Interleukin (IL)-2 activation of p21ras in murine myeloid cells transfected with human IL-2 receptor beta chain.

The T cell growth factor interleukin-2 (IL-2) induces p21ras activation in T lymphocytes. To determine whether the IL-2 receptor (IL-2R) can regulate p21ras when expressed in a non-T cell environment we have examined the ability of IL-2 to activate p21ras in 32D murine myeloid progenitor cells transduced with human IL-2R beta chains. These cells are denoted beta 53 cells. 32D cells normally proliferate in response to IL-3 but the expression of the IL-2R beta chain confers IL-2 responsiveness to the cells. Our data show that IL-3 is able to activate p21ras in the parental 32D cells and both IL-2 and IL-3 can stimulate p21ras in the IL-2R-expressing beta 53 clone of 32D. In T lymphocytes, activation of protein kinase C (PKC) with phorbol esters is sufficient to stimulate p21ras. However, in 32D and beta 53 cells activation of PKC with phorbol esters does not result in p21ras activation even though these cells express functional PKC. It appears, therefore, that a PKC-mediated pathway for p21ras regulation exists in T lymphocytes but not in 32D cells. The IL-2R can couple to p21ras independently of the concomitant presence of the PKC pathway for p21ras regulation. These data imply that multiple intracellular mechanisms may exist to regulate p21ras and that cells of different lineages may differ with regard to p21ras regulation.     

Decreased p21 levels in anti-sense ras transfectants augments NK sensitivity

We have shown that the oncogenic EJ-ras gene, under the control of a metallothionein-I (Mt) promoter, can be induced to cause an increased susceptibility of transfected 10T1/2 fibroblasts to cytolysis mediated by natural killer (NK) cells. This effect may be specific to the ras gene family, since other oncogenes that we have tested here (src) and elsewhere (myc) do not show this effect. We have now examined the effect of modulating the level of p21 in both a positive or negative manner. The level of p21 ras was decreased by two independent mechanisms. First Zn2+ was removed from Mt-EJ-ras transformed cells. In the second approach we transfected 10T1/2 cells with a Mt-anti-sense c-H-ras construct which reduced p21 expression, slowed the growth rate and altered the morphology of 10T1/2 cells when induced with Zn. Surprisingly, the decrease in p21 ras levels by both approaches caused a marked increase in NK susceptibility (NKS) which was equivalent to that observed when the p21 ras levels were increased either by inducing EJ-ras or removing Zn2+ from Mt-anti-sense c-H-ras containing cells. The kinetics of induction of NK sensitivity due to decreasing normal p21 ras levels was identical to that observed for increasing mutated p21 ras levels. Peak enhancement of NKS was observed 24 hr after ras perturbation. These results suggest that either a positive or negative change in the steady-state level of p21 ras is sufficient to induce NK sensitivity, and NK sensitivity is not inextricably linked to cellular transformation by the ras gene.     

Constitutive active p21ras enhances primary T cell responsiveness to Ca2+ signals without interfering with the induction of clonal anergy

Chronic engagement of the T cell receptor mediates the induction of T lymphocyte unresponsiveness called clonal anergy. The development of such unresponsiveness has been suggested as one of the mechanisms that regulate peripheral tolerance to self-antigens and hamper the capacity of tumor antigen-specific T cells to eliminate cancerous cells. In the attempt to enhance the effector function of CD4(+) T lymphocytes and their resistance to clonal anergy induction, we have transduced primary T cells with a retroviral vector encoding active p21(ras) (Ras(Leu61)). Here we show that Ras(Leu61) elicited TCR-independent activation of the Ras-Raf-ERK pathway and conferred primary T cells with the ability to secrete IL-2 in response to stimulation with a Ca(2+) ionophore alone, without altering antigen-, CD3/CD28- and PMA/ionomycin-driven IL-2 secretion and T cell proliferation in vitro. However, chronic engagement of the TCR onthe surface of Ras(Leu61) T cells still led to an inability of the cells to produce IL-2 upon restimulation. These results indicate that enforced p21(ras) functionality enhances primary T cells responses to calcium-generated signals, but is insufficient to prevent TCR-driven T cell unresponsiveness and suggest that additional biochemical mechanisms, independent of p21(ras), negatively regulate IL-2 production in unresponsive T cells.     

Memory T cells of a patient with follicular thyroid carcinoma recognize peptides derived from mutated p21 ras (Gln-->Leu61).

Point mutations in ras genes resulting in substitutions of amino acid Gly in positions 12 and 13, and Gln in position 61 of the ras gene product p21, are commonly found in human tumors. Peptides derived from aberrant p21 may elicit a tumor specific T cell response, provided that these peptides can bind to HLA molecules of the tumor and the patient has T cells able to recognize the corresponding peptide-HLA complex. Here we report that CD4+ T cells of memory type (CD45RO+) from a patient with a follicular thyroid carcinoma respond against a synthetic peptide derived from aberrant p21 ras having a Gln-->Leu substitution at position 61. Such responses were not observed when T cells from healthy volunteers or cancer patients where this mutation does not usually occur were stimulated with this peptide. The responding T cells did not cross-react with the corresponding peptide derived from native p21 ras nor did they recognize peptides carrying other substitutions in position 61. T cells clones were generated which recognized this Leu61 peptide when presented by HLA-DQ8 molecules. These T cell clones also recognized the corresponding intact p21 ras protein. By using several different synthetic peptides, a peptide with optimal stimulatory capacity was defined. Performing polymerase chain reaction and oligonucleotide probing we were, however, not able to detect the p21 ras gene encoding the Gln-->Leu substitution in DNA from tumor biopsies from the patient. This may indicate that tumor cells harboring the mutation leading to the Gln-->Leu substitution had been eliminated and that tumor progression was due to cells that had deleted the mutated ras gene. The finding that ras derived peptides and recombinant mutated p21 ras are immunogenic in man may form the basis for the development of cancer immunotherapy based on synthetic oncogene derived peptides.     

T cell epitopes encompassing the mutational hot spot position 61 of p21 ras. Promiscuity in ras peptide binding to HLA

Activated ras carry a point mutation either in codon 12, 13 or 61 which is tumor specific. Peptides derived from this oncoprotein are therefore potential tumor antigens. Essential for the feasibility of using ras-derived peptides in therapy of cancer is whether p21 ras-derived peptides can be processed, bind to human histocompatibility leukocyte antigen (HLA) and be recognized by T cells. Here we report the fine specificity and HLA restriction of several T lymphocyte clones (TLC) specific for a peptide which is derived from the second mutational hot spot in ras encoding residue 61. These TLC were generated from memory T cells present in the blood of a cancer patient and recognized a ras-derived peptide carrying Leu instead of Gln at residue 61. By sequencing of the T cell receptor (TcR) genes three sets of “sister” TLC carrying highly different TcR were identified. Two of the TLC recognized a peptide carrying the 61 Leu mutation presented by HLA-DQ8 and one recognized the same peptide presented by HLA-DQ4. By using truncated peptides derived from residues 51 to 69 of p21 ras, partially overlapping minimal epitopes could be defined. All three TLC recognized the corresponding recombinant mutant p21 ras oncoprotein carrying Leu at residue 61 presented by autologous B-lymphoblastoid cell lines (B-LCL). This demonstrates that naturally derived ras peptides from this region of p21 ras encompass the three epitopes recognized by the TLC. These results indicate that immunogenic ras-derived peptides may be used in immunotherapy of cancer where transforming ras oncoproteins are involved.     

The carboxy-terminal catalytic domain of the GTPase-activating protein inhibits nuclear signal transduction and morphological transformation mediated by the CSF-1 receptor.

To determine whether ras p21 products are necessary for signal transduction mediated by the colony stimulating factor-1 receptor (CSF-1R, the c-fms proto-oncogene product), we determined whether CSF-1R and ras activate a common nuclear target and whether the interruption of ras action affects CSF-1R signal transduction. Expression of the NVL3 retrotransposon was activated to the same extent in NIH-3T3 cells by both ras and v-fms oncogenes, and the ras-responsive element located in the long terminal repeat of NVL3 was demonstrated to be a common target for oncogene action. Human recombinant CSF-1 stimulated expression of the NVL3 element 30-fold in NIH-3T3 cells that contained human CSF-1R. Expression of the carboxy-terminal 374 amino acid residues of the human ras GTPase-activating protein (GAP) in cells containing CSF-1R was able to inhibit CSF-1 induction of NVL3 expression by 90%. Expression of the catalytic domain of GAP was also able to suppress transformation by either v-fms or ligand-activated CSF-1R. Expression of the c-jun proto-oncogene was activated by CSF-1R but was insensitive to the action of the catalytic domain of GAP. These results provide genetic evidence that in NIH-3T3 cells, ras p21 is involved in signal transduction mediated by CSF-1R.     

T-cell responses against products of oncogenes: Generation and characterization of human T-cell clones specific for p21 ras-derived synthetic peptides

Peptides derived from mutated human proto-oncogenes bound to HLA may represent a novel type of tumor-specific antigen. Mutated ras genes are the oncogenes most frequently identified in human cancer. The transforming genes carry a mutation in codons 12, 13, or 61. We have investigated whether the T-cell repertoire of healthy individuals contains T cells capable of recognizing and responding to oncogene-derived peptides. Synthetic peptides derived from mutated p21 ras proto-oncogenes, covering mutations at codons 12 or 13 were selected. It was feasible to elicit T-cell responses and isolate several new T-cell clones (TCC) with specificity for a number of different mutated ras peptides after repeated in vitro immunization. Fout TCC wer characterized with respect to the fine specificity and HLA restriction. TCC B and I were restricted by HLA-DR molecules, and recognized the mutated p21 ras-derived peptide carrying Arg and Lys at residue 12, respectively. TCC E and F were restricted by HLA-DQ molecules, the former being specific for a mutated p21 ras-derived peptide with Val in position 13 and the latter more broadly reactive. Peptide competition experiments with a panel of ten peptides derived from p21 ras indicated that all could bind to HLA-DQ molecules of the T-cell donor, while several were also able to bind his HLA-DR molecules. These results show that several p21 ras mutations resulting in aa substitutions at residues 12 or 13 could be recognized by T cells derived from precursor T cells of relatively low frequency present in the normal repertoire of a single donor.     

Involvement of p21ras activation in T cell CD69 expression

The involvement of p21^ras in the induction of the early activation antigen CD69 was investigated in T cells. Expression of a v-Ha-ras coding for a constitutively active ras protein in Jurkat cells resulted in CD69 induction on the cell surface. Transfected ras was shown to be constitutively activated and functionally efficient, since it could be immunoprecipitated in the guanosine triphosphate (GTP)-bound form and it induced transactivation of an AP-1 consensus-chloramphenicol acetyltransferase reporter gene. The requirement for ras activation in T cell receptor (TcR) CD3-mediated CD69 induction was also investigated. The expression of a dominant negative c-Ha-ras-N17 mutant markedly reduced the amount of GTP that could be immunoprecipitated from ras proteins after TcR/CD3 triggering in Jurkat cells, and concomitantly decreased TcR/CD3-mediated CD69 induction. These results suggest a central role for ras in TcR/CD3-mediated CD69 expression in T cells.     

Activation of K-p21ras and N-p21ras, but not H-p21ras, is necessary for mitogen-induced human airway smooth-muscle proliferation

Ras proteins (H-, K-, and N-p21ras) play critical roles in the control of normal and neoplastic cell growth. To date, however, little is known about the role of p21ras in regulating mitogen-induced smooth muscle and, specifically, human airway smooth-muscle (HASM) cell growth. We postulate that p21ras is a critical signaling event regulating mitogen-induced HASM cell proliferation. Growth-arrested, confluent HASM cells were treated for 1 h with 10 ng/ml epidermal growth factor (EGF), 1 U/ml thrombin, or 5 microM bradykinin, then cell lysates were immunoprecipitated using anti-p21ras antibody. Immunoblot analysis using a pan p21ras antibody, which recognizes H-, K-, and N-p21ras, found no significant difference in p21ras expression in HASM after stimulation with either agent, as compared with control. In parallel experiments, we characterized that HASM cells express K- and N-p21ras, but not H-p21ras. Further, there was no difference between the levels of each p21ras isoform after stimulation with any of the agonists. The time course of p21ras activation, however, was markedly different among agonists. EGF rapidly activated p21ras within 30 s and was sustained for up to 30 min. Although thrombin also induced a rapid rise in p21ras activity after 2.5 min, the activation was transient. In contrast, bradykinin, which is nonmitogenic for HASM cells, did not activate p21ras. Using single-cell microinjection, the role of p21ras activation in modulating mitogen-induced HASM DNA synthesis was determined by 5-bromo-2'-deoxyuridine (BrdU) incorporation and anti-BrdU immunofluorescent staining. Thrombin- and EGF-induced DNA synthesis in cells microinjected with Y13-259, a neutralizing p21ras antibody, was significantly inhibited as compared with those microinjected with isotype-matched rat immunoglobulin G(1) or a vehicle control. These data suggest that activation of p21ras appears to be necessary for EGF and thrombin-induced HASM cell proliferation and that activation of K- and N-p21ras, but not H-p21ras, mediates smooth-muscle cell growth.     

Defective activation of p21ras in peripheral blood mononuclear cells from patients with insulin dependent diabetes mellitus.

We previously reported that a decreased TCR mediated activity of the GTP-GDP binding p21ras protooncogene is associated with prediabetes in non-obese diabetic (NOD) mice. Furthermore, prevention of autoimmune diabetes is associated with reversal of the p21ras signaling defect in NOD T cells. Based on these animal studies we determined the activation of p21ras in PBMC from patients with Insulin Dependent Diabetes Mellitus (IDDM), Non-Insulin Dependent Diabetes Mellitus (NIDDM) and normal healthy controls. Stimulation by PHA induced a decrease of 3.7 +/- 1.4% and an increase of 2.44 +/- 2.3%, p < 0.02 and 2.6 +/- 1.6%,p < 0.003 in the basal unstimulated p21ras activity in the IDDM, NIDDM and normal control groups, respectively. Expression of p21ras and its regulatory elements, the GTPase activating protein p120ras-GAP and the guanine nucleotide releasing factor (GNRF) hSOS, was comparable in the three groups. The in vitro proliferative response to PHA was comparable in the IDDM and control groups: stimulation index (SI) of 8.6 +/- 2.5 and 9.4 +/- 3.5 respectively, p < 0.44. No correlations were found in the IDDM patients between the degree of p21ras activation and the mitogen induced in vitro proliferative response or the various clinical parameters including age, gender, disease duration, daily insulin requirements and metabolic control. Taken together these data indicate that PBMC from IDDM patients are characterized by a persistent impairment in the activation of their p21ras. They also suggest that p21ras stimulated activity is a sensitive and independent parameter of PBMC activation in these patients.     

Optimal preservation of p21 ras immunoreactivity and morphology in paraffin-embedded tissue

Specific immunostaining of p21 ras protein by the well-characterized pan-ras antibody Y13-259 is achieved in paraffin sections of human and animal tissues fixed in periodate-lysine-paraformaldehyde-dichromate (PLPD). Intensity of staining is as good as in cryostat sections, with superior histological detail. Localization to plasma membrane is demonstrated in rodent cells genetically manipulated to express abundant p21 ras (the FHO5T1 cell line), both in preparations suspended in agar after culture in vitro and in those growing as tumour in vivo. Strong positive staining is observed in neoplasms of human breast and colon, tissues in which there is independent evidence of elevated ras gene expression. The superior morphology afforded by this technique allows clear characterization of p21 ras expression in small premalignant lesions for which other methods of detection of oncogene expression are not appropriate.     

The role of protein kinase C in the regulation of extracellular signal-regulated kinase by the T cell antigen receptor

The aim of this study was to explore the role of protein kinase C (PKC) in the activation of mitogen-activated protein kinases (MAPK) in T lymphocytes. The MAPK extracellular signal-regulated kinase-2 (ERK2) is activated in response to phorbol esters which stimulate PKC, by transient expression of a constitutively active ras mutant, by cell activation via the G protein-coupled type 1 muscarinic acetylcholine receptor (HM1R) or in response to triggering of the T cell antigen receptor (TCR). The relative contribution of PKC to TCR and HM1R regulation of ERK2 was explored by examining the effects of a PKC inhibitor (Ro 31-8425) on ERK2 activation. The data demonstrate that phorbol ester and HM1R regulation of ERK2 was prevented by the PKC inhibitor, but that the inhibitor had no effect on ERK2 activation induced by expression of a constitutively active ras mutant p21v-Ha-ras. Furthermore, the TCR stimulates both PKC and p21^ras but TCR regulation of ERK2 was only weakly suppressed by the PKC inhibitor. These data indicate that PKC has a potential but not a predominant role in TCR regulation of ERK2.     

T cell clones specific for p21 ras-derived peptides: Characterization of their fine specificity and HLA restriction

Peptides corresponding to the mutated regions of the oneoprotein p21 ras are immunogenic and capable of eliciting HLA class II-restricted T cell responses. Here we report studies on the fine specificity of four T lymphocyte clones (TLC) from a single donor, using various truncated peptides derived from the residues 6–19 of p21 ras and a panel of well-characterized HLA homozygous cells as antigen-presenting cells. Putative minimum peptides of nine or ten amino acids could be defined for each TLC. Two of the TLC recognized peptides presented by DR2, and the two others recognized peptides presented by DQ6. Some notable differences in the requirement for certain amino acids were seen between the DR-and DQ-restricted TLC. Thus, Ser at residue 17 was required for stimulation of the DQ6-but not the DR2-restricted TLC. Val at residue 8 was essential for stimulation of all TLC, whereas one of the DR2-restricted TLC also required Val at residue 7. Some peptides which were nonstimulatory were still capable of binding to DQ6 molecules in peptide competition experiments. The results may be of importance for potential immunotherapy of cancer where transforming ras oncoproteins are involved.     

EXPRESSION OF Ha-ras ONCOGENE PRODUCT IN RAT GASTROINTESTINAL CARCINOMAS INDUCED BY CHEMICAL CARCINOGENS

The expression of Ha ras oncogene product in rat gastrointestinal carcinomas induced by N-methyl-N-nitro-N-nitrosoguanidine (MNNG) or 1, 2-dimethylhydrazine (DMH) was studied by Western blotting and immunohistochemistry using anti-Ha ras p 21 oncoprotein antibody. In Western blotting, high levels of c-Ha ras p 21 were found in serially transplantable rat duodenal carcinomas induced by MNNG and rat colon carcinomas induced by DMH. mmunohistochemically, c-Ha ras p21 immunoreactivity was detected in 3 (16.7%) of 17 MNNG-induced stomach carcinomas and in 21 (63.6%) of 33 DMH-induced colon carcinomas, respectively. In the colon carcinomas, c-Ha- ras p 21 immunoreactivity in deeply invasive tumors was stronger than that in superficially invasive tumors and was expressed in all subserosal tumors. Moreover, all of the metastatic colon carcinomas had c-Ha- ras p 21 immuno-reactive tumor cells. These findings suggest that c-Ha- ras p21 expression plays an important role in tumor proliferation, invasion and metastasis of DMH-induced colon carcinoma. ACTA PATHOL. JPN. 37: 1731–1741, 1987.     

Microinjection of a p21ras antibody into PC12 cells inhibits neurite outgrowth induced by nerve growth factor and basic fibroblast growth factor

The role of p21ras in signal transduction in PC12 cells was studied using an antibody that blocks its function. Native cells were microinjected with either a control solution or a solution containing the monoclonal antibody Y13-259. Treatment of the cells with growth factors appeared to enhance the ability of the cells to survive the microinjection procedure. Of the cells microinjected with the control solution 66-69% of those treated with either nerve growth factor (NGF) or basic fibroblast growth factor (bFGF) were still present 24 h post-injection, compared with only 57% for those not treated with growth factor after microinjection. This effect of the growth factors was inhibited by introduction of the Y13-259 antibody, suggesting that it occurs through a pathway that involves p21ras. Similarly, introduction of the Y13-259 antibody into cells also resulted in a statistically significant decrease in the percentage of neurite-bearing cells; 25-36% of the cells microinjected with the control solution had neurites, whereas 12-14% of the cells microinjected with the antibody solution had neurites. This decrease suggests that the induction of neurite outgrowth and the maintenance of established neurites by these growth factors is dependent on a functional p21ras pathway. As well as complementing the finding that p21ras is apparently involved in the mechanism of action of NGF in PC12 cells, these results further establish (1) that p21ras is also involved in the mechanism of action of bFGF, and (2) that the effect of NGF and bFGF on the number of labeled cells still present 24 h postinjection requires a functional p21ras protein.     

Allicin stimulates lymphocytes and elicits an antitumor effect: a possible role of p21ras

Allicin, the main organic allyl sulfur component in garlic, exhibits immune-stimulatory and antitumor properties. Allicin stimulated [(3)H]thymidine incorporation in mouse splenocytes and enhanced cell-mediated cytotoxicity in human peripheral mononuclear cells. Multiple administration (i.p.) of allicin elicited a marked antitumor effect in mice inoculated with B-16 melanoma and MCA-105 fibrosarcoma. The immune-stimulatory and antitumor effects of allicin are characterized by a bell-shaped curve, i.e. allicin at high, supra-optimal concentrations is less effective or inhibitory. Allicin induced activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2) in human peripheral mononuclear cells, and also in wild-type Jurkat T-cells. Allicin failed to activate ERK1/2 in Jurkat T cells that express p21(ras), in which Cys118 was replaced by Ser. These cells are not susceptible to redox-stress modification and activation. We postulate that the immune stimulatory effect of allicin is mediated by redox-sensitive signaling such as activation of p21(ras). It is suggested that the antitumor effect of allicin is related to its immune-stimulatory properties.     

Induction in transgenic mice of HLA-A2.1-restricted cytotoxic T cells specific for a peptide sequence from a mutated p21ras protein.

Cytotoxic T cells (CTL) recognize short peptides that are derived from the proteolysis of endogenous cellular proteins and presented on the cell surface as a complex with MHC class I molecules. CTL can recognize single amino acid substitutions in proteins, including those involved in malignant transformation. The mutated sequence of an oncogene may be presented on the cell surface as a peptide, and thus represents a potential target antigen for tumour therapy. The p21ras gene is mutated in a wide variety of tumours and since the transforming mutations result in amino acid substitutions at positions 12, 13 and 61 of the protein, a limited number of ras peptides could potentially be used in the treatment of a wide variety of malignancies. A common substitution is Val for Gly at position 12 of p21ras. In this study, we show that the peptide sequence from position 5 to position 14 with Val at position 12-ras p5-14 (Val-12)-has a motif which allows it to bind to HLA-A2.1. HLA-A2.1-restricted ras p5-14 (Val-12)-specific CTL were induced in mice transgenic for both HLA-A2.1 and human beta2-microglobulin after in vivo priming with the peptide. The murine CTL could recognize the ras p5-14 (Val-12) peptide when they were presented on both murine and human target cells bearing HLA-A2.1. No cross-reactivity was observed with the native peptide ras p5-14 (Gly-12), and this peptide was not immunogenic in HLA-A2.1 transgenic mice. This represents an interesting model for the study of an HLA-restricted CD8 cytotoxic T cell response to a defined tumour antigen in vivo.     

Production and characterization of anti-RAS p21 monoclonal antibodies

Monoclonal antibodies (MAb) Ras 10 and Ras 11 were raised to an activated human Harvey-ras p21 and shown to react with recombinant p21 as well as p21 derived from human and rodent cells. Characterization studies by ELISA, immunoprecipitation and Western blot procedures demonstrated that MAb Ras 10 (IgG2a) and Ras 11 (IgG2b) react with normal p21, activated p21, and p21 from each of the Harvey, Kirsten and N-ras families. Studies illustrated that MAb Ras 10 and Ras 11 can also be used in flow cytometry and immunohistochemistry to specifically detect cellular p21. ELISA, immunoprecipitation and Western blot studies comparing rat anti-p21 MAb Y13-259 with Ras 10 and Ras 11 demonstrated that Ras 10 and Ras 11 had a greater sensitivity for ras protein detection than Y13-259. Collectively, these studies illustrate that MAb Ras 10 and Ras 11 can be applied to a variety of assay formats to detect ras proteins and, therefore, may be valuable tools in detecting and measuring of ras protein expression in normal, neoplastic and pre-neoplastic cells.     

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

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 p56(lck), p59(fyn), ZAP-70, p95 (vav), phospholipase C-gamma1, 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 NH(2)-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.