Rantes activates Jak2 and Jak3 to regulate engagement of multiple signaling pathways in T cells.

The chemokine RANTES (regulated on activation normal T cell expressed and secreted) and its cognate receptor CC chemokine receptor 5 (CCR5) have been implicated in regulating immune cell function. Previously we reported that in T cells, RANTES activation of CCR5 results in Stat1 and Stat3 phosphorylation-activation, leading to Stat1:1 and Stat1:3 dimers that exhibit DNA binding activity and the transcriptional induction of a Stat-inducible gene, c-fos. Given that RANTES and CCR5 have been implicated in T cell activation, we have studied RANTES-induced signaling events in a CCR5-expressing T cell line, PM1. RANTES treatment of PM1 T cells results in the rapid phosphorylation-activation of CCR5, Jak2, and Jak3. RANTES-inducible Jak phosphorylation is insensitive to pertussis toxin inhibition, indicating that RANTES-CCR5-mediated tyrosine phosphorylation events are not coupled directly to Galpha(i) protein-mediated events. In addition to Jaks, several other proteins are rapidly phosphorylated on tyrosine residues in a RANTES-dependent manner, including the Src kinase p56(lck), which associates with Jak3. Additionally our data confirm that the amino-terminally modified RANTES proteins, aminooxypentane-RANTES and Met-RANTES, are agonists for CCR5 and induce early tyrosine phosphorylation events that are indistinguishable from those inducible by RANTES with similar kinetics. Our data also demonstrate that RANTES activates the p38 mitogen-activated protein (MAP) kinase pathway. This is evidenced by the rapid RANTES-dependent phosphorylation and activation of p38 MAP kinase as well as the activation of the downstream effector of p38, MAP kinase-activated protein (MAPKAP) kinase-2. Pharmacological inhibition of RANTES-dependent p38 MAP kinase activation blocks MAPKAP kinase-2 activity. Thus, activation of Jak kinases and p38 MAP kinase by RANTES regulates the engagement of multiple signaling pathways.

Chemokines are 8-to 10-kDa inducible and secreted proteins that comprise the largest mammalian cytokine superfamily. Over 50 chemokines have been identified to date and have been subdivided into four families on the basis of the relative position of their cysteine residues (1)(2)(3). Chemokines were named originally for their ability to act as chemotactic cytokines for functionally mature blood cell types, thereby regulating inflammatory events (4 -9), and have been implicated more recently in the regulation of hematopoiesis (10) and in the clearance of infectious organisms (7). Chemokines can also modulate angiogenesis and tumor growth and inhibit stem cell proliferation (11)(12)(13)(14)(15)(16). Chemokines induce their effects by binding to specific seven-transmembrane-spanning, pertussis toxin-sensitive, G protein-coupled receptors on target cells (10,17,18) that are expressed on a wide range of leukocytes, as well as epithelial cells, endothelial cells, neurons, astrocytes, and smooth muscle cells.
RANTES is a CC chemokine that induces both the migration and activation of specific leukocyte subsets by binding to the receptors CCR1, CCR3, CCR5, and CCR9. A number of studies have examined chemokine-induced signal transduction. There is increasing evidence that RANTES may act as an antigenindependent activator of T cells, stimulating protein tyrosine phosphorylation (19). RANTES-induced T cell activation is apparently mediated via two distinct signal transduction cascades: one linked to recruitment of pertussis toxin-sensitive G proteins and the other to protein-tyrosine kinase activation. RANTES-induced T cell activation apparently requires CD3 expression (20), implying that RANTES may engage the T cell receptor complex as a way of effecting cellular activation. Indeed CCR5 is constitutively associated with membrane raft microdomains (21). RANTES has been show to induce the activation of the tyrosine kinases -associated protein 70 and p125 FAK and their association with paxillin as well as the tyrosine phosphorylation of the related Pyk2 kinase (22,23). RANTES-induced activation of phospholipase D is dependent on GTP-binding proteins (ADP-ribosylation factor(s) and RhoA) mediated by interactions with the receptor-coupled G proteins and not protein-tyrosine kinases (24).
Many cytokines and growth factors mediate their effects by activation of a common signal transduction pathway, the STAT pathway. Binding of the ligand to its specific transmembrane receptor results in receptor aggregation, which may involve single or multiple receptor chains. Receptor aggregation leads to the catalytic activation of receptor-associated cytoplasmic protein-tyrosine kinases, JAKs, and phosphorylation-activation of latent monomeric STAT proteins. In our earlier investigations of RANTES-mediated signal transduction, we demonstrated the activation of Stat1 and Stat3 by RANTES (25). In this study we report that RANTES treatment of human PM1 T cells that express cell surface CCR5 results in the rapid and transient phosphorylation of CCR5 on tyrosine residues and the activation of CCR5-associated Jaks. Furthermore, we provide evidence for the RANTES-dependent association of the Src family kinase, p56 lck , with Jak3. Our data reveal that these RANTES-CCR5-mediated tyrosine phosphorylation events are pertussis toxin-insensitive and therefore are not coupled to G␣ i protein-mediated events. In comparative experiments we show that the amino-terminally modified RANTES proteins, Met-RANTES and aminooxypentane-RANTES (AOP-RANTES), 1 exhibit agonist activity on CCR5 in PM1 cells in the context of tyrosine phosphorylation events with similar kinetics to RANTES.
The p38 mitogen-activated protein (MAP) kinases are serinethreonine protein kinases that are activated by diverse stimuli including physical and chemical stresses and by various hemopoietic and pro-inflammatory cytokines (reviewed in Refs. 26 -30). Signal transduction mediated via the p38 MAP kinase pathway seems to play an important role in regulating inflammatory responses including cytokine secretion and apoptosis in a number of different biological systems. p38 MAP kinase activation is regulated by its phosphorylation on threonine and tyrosine residues. Focal adhesion kinase and Pyk2 kinase are nonreceptor protein-tyrosine kinases that are phosphorylatedactivated upon T cell activation and after stimulation of G protein-linked receptors (31-33). As indicated above RANTES will stimulate the phosphorylation of focal adhesion kinase, the tyrosine kinase -associated protein 70, and the focal adhesion protein paxillin in human T cells (22). Moreover, there is evidence that the focal adhesion kinase-related tyrosine kinase, Pyk2, will activate p38 MAP kinase (34). Indeed, the activation of p38 MAP kinase has been implicated in chemokine-induced responses (35). Viewed together, these observations raise the possibility that RANTES activation of CCR5 in T cells may invoke p38 MAP kinase activation. In this report we provide the first evidence that RANTES-CCR5 interactions result in the rapid phosphorylation of p38 and activation of its catalytic activity. Overall, our data establish that RANTES activation of CCR5 leads to the rapid phosphorylation of distinct signaling intermediates on tyrosine residues that invoke discrete signaling pathways.

EXPERIMENTAL PROCEDURES
Cells and Reagents-Human PM1 T cells expressing CCR5 (36) were obtained from the National Institutes of Health AIDS Research and Reference Reagent Program and maintained in RPMI 1640 medium with 10% fetal calf serum, 100 units/ml penicillin, and 100 mg/ml streptomycin. Recombinant RANTES, Met-RANTES, and AOP-RANTES were provided by Serono Pharmaceutical Research Institute. Polyclonal antibodies against Jak2, Jak3, p38, and CCR5 and a monoclonal antibody against p56 lck were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Polyclonal antibodies recognizing p38, the phosphorylated-activated form of p38, and the phosphorylated-activated form of ATF-2 were obtained from New England Biolabs. Antibodies against MAP kinase-activated protein (MAPKAP)-kinase-2 and phosphorylated tyrosine (4G10) were obtained from Upstate Biotechnology, Inc. The SB203580 inhibitor and pertussis toxin were obtained from Calbiochem.
Cell Lysis and Immunoblotting-Actively growing cells at a concentration of 1 ϫ 10 7 cells/ml were stimulated with RANTES as indicated, and the cells were lysed as described previously (37). Immunoprecipitations and immunoblotting using an enhanced chemiluminescence method were performed as described previously (37).
In Vitro Kinase Assays-In vitro kinase assays for Jaks, p38 MAP kinase, and MAPKAP kinase-2 were carried out as previously described (30,38,39). In some experiments the immunoblot membranes were incubated for 1 h in 1 M KOH at 70°C to select for tyrosine-phosphorylated proteins (37).
Flow Cytometric Analysis of Antibody Binding to Native CCR5 on PM1 Cells-FACScan analyses of CCR1 and CCR5 expression on PM1 cells was performed using monoclonal antibodies against CCR1 (provided by R. Horuk, Berlex Laboratories, Inc.) and CCR5 (National Institutes of Health AIDS Research and Reference Reagent Program) as previously described (40).

RESULTS AND DISCUSSION
The CD4ϩ clonal PM1 cells used in these studies were derived from the human T cell line Hut78 (36). At the onset we determined that PM1 cells express cell surface CCR5 (Fig. 1).
To determine whether RANTES induced the phosphorylation of CCR5 in PM1 cells, CCR5 was immunoprecipitated from lysates from RANTES-treated cells, and the solubilized immunoprecipitate was resolved by SDS-PAGE and then immunoblotted for tyrosine phosphorylation and CCR5. The data in Fig. 2A reveal that CCR5 is phosphorylated on tyrosine residues after RANTES treatment of PM1 cells. It is known that the chemokine monocyte chemotactic protein-1 triggers Jak2 activation and tyrosine phosphorylation of CCR2 in the human monocytic cell line Mono Mac 1 (41) and that RANTES induces tyrosine phosphorylation of CCR5 in CCR5-transfected human embryonic kidney HEK-293 cells and association with Jak1 (42). We therefore examined PM1 cells for RANTES-dependent CCR5 and Jak phosphorylation. In time-course studies when cell lysates from RANTES-treated cells were immunoprecipitated with anti-CCR5 antibodies, resolved by SDS-PAGE, and immunoblotted with anti-Tyr(P) antibodies, phosphorylated bands that were identified subsequently as CCR5 and Jak2 appeared within 1 min of RANTES stimulation (Fig. 2B). Apparently phosphorylated Jak2 rapidly associates with CCR5 in a RANTES-dependent manner.
To determine whether RANTES-CCR5 interactions result in the activation of other Jaks, we immunoprecipitated RANTESinduced cell extracts with different anti-Jak antibodies, resolved the immunoprecipitates by SDS-PAGE and then immunoblotted with anti-Tyr(P) antibodies. Although we were unable to detect Jak1 activation, the results in Fig. 3 show RANTES-inducible Jak 2 and Jak3 phosphorylation. Interestingly, the inclusion of pertussis toxin did not affect RANTESinducible Jak phosphorylation, suggesting that G␣ i protein signaling events are not coupled to Jak activation. In subsequent experiments we examined whether the kinase activity of Jak2 and Jak3 is induced by RANTES. PM1 cells were treated with RANTES, cell lysates were immunoprecipitated with anti-Jak2 or anti-Jak3 antibodies, and in vitro kinase assays were performed on the immunoprecipitates. The data in Fig. 4 indicate that RANTES-inducible phosphorylation of Jak2 and Jak 3 results in activation of their catalytic domains. The rapid phosphorylation-activation of Jak2 and Jak3 and the rapid association of Jak2 with CCR5 suggests that these kinases may effect early receptor tyrosine phosphorylation.
When cell lysates from RANTES-treated cells were immunoprecipitated with anti-Jak3 antibodies and Western blots were developed with anti-Tyr(P) antibodies, we observed a 56-kDa phosphorylated protein that associated with Jak3 in a RANTESdependent manner (Fig. 5). Stripping and reprobing the membrane identified this phosphorylated protein as the Src kinase p56 lck . Moreover, treatment of PM1 cells with the amino-terminally modified RANTES proteins, AOP-RANTES and Met-RANTES, resulted in similar Jak3 tyrosine phosphorylation and p56 lck association (Fig. 5). Clearly, despite the fact that both Met-RANTES and AOP-RANTES inhibit RANTESinduced activities both in vitro (43) and in vivo (44 -47), both exert agonist activities on CCR5 in the context of early tyrosine phosphorylation events. Notably, RANTES-, Met-RANTES-, and AOP-RANTES-dependent association of phosphorylated p56 lck with Jak3 follows ligand-stimulated Jak3 activation.
Because we observe that Jaks are rapidly associated and activated with ligand-stimulated CCR5, followed by the sequential recruitment of p56 lck , the implications are that p56 lck is re- cruited to the activated receptor complex, perhaps via an interaction with Jak3. This may occur through CCR5 interaction with CD4 (48). Indeed, Jak3 may function to phosphorylate p56 lck . Interactions between Src kinases and activated Jak kinases have been reported. Specifically, both Lyn and p59 fyn associate via SH2 domains with interferon ␣-activated Tyk2 in hemopoietic cells (49,50). Furthermore, there is some evidence to suggest that association of p56 lck with the activated interferon-receptor complex influences the antiproliferative action of interferon in T cells (51). It has been reported that Src kinases can associate with the ␣ subunits of heterotrimeric G protein complexes (reviewed in Refs. 52 and 53), and a recent report describes a functional interaction between the folate receptor and the associated signaling molecules Lyn and G␣ i-3 (54). Thus, RANTES-dependent recruitment of p56 lck to CCR5 may result in complex patterns of interactions among signaling molecules that may allow for cross-talk between G proteincoupled signaling cascades and non-G protein-linked cascades.
RANTES-inducible activation of T cells influences their proliferation and differentiation, adhesion molecule expression, and cytokine release (reviewed in Ref. 49). The precise biochemical pathways that determine specific biological consequences are unknown. The preceding provides compelling evidence for protein-tyrosine kinase involvement in mediating RANTES-CCR5 signal transduction. Apparently, non-CCR5-associated protein-tyrosine kinases such as Jaks and p56 lck can be recruited to the activated receptor.
The implications are that membrane-localized protein-tyrosine kinases are recruited to the ligand-stimulated receptor where they are activated and act in concert to initiate intracellular signaling cascades. MAP kinases are included among the signaling kinases regulated by chemokines (50,51) and are known to be activated by phosphorylation on tyrosine and threonine residues. Recently, we reported that the p38 MAP kinase pathway regulates interferondependent gene transcription without affecting DNA binding of Stat proteins, suggesting a cooperation between the p38 MAP kinase pathway and the Jak-Stat pathway in transcriptional regulation (30). Therefore, we examined whether RANTES activation of CCR5 in PM1 T cells leads to p38 MAP kinase activation. In time-course studies, whole-cell lysates from untreated and RANTES-treated PM1 cells were analyzed by SDS-PAGE and immunoblotted with an antibody against the phosphorylated form of p38 MAP kinase. Maximum phosphorylation of p38 occurred at 15 min post-RANTES treatment (Fig. 6A). In subsequent experiments, we demonstrated that the kinase activity of p38 is inducible by RANTES. In vitro kinase assays were performed on whole-cell lysates, from untreated and RANTEStreated cells that had been immunoprecipitated with an anti-p38 antibody, using a glutathione fusion protein encoding for ATF-2 as an exogenous substrate. Stimulation of PM1 cells resulted in the phosphorylation of ATF-2 with maximal p38 kinase activity exhibited at 30 min after RANTES treatment (Fig. 6B).
In vivo, p38 phosphorylates and activates mitogen-activated protein kinase-activated protein kinase-2, which in turn phosphorylates heat-shock proteins (Hsp) 25 and 27 (55). Accordingly, lysates from untreated and RANTES-treated cells were immunoprecipitated with an antibody against MAPKAP ki- FIG. 4. RANTES induction of the kinase activities of Jak2 and Jak3. PM1 cells were either left untreated (Ϫ) or treated (ϩ) with RANTES for the times indicated. Cell lysates were immunoprecipitated with either an anti-Jak2 (A) or an anti-Jak3 (B) antibody. Immunoprecipitates were subjected to an in vitro kinase assay, the proteins were resolved by SDS-PAGE, and phosphorylated Jak2 and Jak3 were detected by autoradiography. A, cells were either left untreated (Ϫ) or treated (ϩ) with RANTES for the times indicated. Cell lysates were resolved by SDS-PAGE and immunoblotted (WB) with an antibody against the phosphorylated form of p38 MAP kinase (pp38). The blot was then stripped and reprobed with an antibody against p38 MAP kinase (p38). B, cells were either left untreated (Ϫ) or treated (ϩ) with RANTES for the times indicated. Cell lysates were immunoprecipitated with an antibody against p38 MAP kinase, and immunoprecipitates were subjected to an in vitro kinase assay using glutathione S-transferase-AFT-2 as substrate. Proteins were resolved by SDS-PAGE, and phosphorylated ATF-2 was detected by immunoblotting with an antiphospho-ATF-2 antibody. C, cells were either left untreated (Ϫ) or treated (ϩ) with RANTES for the times indicated in the presence (ϩ) or absence (Ϫ) of SB203580 (30 min at 10 M). Cell lysates were immunoprecipitated with an antibody against MAPKAP kinase-2 and then an in vitro kinase assay was performed using Hsp25 as substrate. Proteins were analyzed by SDS-PAGE, and phosphorylated Hsp25 was detected by autoradiography. nase-2, and in vitro kinase assays were performed on the immunoprecipitates using Hsp25 as the exogenous substrate. The results in Fig. 6C indicate that MAPKAP kinase-2 is activated by RANTES treatment. The data reveal a concordance between the kinetics of maximal inducible kinase activity of p38 and MAPKAP kinase-2 at 30 min post-RANTES stimulation. Moreover, using a p38-specific inhibitor, the pyridinyl imidazole compound SB203580, we provide evidence that MAPKAP kinase-2 is indeed a downstream effector of the p38 MAP kinase pathway in RANTES-stimulated T cells (Fig. 6C).
The specific role(s) of p38 MAP kinase in RANTES-inducible biological responses remains unknown. A recent report describes a role for p38 MAP kinase in the serine phosphorylation of paxillin and the concurrent disassembly of focal adhesion complexes (56). The implications are that p38 MAP kinase signaling may function to negatively regulate RANTES-inducible T cell activation in the context of disassembling T cell focal adhesions (22). Certainly, the kinetics of RANTES activation of p38 kinase activity are consistent with a role for this MAP kinase in negative feedback inhibition.
In conclusion, our findings provide direct evidence for the RANTES-CCR5-dependent recruitment and activation of distinct protein kinases in T cells: the Jaks, Jak2 and Jak3; the Src kinase p56 lck ; and the MAP kinases p38 and MAPKAP kinase 2. Whereas activation of Jak2, Jak3, and p56 lck requires their phosphorylation on tyrosines, activation of p38 requires both threonine and tyrosine phosphorylations. Furthermore the hierarchical p38 signaling pathway invokes serine phosphorylation of target substrates. Clearly the RANTES-dependent sequestering of different signaling molecules to CCR5 provides for signal integration or reciprocal modulation of interacting signaling pathways. The specific roles of these interacting pathways during RANTES activation of CCR5 remain to be elucidated and are the subject of our ongoing investigations.