by costimulatory molecules [46 ]; an additional calcium signal is also required to activate JNK [46 ], indicating the importance of the calcium pathway in the regulation of JNK. JNK family members are regulated and activated by MEK. Two upstream MEK, MKK4 and MKK7, have been found to be the primary activators of JNK [47 ].

As jacalin is a selective mitogen for CD4+ T cells, and CD4 molecules are the primary receptors for HIV-1 envelope glycoprotein gp120, we examined the inhibitory effects of HIV-gp120 on jacalin-induced T cell signaling. We demonstrated that pretreatment of CD4+ T cells with HIV-1 gp120 leads to decreased tyrosine phosphorylation of PLC-{gamma}1 and decreased ras activation, in addition to inhibition of molecules, described previously, which are proximal to the cell surface, namely p59fyn and ZAP-70 [34 ]. The failure of CD4 signaling via preligation of CD4 with HIV-gp120 and subsequent stimulation with jacalin to elicit PLC-{gamma}1 phosphorylation marks a clear distinction from direct stimulation with jacalin or TCR signaling, wherein PLC-{gamma}1 is phosphorylated and plays a key role in PTK- and PKC-mediated signaling, calcium mobilization, and IL-2 secretion. Stimulation with jacalin resulted in tyrosine phosphorylation of p95 vav, which has been implicated to be a substrate for tyrosine kinase p56lck [6 ], and in activation of ras, as measured by conversion of rasGDP to rasGTP. Structurally, vav is a likely candidate as an exchange protein for the ras-related rho/rac proteins [48 ]. Furthermore, vav has also been shown to induce activation of JNK via the Rac-1 pathway [49 ]. ras activation is critical, as it appears to be significantly important for many functional responses of T cells [50 , 51 ]. The failure of ras activation in the present study in gp120-pretreated, jacalin-stimulated cells is likely a result of the failure of activation of upstream regulators such as PLC-{gamma}1, resulting in impaired nucleotide exchange or in the regulation of GTPase activity. The block in the ras activation may lead to the defective transactivation of AP-1 sites in the IL-2 promoter and impaired IL-2 gene transcription [52 ]. It has been shown that ERK2 and JNK activation is critical for IL-2 gene transcription [51 , 52 ].

Previously, it has been shown that pretreatment of CD4+ T cells with anti-CD4 mAb suppressed the CD3-mediated induction of ERK2 activity in murine CD4+ T cells as well as inhibited the activation of transcription factors NF-AT and AP-1 and IL-2 secretion. [53 ].

It is clear from these studies that partial activation of CD4+ T cells by preligation interferes with MAPK pathways. These studies support the role of these transcription factors and the role of MAPK family members on the activation of the transcription factors and subsequent IL-2 secretion. At the transcriptional level, the expression of the IL-2 gene is controlled by several inducible, transcription factors [54 ]. Furthermore, these studies also indicated that CD3 + CD28 mAb-mediated action of JNK remained resistant to the anti-CD4 mAb pretreatment [53 ].

It should be noted that the nature of CD4 signaling depends on the site of the CD4 molecule being activated [55 56 57 ]. Baldari et al. [55 ] identified functionally distinct CD4 epitopes with a different ability to mobilize calcium and induce activation of the T cell-specific transcription factor NF-AT. As it has been demonstrated that distinct signaling properties identify functionally different CD4 epitopes, one can hypothesize that CD4 epitopes recognized by jacalin allow cell signaling, and this is not the case upon lectin binding to the CD8 molecule. Lafont et al. [58 ] examined the binding capacities of jacalin toward glycosylated, deglycosylated, and unglycosylated CD4 and provided evidence that jacalin binds to CD4 through a specific protein–protein interaction. This is an important finding in understanding jacalin-induced CD4+ T cell activation and its role in the context of HIV infection [32 ].

Previously, we [32 , 33 ] have shown that deglycosylated gp120 fails to block jacalin–CD4 interaction. Therefore, it is possible that glycosylated gp120 binds to CD4 on overlapping jacalin-binding sites. These findings indicate that gp120 exerts its inhibitory effects on jacalin-induced CD4+ T cell activation through interaction with CD4 molecules [32 ]. Jacalin may be used as a possible tool for the study of CD4-mediated signal transduction and HIV-impaired CD4+T cell activation.


   ACKNOWLEDGEMENTS
 
A grant from C. W. Post Campus Research Committee of Long Island University supported this work. We acknowledge Dr. Thoedora Grauer and Dr. Ellen Duffy, School of Health Professions and Nursing, C. W. Post Campus, for helpful suggestions. We are extremely grateful to Dr. Surya Tetali, North Shore University Hospital, for helping with densitometric readings. Ms. Louise Miller, Nutrition Department, and Ms. Daniela Tine, Department of Biomedical Sciences, School of Health Professions and Nursing, C. W. Post Campus, also deserve recognition for their help.

Received November 7, 2002; revised December 23, 2002; accepted January 20, 2003.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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