Protein tyrosine kinases couple the interleukin-2 receptor to p21ras

The T cell growth factor interleukin-2 (IL-2) induces p21^ras activation in T lymphocytes. We have previously shown that a protein kinase C (PKC)-mediated pathway for p21^ras regulation exists in T cells and that the IL-2 receptor (IL-2R) can couple to p21^ras independently of the presence of the PKC pathway for p21^ras regulation. Our data show that in conditions where cellular protein tyrosine kinases (PTK) were efficiently down-regulated by pretreatment with the specific PTK inhibitor herbimycin, the IL-2-induced activation of p21^ras was blocked. Herbimycin did not inhibit the PKC-mediated pathway for p21^ras regulation. Thus, the data indicate that PTK are involved in the coupling of the IL-2R to p21^ras.     

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.     

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.     

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.     

IL-2 signalling in T and natural killer (NK) cells associated with their class I-non-restricted killing activity

The signal transduction of IL-2 in NK cells and T cells was compared. On 5 min incubation of these cells with IL-2, we observed tyrosine phosphorylation of 105-kD and 110-kD proteins in NK cells and of 95-kD and 110-kD proteins in T cells. The phosphorylation reached maximal levels in 15 min in both NK and T cells, but the levels were higher in NK cells, which showed superior killing against Daudi cells. With this phosphorylation, p52^shc was also tyrosine-phosphorylated and p21^ras was activated by the short term (10 min) treatment of NK and T cells with IL-2. These signals were completely suppressed by anti-IL-2Rβ MoAb, but only slightly suppressed by anti-IL-2Rα MoAb, correlated with the suppression of the class-I-non-restricted cytotoxic activity of NK and T cells by these MoAbs. When tyrosine phosphorylation was inhibited by herbimycin A and genistein, the cytotoxic activities of NK and T cells were nearly completely suppressed. In addition, the tyrosine phosphorylation of JAK3 by IL-2 was more prominent in NK cells than in T cells, but JAK1, JAK2, STAT1α, STAT2 and STAT3 were not phosphorylated. These results indicate that the IL-2 signal flows downstream via both ras-dependent and ras-independent pathways and that the superior killing activity of NK cells depends on their high susceptibility to protein tyrosine phosphorylation by IL-2.     

Interleukin-6 induces tyrosine phosphorylation of the Ras activating protein Shc,and its complex formation with Grb2 in the human multiple myeloma cell line LP-1

Like many other cytokines and growth factors, interleukin-6 (IL-6) activates p21^ras. However, the precise biochemical mechanisms inducing this activation are unknown. Therefore, we investigated the effects of IL-6 on some recently identified signaling intermediates, Shc (Src homology and collagen) and Grb2 (growth factor receptor bound protein 2), known to activate p21^ras. In the multiple myeloma cell line LP-1, IL-6 stimulated the tyrosine phosphorylation of Shc in a time- and concentration-dependent manner. This led to the complex formation of Shc with Grb2, an adaptor protein known to relocate a p21^ras-GDP exchange factor, Sos1 (Son-of-sevenless), to the cell membrane. Taken together, these findings suggest that IL-6 might activate the Ras signaling pathway via tyrosine phosphorylation of Shc and subsequent recruitment of Grb2. Further studies will elucidate which of the IL-6 receptor associated non-receptor tyrosine kinases of the Src kinase or Janus kinase family, mediate these effects.     

Multiple signal transduction pathways activated through the T cell receptor for antigen.

The T cell receptor for antigen (TCR) is a multichain complex on the surface of T lymphocytes which binds peptide antigen and transduces a transmembrane signal leading to IL-2 secretion. Engagement of the TCR leads to activation of a tyrosine phosphorylation pathway and a phospholipase C (PLC) pathway leading to activation of protein kinase C (PCK). Currently available data suggest that the primary event in signal transduction is tyrosine kinase activation, since when this pathway is inhibited, PLC activation is blocked and there is no production of IL-2. The nature of the tyrosine kinase which initiates the signaling cascade is currently unknown. The CD4/CD8 associated kinase p56lck clearly plays a role in tyrosine phosphorylation, but it is clearly not the only tyrosine kinase involved. Studies demonstrating physical association of p59lyn with the TCR implicate fyn as an important candidate for the TCR tyrosine kinase. The protein tyrosine phosphatase CD45 also plays a critical early role in signal transduction since in cells where it is deficient, neither tyrosine kinase activation nor later signaling events are seen. The importance of the PLC/PKC pathway is illustrated by the fact that activation of this pathway alone may lead to IL-2 production. However, there may also be other mechanisms which can generate an IL-2 response. Two proteins known to be involved in growth regulation--p21ras and c-raf--have now been shown to be downstream targets of the PLC/PKC pathway.     

Interleukin-7 induces T cell proliferation in the absence of Erk/MAP kinase activity

Interleukin (IL)-7 and IL-2 are important lymphoproliferative cytokines which both use the γc chain as part of their respective receptors. To learn more of their signaling mechanisms a comparison was made of the patterns of intracellular tyrosine phosphorylated proteins induced by these cytokines in the murine T cell line, CT6. Several similarities were revealed in the tyrosine phosphorylated proteins induced. However, a notable subset of proteins of mainly < 60 kDa were only phosphorylated by IL-2. Characterization of the two most prominent bands of this subset, pp54 and pp42, revealed these to contain Shc and p42^MAP/Erk kinase, respectively. Further studies confirmed that IL-7 was unable to induce the phosphorylation of either the p44^MAP/Erk or p42^MAP/Erk or activation of the kinases. Shc is involved in activation of p21^ras, a key event in the signaling cascade, via p72^raf and MEK, leading to MAP/Erk kinase (MAPK) activation. These data indicate that this pathway is not utilized by IL-7 and may not, therefore, be essential for cytokine-driven T cell proliferation. This possibility was supported by studies with the MEK inhibitor PD098059, which had no selective effect on CT6 proliferation induced by IL-2 as compared with IL-7, although the drug completely inhibited MAP/Erk phosphorylation induced by IL-2.     

B cell antigen receptor cross-linking induces tyrosine phosphorylation and membrane translocation of a multimeric Shc complex that is augmented by CD19 co-ligation

The SH2 domain-containing transforming Shc protein has been implicated in mitogenic signaling via several surface receptors through p21^ras. Following tyrosine phosphorylation by either receptor or non-receptor tyrosine kinases, Shc may interact with the adaptor protein Grb2, which is linked to Sos1, a guanine nucleotide exchange factor for human ras. Ligation of the antigen receptor complex on B cells (BCR) is known to activate various intracellular signaling pathways, which may accumulate in mitogenic responses. With respect to the initial steps, the activation of BCR-associated non-receptor tyrosine kinases appears to be indispensible. In this report we show that Shc proteins become tyrosine phosphorylated after BCR ligation on both transformed and normal human B cells. This is accompanied by the association of Shc with Grb2 proteins and a yet unidentified 145-kDa tyrosine phosphorylated protein. Subcellular fractionation revealed that this activation-induced multimeric Shc complex rapidly translocates towards the plasma membrane. Co-ligation of the BCR with the CD19 molecule results in a marked increase of these events, whereas CD19 cross-linking alone does not induce Shc tyrosine phosphorylation or translocation. Thus, in B cells the Shc complex may represent a molecular junction between the BCR and the mitogenic p21^ras cascade.     

Ligands of CD4 inhibit the association of phospholipase Cγ1 with phosphoinositide 3 kinase in T cells: regulation of this association by the phosphoinositide 3 kinase activity

We have previously shown that CD4 ligands inhibit interleukin-2 (IL-2) production and T cell proliferation in human peripheral CD4⁺ T lymphocytes, in an MHC-independent way. Two major pathways implicated in T cell activation are inhibited by binding of CD4 ligands to the CD4 molecule, i. e. Ca²⁺ signaling by phospholipase Cγ1 (PLCγ1), and ERK-2 activation, suggesting a p21^ras inhibition. We have correlated these inhibitions with the disruption of multifunctional complexes containing PLCγ1, p120GAP and Sam68, induced by T cell activation. We report here that T cell activation through the TCR/CD3 induces an association of the phosphoinositide 3 kinase (PI3 kinase) with PLCγ1, both in peripheral CD4⁺ T lymphocytes and the HUT-78 CD4⁺ T cell line. PI3 kinase is present in the multifunctional complexes that we have described previously. Preincubation of human peripheral CD4⁺ T cells and HUT-78 CD4⁺ T cells with gp160 or a peptide analogue of the HLA class II DR molecule precludes the association of PLCγ1 with PI3 kinase. We also demonstrate, using two specific inhibitors of PI3 kinase activity (LY294002 and wortmannin), that this activity plays a key role in the association of PLCγ1 with PI3 kinase. Moreover, we demonstrate the implication of the PI3 kinase activity in the negative signal mediated by HIV gp160 binding to CD4 molecules. We propose that the products of the PI3 kinase are important mediators of the negative signaling induced by the binding of CD4 ligands to the CD4 molecule implicated in the regulation of the formation of multifunctional complexes.     

ras Transfection and expression does not induce progression from tumorigenicity to metastatic ability in mouse LTA cells

Studies testing the ability of a transfected ras oncogene to confer metastatic properties on non-metastatic cells have yielded conflicting results. Most of these studies have used recipient cells at early stages of progression (primary or immortalized, non-tumorigenic lines). In this study we tested the ability of the T24-H-ras oncogene to induce progression of tumorigenic, non-metastatic, murine LTA cells to a metastatic phenotype. Metastatic ability was assessed in complementary assays in two immune-deficient hosts, nude mice (after s.c. injection) and chick embryos (after i.v. injection), to determine if ras transfection affected metastatic properties in hosts lacking an intact immune system. Even with greatly elevated levels of ras p21 protein, pools of ras-transfected cells as well as individual clonal populations remained non-metastatic in both hosts. Serial in vivo passaging did not consistently enhance for either ras expression or metastatic ability. We conclude that expression of an activated ras oncogene in LTA cells does not induce progression from a tumorigenic to a metastatic phenotype. These results are in marked contrast to those obtained for ras expression in most other cell types. High levels of expression of an activated ras oncogene thus do not always promote progression from tumorigenicity to metastatic ability.     

Peptide-specific activation of cytolytic CD4+ T lymphocytes against tumor cells bearing mutated epitopes of K-ras p21

Alterations in the ras p21 protein have been associated with both rodent and human neoplasia. Thus, mutated ras p21 proteins may bear unique antigenic epitopes for immune recognition, such as by T cells, which have been implicated in host antitumor activity. Synthetic peptides that mimic segments of mutated ras p21 have been reported to be immunogenic in mice in vivo, although detailed functional analyses remains undefined. Here, in a murine model, we explored and characterized distinct effector properties of host-derived T lymphocytes reactive to mutated ras peptides, which was consistent with the CD4⁺ T helper type 1 (T_h1) subset. BALB/c mice (H-2^d) were immunized with a purified peptide, 13 amino acids in length, containing the substitution of Gly (G12) to Val (V12) at position 12, which is commonly found in human carcinomas. An αβ T cell receptor-positive, CD3⁺, CD4⁺, CD8^? T cell line was established, which expressed peptide-specific proliferation. Cytokine assays revealed the production of interleukin-2, interferon-γ, tumor necrosis factor and granulocytemacrophage colony-stimulating factor. Moreover, antigen-specific cytotoxicity was demonstrable against: (1) Ia^d-bearing A20 tumor cells incubated with exogenously bound V12 peptide; and (2) A20 tumor cells transduced with the K-ras p21 oncogene encoding the corresponding point mutation. CD4⁺-mediated cytotoxicity was major histocompatibility complex (MHC) class II-restricted, as revealed by the absence of lysis against MHC class II^? P815 targets, inhibition of A20 lysis with anti-Ia^d monoclonal antibodies, and induction of lysis against L cell targets transfected with EαAβ^d. Independent isolation of a second CD4⁺ V12 line revealed a very similar cytolytic and MHC class II-restricted profile. Overall, these data demonstrated that peptide immunization produced a CD4⁺ T_h1 response that specifically recognized tumor cells expressing endogenous activated K-ras epitopes, which may have implications for the development of peptide-based active immunotherapies.     

Engagement of Toll-like receptor 2 enhances interleukin (IL)-17+ autoreactive T cell responses via p38 mitogen-activated protein kinase signalling in dendritic cells

Functional analysis of single Toll-like receptors (TLRs) in vivo is necessary to understand how they shape the ocular inflammation involved in uveitis. In this study we explored the role and mechanisms of TLR-2 agonists on the autoreactive T helper type 17 (Th17) response in experimental autoimmune uveitis (EAU). Treatment by peptidoglycan (PGN), a specific TLR-2 agonist, remarkably increased mRNA levels of Th17-lineage genes interleukin (IL)-17A, IL-21 and RAR-related orphan receptor (ROR)γt and promoted antigen-specific Th17 response in EAU mice. A mixture of PGN and interphotoreceptor retinoid-binding protein peptide (IRBP_161–180) could effectively induce EAU in the absence of complete Freund''s adjuvant (CFA). PGN treatment also enhanced the pathogenic activities of activated antigen-specific Th17 cells in vivo. PGN significantly increased the production of IL-1β, IL-6 and IL-23 of dendritic cells (DCs) and enhanced their ability to promote IL-17⁺ uveitogenic T cells. Enhanced immunostimulatory activities of PGN-DCs depend upon p38 activation. Inhibition of p38 mitogen-activated protein kinase (MAPK) activity dramatically decreased IL-17 gene expression and antigen-specific Th17 responses stimulated by PGN-DCs. Our findings suggest that PGN treatment dramatically promotes the IL-17⁺ uveitogenic T cell responses via enhancing the immunostimulatory activities of DCs. This effect may be mediated, at least in part, by activation of the p38 signalling pathway in DCs.