Interleukin (IL)-7 Induces Rapid Activation of Pyk2, Which Is Bound to Janus Kinase 1 and IL-7Rα*

Interleukin-7 (IL-7) receptor signaling begins with activation of the Janus tyrosine kinases Jak1 and Jak3, which are associated with the receptor complex. To identify potential targets of these kinases, we examined Pyk2 (a member of the focal adhesion kinase family) using an IL-7-dependent murine thymocyte line, D1. We demonstrate that stimulation of D1 (or normal pro-T) cells by IL-7 rapidly increased tyrosine phosphorylation and enzymatic activity of Pyk2, with kinetics slightly lagging that of Jak1 and Jak3 phosphorylation. Conversely, IL-7 withdrawal resulted in a marked decrease of Pyk2 phosphorylation. Pyk2 was found to be physically associated with Jak1 prior to IL-7 stimulation and to increase its association with IL-7Rα chain following IL-7 stimulation. Pyk2 appeared to be involved in cell survival, because antisense Pyk2 accelerated the cell death process. Activation of Pyk2 via the muscarinic and nicotinic receptors using carbachol or via intracellular Ca2+ rise using ionomycin/phorbol myristate acetate promoted survival in the absence of IL-7. These data support a role for Pyk2 in coupling Jak signaling to the trophic response.

IL-7, 1 a cytokine produced by thymic epithelial cells, is required for normal thymocyte development (reviewed in Ref. 1). This IL-7 requirement occurs at the pro-T1, T2, and T3 stages (and possibly before) and includes activities on cell survival (2)(3)(4) and VDJ recombination (5,6). IL-7 acts by binding to IL-7R␣ (7) and inducing its association with the ␥ c chain (8,9), bringing their intracellular domains together bearing the kinases Jak1 and Jak3 (10,11), respectively. Knockouts of IL-7 (12), IL-7R␣ (13), ␥ c (14,15), Jak3 (16 -18), and Jak1 (19) show similar blocks at a very early stage in T cell development, verifying that each of these components is important in IL-7 signaling. However, the subsequent steps in the signal transduction cascade are unclear. Whereas IL-7 stimulation has been shown to activate pI3-kinase (20) and Src family kinases (21,22) and to activate the transcription factor Stat5 (11), knockouts of these genes (23)(24)(25)(26) have not verified a critical role for these signaling molecules in the pathway downstream of the IL-7 receptor. Thus, the physiologically relevant substrates for the Janus kinases remain to be determined for this pathway.
Because Pyk2 was implicated in other types of signals in hematopoietic cells and neurons we analyzed its role in IL-7 signaling in an IL-7-dependent murine thymocyte line. Pyk2 was shown to be rapidly activated by IL-7 signaling and to mediate survival signals.
Normal thymus was obtained by performing timed breeding of C57BL/6 mice maintained at the NCI-Frederick Cancer Research and Development Center facility (Frederick, MD). Embryonic thymocytes were obtained from embryos at day 14 of gestation.
Antisense Treatments-To block Pyk2 synthesis, antisense oligonucleotides were used. The sequence of antisense was as follows: 5Ј-GGC-TCGGACACCCCAGACAT-3Ј. We used as controls the sense sequence 5Ј-ATGTCTGGGGTGTCCGAGCC-3Ј and an irrelevant sequence of the same nucleotide composition as the sense oligonucleotide 5Ј-TTCGG-TCGAGGTCCGGAGCT-3Ј. Cells were treated with antisense oligonucleotides (20 M) in the absence of IL-7 and then received 50 nM IL-7 12 h later. Cell viability was assessed at different times thereafter.
Immunoprecipitation and Immunoblotting-After washing twice in RPMI 1640, D1 cells were cultured in IL-7-free medium for 12 h and stimulated with recombinant murine IL-7 (50 ng/ml) for various times. The reaction was stopped by adding ice-cold phosphate-buffered saline in the presence of 100 M Na 3 VO 4 , and cells were immediately pelleted and lysed in ice-cold buffer containing 150 mM Nacl, 50 mM Tris-Hcl, pH 7.4, 1% Nonidet P-40 (or 1% digitonin for coimmunoprecipitation as-says), 100 M Na 3 VO 4 , 1 mM NaF, 1 mM phenylmethylsulfonyl fluoride, 10 g/ml leupeptin, 10 g/ml aprotinin. Cell lysates were clarified by centrifugation.
For immunoprecipitation, cell lysates were incubated for 2 h in the presence of rabbit anti-Pyk2 antiserum (Upstate Biotechnology), rabbit anti-Jak3 (Upstate Biotechnology), or mouse monoclonal anti-Jak1 (Transduction Laboratories). Immune complexes were captured using protein G/protein A-agarose (Roche Molecular Biochemicals) for 1 h, then washed three times with ice-cold lysis buffer, and denatured in 2ϫ Laemmli sample buffer. Cell lysates were resolved by 8% SDS-polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. Tyrosine phosphorylation or the presence of immunoprecipitated proteins was detected by protein immunoblotting. Phosphotyrosine was detected using the specific antibody 4G10 (Upstate Biotechnology) followed by incubation with a peroxydase-conjugated anti-mouse IgG1 as a secondary antibody (Amersham Pharmacia Biotech). Membranes were stripped and reprobed using monoclonal antibodies to Pyk2, Jak1 (Transduction Laboratories), Jak3 (Upstate Biotechnology), or mouse monoclonal anti-IL-7R␣. Bound antibodies were visualized using the chemiluminescence ECL system (Amersham Pharmacia Biotech).
Analysis of PYK2 Kinase Activation-Cell lysates were immunoprecipitated with polyclonal anti-Pyk2, anti-Jak1, or anti-Jak3 antibody, and the immunoprecipitates were subjected to an in vitro kinase assay in the presence of the biotinylated exogenous substrate, poly(Glu-Tyr) (Pierce), previously fixed for 30 min in avidin-coated plates. The kinase reaction was conducted for 15 min at 37°C in a buffer containing 20 mM Tris, pH 7.5, 10 mM MgCl 2 , 10 mM MnCl 2 , 2 mM ATP. The plates were then washed five times with phosphate-buffered saline containing 1% bovine serum albumin. Phosphorylation of peptide was detected by using 1/500 diluted peroxydase-labeled anti-PY20 and 3,3Ј,5,5Ј-tetramethylbenzidine as the peroxydase substrate. The OD was measured at 450 nm. A standard phosphopeptide was run in the same plate.

IL-7 Stimulation Induces Tyrosine Phosphorylation of
Pyk2-To evaluate the role of Pyk2 in the IL-7 signaling pathway, we first analyzed whether Pyk2 was phosphorylated on tyrosine following stimulation of the D1 cell line by IL-7. D1 cells were cultured without IL-7 for 12 h and then stimulated with IL-7 for various times. Immunoblotting using anti-phosphotyrosine antibody revealed that phosphorylation of Pyk2 underwent a marked, and transient, enhancement. As shown in Fig. 1A, the Pyk2 phosphorylation level increased within 2 min and declined 5 min after IL-7 stimulation. In the absence of IL-7, a basal level of Pyk2 tyrosine phosphorylation was also always evident upon longer exposure of the membrane. Equal amounts of Pyk2 protein were immunoprecipitated at all time points as indicated by the loading control (Fig. 1A). In parallel, using the same cell extracts, phosphorylation of Jak3 (Fig. 1B) and Jak1 (Fig. 1C) was examined and showed a somewhat different pattern compared with Pyk2. Jak1 and -3 showed less basal phosphorylation in the absence of IL-7 than Pyk2. Phosphorylation of Jak1 and -3 was slightly faster than Pyk2 and did not show the decline after 2 min observed with Pyk2.
We analyzed whether the PYK2 signaling pathway that we observed in the D1 cell line also occurred in normal pro-T cells. Data presented in Fig. 2 demonstrates that PYK2 protein is highly expressed in day 14 embryonic thymus, which contains a mixture of IL-7-responsive pro-T1 and T2 cells (4). Furthermore, IL-7 induced a rapid tyrosine phosphorylation of PYK2 in vitro after depriving the cells of IL-7 for 6 h (Fig. 2). These data verify that PYK2 is involved in IL-7 signaling during T cell development.
We examined the state of Pyk2 phosphorylation during IL-7 withdrawal and observed (Fig. 3) that by 2 h after IL-7 withdrawal, phosphorylation had declined to a basal level that persisted until 18 h. Tyrosine phosphorylation of Jak1 also decreased very rapidly, disappearing by 4 h, whereas phosphorylation of Jak3 declined much more slowly (Fig. 3). Thus the initial decline in Pyk2 phosphorylation would be compatible with its being a direct substrate of Jak1, which showed a parallel decline in phosphorylation following IL-7 withdrawal. The data also suggest that a kinase other than Jak1 maintained Pyk2 phosphorylation between 2 and 18 h following IL-7 withdrawal.
The enzymatic activity of Pyk2 was measured using in vitro kinase assays with poly(Glu-Tyr) as an exogenous substrate. As shown in Fig. 4A, stimulation by IL-7 induced a marked increase of the kinase activity of Pyk2. The onset of this in- FIG. 2. PYK2 is activated in embryonic thymuses. Embryonic thymuses were used at day 14 of gestation. Thymocytes were obtained after treatment of thymic lobes with collagenase 0.2%, and deprived of IL-7 for 6 h in medium containing 2% fetal bovine serum in the absence of IL-7. Cells were stimulated with IL-7 for 3 min, and cell extracts were prepared. PYK2 immunoprecipitates (IP) were blotted with antiphosphotyrosine. The membranes were stripped and reprobed with anti-PYK2 monoclonal antibody for the total protein level. crease correlated with the increased tyrosine phosphorylation of Pyk2 (Fig. 1A). Both Jak1 and Jak3 showed similar increases in kinase activity following IL-7 stimulation as shown (Fig. 4), which correlated with their tyrosine phosphorylation status (Fig. 1, B and C).
Selective Association of Pyk2 with Jak1 and IL-7R␣ Chain-Early events in IL-7 signaling involve cross-linking IL-7R␣ with ␥ c whose intracellular domains are associated with Jak1 and Jak3, respectively. To determine whether there was a physical association of Pyk2 with these other signaling components, we examined whether Pyk2 coimmunoprecipitated with IL-7R␣, Jak3, or Jak1. We found that Pyk2 was constitutively associated with Jak1 before and after IL-7 stimulation (Fig. 5). In contrast, no association of Pyk2 with Jak3 was detected. We also observed that IL-7R␣ coimmunoprecipitated with Pyk2 (Fig. 5); a basal association was observed, and then an increase occurred upon IL-7 stimulation. This would be consistent with Pyk2 directly binding Jak1, the latter increasing its association with IL-7R␣ following IL-7 stimulation.
Whether PYK2 is a direct enzymatic substrate of Jak1 remains to be investigated. Our present data suggest that Pyk2 could be a possible target of Jak1 because the two are physically associated and because phosphorylation of Pyk2 slightly lags that of Jak1 (Fig. 1).
The Functional Role of Pyk2 on the D1 Cell Line-The D1 cell line is dependent on IL-7 for survival, IL-7 withdrawal resulting in complete cell death by 30 h. Up to 18 h after IL-7 withdrawal, D1 cells can be rescued by the readdition of IL-7. To determine whether Pyk2 played a role in cell survival, the effect of antisense oligonucleotide against Pyk2 was compared with control oligonucleotides (sense sequence and a control irrelevant sequence). D1 cells were treated with oligonucleotides in the absence of IL-7; then after 12 h, IL-7 was added. Although IL-7 readdition could rescue sense-and controltreated cells from death, Pyk2 antisense markedly inhibited cell survival for at least 48 h (Fig. 6A). Following antisense Pyk2, we observed about 50% dead cells after 18 h and over 70% after 30 h (compared with 9 and 6% for the sense control oligonucleotide). As shown in Fig. 6B the level of Pyk2 is inhibited in the presence of antisense Pyk2, whereas the sense sequence does not have an effect. These data indicate that Pyk2 is critical for the survival of D1 cells.
Because Pyk2 is also activated by G protein-coupled recep- tors, we examined whether the IL-7 survival signal could be mimicked by other stimuli that also activate Pyk2. Carbachol, a pharmacological agonist of the muscarinic and nicotinic receptors, improved cell survival in the absence of IL-7 (Fig. 7A) and induced tyrosine phosphorylation of Pyk2 (Fig. 7B).
Ca 2ϩ influx has also been shown to activate Pyk2 in many cell types. Therefore calcium ionophore (together with phorbol myristate acetate) at suboptimal concentrations was tested and found to improve cell survival in the absence of IL-7 (Fig. 7C) as well as inducing Pyk2 phosphorylation (Fig. 7D). The kinetics of activation using ionomycin and phorbol 12-myristate 13acetate were similar to that observed in the presence of IL-7 with a peak of phosphorylation observed by 2-3 min, which decreased afterward.
In addition, the use of either carbachol or suboptimal concentrations of ionomycin/phorbol 12-myristate 13-acetate did not show any significant increase of [ 3 H]thymidine incorporation (data not shown). These data suggest that PYK2 is involved in cell survival rather than in proliferation, but it remains possible that it also has replication activities that cooperate with other IL-7-induced activities. Thus, two alternative methods of activating Pyk2 partially mimic the IL-7 survival stimulus in D1 cells, suggesting that one survival pathway from the IL-7 receptor incorporates Pyk2, but that other pathways are also required for long term survival.

DISCUSSION
Protein-tyrosine kinases have been shown to mediate a variety of cytokine signals. The present study implicates the protein-tyrosine kinase Pyk2 in the pathway leading from the IL-7 receptor to cell survival. Following IL-7 stimulation, Pyk2 became tyrosine-phosphorylated and increased its enzymatic activity. Blocking Pyk2 synthesis inhibited cell survival, and activating Pyk2 (by alternative stimuli) promoted survival. Pyk2 was physically associated with Jak1 and IL-7R␣.
The initial events in IL-7 receptor signaling are presumed to resemble that of other cytokine receptors sharing ␥ c (44), IL-2 (45), and IL-4 (46) receptors being the best studied. Thus, IL-7 would first bind to the IL-7R␣ chain and then that complex would attract ␥ c , which carries Jak3 on its intracellular domain. Jak3 would then phosphorylate IL-7R␣, and Jak1, which is associated with it, activating the latter. The phosphorylated IL-7R␣ chain would then bind other proteins, such as Stat5, via their SH2 domains, and they would in turn become phosphorylated by Jak1 and Jak3. Phosphorylated Stats would then translocate to the nucleus and serve as transcription factors.
The preceeding model of IL-7 receptor signaling is partly based on presumed parallels in the IL-2 and -4 pathway, as well as on several other lines of evidence. IL-7 has been shown to activate Jak1, Jak3, and Stat5 (11). Jak3 indeed appears to be essential for IL-7 signaling, because its knockout (16 -18) closely resembles that of IL-7 (12) and IL-7R␣ (13). Moreover, human Jak3 deficiency (47) resembles an IL-7 deficiency in mice. A Jak1 requirement is also verified by its knockout, which shows a defect in IL-7 signaling (19) (although there is evidence against a Jak1 requirement for IL-2 (48) or IL-4 (49) signaling). On the other hand, Stat5 deficiency (26) does not show a defect in the IL-7 receptor signaling pathway. What then are the substrates of the Jaks in the IL-7 receptor system? Whereas other Stats may be substrates, our current findings suggest another type of potential Jak substrate, the proteintyrosine kinase Pyk2, which we show undergoes a rapid rise in tyrosine phosphorylation following IL-7 stimulation. We suggest that Pyk2 could be a Jak1 (rather than Jak3) substrate because Jak1 and Pyk2 were shown to be physically associated (Fig. 3), although the basal phosphorylation of Pyk2 (in the absence of IL-7) could be because of a different kinase.
Pyk2 is related to p125-Fak and is expressed primarily in cells of the neuronal and hematopoietic lineages (28). The knockout of Fak is embryonic lethal (50); its role being thought to include signaling from integrins, turnover of focal adhesion, and anti-apoptotic effects of cell contact with the extracellular matrix in the adult nervous system. Pyk2 displays a high degree of sequence similarity with Fak over most of its se- quence, and perhaps its anti-apoptotic action in the IL7 receptor pathway is similar to that of Fak. Pyk2 was shown to be phosphorylated on tyrosine after ligand binding to G proteincoupled receptors or treatment with agents that elevate intracellular Ca 2ϩ concentration (28,37) stimuli that partly mimicked the trophic action of IL-7 in the current studies.
Whereas we observed a physical association of Pyk2 with Jak1, others have previously observed that Pyk2 can also associate with other kinases and other types of proteins, possibly via parts of its noncatalytic domains including its phosphotyrosine residues being recognized by the SH2 domains of other proteins. Pyk2 does not itself contain SH2 or SH3 binding domains, but it contains the cannonical binding site (Tyr-881) for the SH2 domains of Src and Grb2. Associations of Pyk2 with SH2-containing Src kinases and Grb2 were reported (31,37,(51)(52)(53).
Recent studies concluded that, in interferon ␥ or IL-2 signaling, Pyk2 was preassociated with Jak2 (35) or with Jak3 (34), respectively. Those studies did not detect significant complexing of Jak1 with Pyk2, as we observe here, nor did we detect significant Pyk2⅐Jak2 or Pyk2⅐Jak3 complexes. Perhaps cell types differ in this regard, or introducing proteins by overexpression (in those studies) influences these protein interactions.
We also noted an increasing association of Pyk2 with IL-7R␣ following IL-7 stimulation. This association could be explained by a preassembled Jak1⅐Pyk2 complex becoming associated with IL-7R␣ via Jak1, even a third party could be involved, or could also represent a direct association of Pyk2 with IL-7R␣. There is a region in Pyk2 (54) that is homologous to the region in Janus kinases (4.1/JEF) (55) that directs their association with cytokine receptors. If there is a direct Pyk2⅐IL-7R␣ complex, perhaps it positions Pyk2 so that it can become phosphorylated.
The trophic action of IL-7 is partly based on the induction of Bcl-2 (4) and partly on effects on Bax; 2 how Pyk2 relates to these or other survival functions has not yet been determined. A number of pathways could be involved downstream of Pyk2. The association of Pyk2 with the Grb2⅐Sos1 complex may link Pyk2 to the small GTPase Ras signaling pathway as well as the signaling pathways of the Rho family GTPases CDC42 and Rac1 (33). These GTPases are regulated by guanine nucleotide exchange factors such as Vav and by GTPases such as Ras (56 -58). In addition, it has been shown that Pyk2 can function as an upstream mediator of the extracellular signal-regulated kinase and/or JNK signaling pathways. Pyk2-induced activation of Src kinase is necessary for phosphorylation of Shc and p130 Cas , and their association with the adaptor proteins Grb2 and Crk, respectively, and for the activation of extracellular signal-regulated kinase and JNK cascades (59). The Grb2⅐Sos complex connects Pyk2 to the activation of extracellular signalregulated kinase, whereas adaptor proteins p130 Cas and Crk link Pyk2 with the JNK pathway (59). A recent report also indicated the activation of p38 MAPK by overexpression of Pyk2, and the use of a dominant negative mutant of MKK3, an upstream component of p38, inhibits Pyk2-induced p38 MAPK activity (60). Additionally, several recently defined cellular proteins, such as the GTPase-activating protein for RhoA and Cdc42, amino-terminal domain-interacting receptors, and Pap proteins, were also shown to constitutively associate with Pyk2 and to be potential target substrates for Pyk2 (61-64). Whether they directly or indirectly participate in Pyk2 downstream signaling in D1 cells remains to be investigated.