(Received for publication, March 7, 1995; and in revised form, July 26, 1995)
From the
Protein tyrosine phosphorylation and thus dephosphorylation are part of the interleukin (IL)-11 response in mouse 3T3-L1 cells. We report here for the first time the involvement and interactions of the SH2-containing protein tyrosine phosphatase Syp in the IL-11 signal transduction pathway. Addition of IL-11 to 3T3-L1 cells resulted in an increase in the tyrosine phosphorylation of Syp. When cell lysates were precipitated with glutathione S-transferase fusion products of Syp, the C-terminal SH2 domain of Syp was shown to precipitate several proteins of 70, 130, 150, and 200 kDa that were tyrosine phosphorylated in response to IL-11. Reciprocal immunoprecipitation experiments showed that Syp was inducibly associated with both gp130 and Janus kinase 2 (JAK2). A phosphopeptide containing the sequence for a potential Syp binding site (YXXV) was used to compete with the associations of Syp with gp130 and JAK2. The phosphopeptide reduced the Syp association with both gp130 and JAK2. To summarize, Syp has multiple interactions in IL-11 signal transduction. In addition to the IL-11-induced tyrosine phosphorylation of Syp, Syp coprecipitated with gp130, JAK2, and other tyrosine-phosphorylated proteins in response to IL-11. These findings may have extensive significance to IL-11 and related cytokine signal transduction, suggesting new pathways and mechanisms.
IL-11 ()is a stromal fibroblast-derived cytokine
involved in lymphopoiesis and hematopoiesis and is also important in
proliferation and differentiation of a variety of cell
types(1) . IL-11 binds to a specific receptor, which requires
the signal transducer gp130 for transmission of IL-11 signals into the
cell(2) . gp130 is the common signal transducer for
IL-11(3) , ciliary neurotrophic factor(4) , leukemia
inhibitory factor(4) , IL-6(5) , and oncostatin
M(6) . It has been shown that gp130 associates with Janus
kinase 2 (JAK2) constitutively and that JAK2 is activated upon IL-11,
IL-6, ciliary neurotrophic factor, oncostatin M, or leukemia inhibitory
factor stimulation of cells(7, 8) . JAK2 is a 130-kDa
tyrosine-phosphorylated kinase whose response to IL-6 is blocked by
point mutations or deletions of the membrane proximal portion of gp130 (9) .
Tyrosine phosphorylation and dephosphorylation is a common means for cytokine-mediated intracellular signal transduction. Induction with IL-2(10) , IL-6(11) , IL-7(12) , or IL-11 (13) results in the activation of kinases leading to protein tyrosine phosphorylation. IL-6 also stimulates tyrosine dephosphorylation by unspecified phosphatases leading to the reduction of tyrosine phosphorylation(14) . Recently, it was reported that ciliary neurotrophic factor can induce the tyrosine phosphorylation of the protein-tyrosine phosphatase Syp (also named SH-PTP2, PTP1D, and PTP2C)(15) . Syp is a recently discovered protein-tyrosine phosphatase containing two SH2 domains(16, 17) . Syp is ubiquitously expressed and found to be involved in platelet-derived growth factor (PDGF) and epidermal growth factor signal transduction by direct association with the activated receptors(18, 19, 20) . Syp is also involved in insulin receptor substrate 1 signal transduction(21, 22, 23) . In addition, Syp functions as an adaptor protein for the signal transduction from PDGF through GRB2-Sos, leading to the activation of the Ras signaling pathway(24) .
In this study, we identify and characterize a new member of the IL-11 signal transduction mechanism, Syp, and determine its interactions with established components of this pathway, gp130, and JAK2. Both gp130 and JAK2 have potential binding sites for Syp(25, 26) . We suggest that the interactions of Syp and these components are integral parts of the IL-11 signaling pathway.
Figure 1: IL-11 time course and concentration effect on Syp tyrosine phosphorylation. 3T3-L1 cells were stimulated with IL-11 (500 ng/ml) for various amounts of time (A) or various concentrations of IL-11 (B) as indicated. The lysates were immunoprecipitated with anti-Syp, and the immunoblot was probed with clone 4G10-derived antiphosphotyrosine (APT). Blots were stripped and reprobed with anti-Syp as a control (lower bands).
Figure 2:
Purified GST-Syp proteins. Syp cDNA was
cloned into the pGEX-K2 vector and induced with
isopropyl-1-thio--D-galactopyranoside in E.
coli. Approximately 1 µg of glutathione-Sephadex-purified
GST-Syp protein was loaded onto each lane, separated by
SDS-polyacrylamide gel electrophoresis, and stained with Coomassie
Blue.
Figure 3: C-terminal of SH2 domain GST-Syp precipitates tyrosine-phosphorylated proteins from cells stimulated with IL-11. 3T3-L1 cells were either stimulated with IL-11 (500 ng/ml, 7 min) or unstimulated before being lysed and precipitated with various combinations of GST-Syp-SH2 domains as indicated. Protein bands were resolved with clone 4G10 antiphosphotyrosine antibodies. Arrows indicate induced tyrosine-phosphorylated protein bands with approximate molecular masses of 200, 150, 130, and 70 kDa.
Figure 4: gp130 immunoprecipitation coprecipitates Syp. Anti-gp130 was used to immunoprecipitate lysates from 3T3-L1 cells treated with and without IL-11 (500 ng/ml, 3 min). The resulting immunoblot was screened with clone 4G10 antiphosphotyrosine (A), anti-Syp (B), and anti-gp130 (C).
To investigate JAK2 association with Syp, JAK2 immunoprecipitations were carried out. JAK2 immunoprecipitations coprecipitated Syp transiently in response to IL-11 with the highest level of association at 3 min (Fig. 5A), which corresponds to the peak level of Syp tyrosine phosphorylation (Fig. 1). JAK2 was shown to be present in equal amounts in each lane in the same experiment (Fig. 5B). The association between Syp and JAK2 appears to be constitutive in unstimulated cells but can be further induced by addition of IL-11.
Figure 5: Syp is transiently associated with JAK2 in response to treatment of cells with IL-11. 3T3-L1 cells were treated with IL-11 (500 ng/ml) for various amounts of time (min) as indicated and immunoprecipitated with anti-JAK2. The immunoblots shown have been resolved with anti-Syp (A) and anti-JAK2 (B).
Figure 6: Syp associates with gp130 and JAK2, but association with gp130 is disrupted in the presence of the YSTV peptide. Unstimulated and stimulated 3T3-L1 cells (IL-11 500 ng/ml, 3 min) were lysed and immunoprecipitated with anti-Syp. Two of the three equally stimulated batches of cells were treated with 1 µM of either the unphosphorylated YSTV peptide or the tyrosine-phosphorylated Y*STV-containing peptide as indicated. A representative immunoblot was tested with clone 4G10 antiphosphotyrosine (A), anti-JAK2 (B), anti-gp130 (C), and anti-Syp (D). Arrowheads indicate induced tyrosine-phosphorylated protein bands seen with antiphosphotyrosine probing of the immunoblot. Approximate molecular masses of marked protein bands are 200, 150, 130, and 80 kDa.
The Syp immunoprecipitation was assayed for the coprecipitation of JAK2 and showed that IL-11 induced the increased association of JAK2 to Syp (Fig. 6B). Further analysis of the Syp immunoprecipitation involved testing for the coprecipitation of gp130. gp130 was seen coprecipitating with Syp when the cells were induced with IL-11 (Fig. 6C). In addition, peptides corresponding to the putative Syp binding site (YSTV) on gp130 were used in a peptide competition experiment to study this association. Immunoprecipitation of Syp in the presence of the unphosphorylated YSTV-containing peptide reduced the association of Syp to gp130 (Fig. 6C, lane 3) but did not decrease the induced association of JAK2 to Syp significantly (Fig. 6B, lane 3). The phosphorylated YSTV-containing peptide blocked significant association of JAK2 and gp130 to Syp. A control assay with anti-Syp was done on the same membrane to test for the amount of Syp with each variable and demonstrated equal quantities of Syp immunoprecipitated (Fig. 6D). Peptide addition before or after lysis coupled with the continued peptide presence throughout the immunoprecipitation gave similar results (data not shown).
Our current understanding of IL-11 signal transduction is that after IL-11 joins with its cognate receptor, it then complexes with the signal transducer gp130. gp130 increases its association with JAK2, and JAK2 shows an increase in activity and tyrosine phosphorylation (7) along with an increase in the tyrosine phosphorylation of gp130(3) . IL-6, which has a similar mechanism to IL-11, causes dimerization of gp130(5) , and it has been shown with IL-6 that gp130 tyrosine phosphorylation occurs after JAK2 activation(28) . JAK2 is a relatively large (130 kDa) tyrosine-phosphorylated cytoplasmic signaling protein involved in many signaling pathways(29) . JAK2 is known to activate signal transducers and activators of transcription proteins (30) and associate with receptors(31, 32) . In the present study, we have identified a component of the IL-11 pathway that may be equally pervasive. We have shown that the ubiquitously expressed phosphotyrosine phosphatase Syp associates with gp130 and JAK2 and that IL-11 stimulation increases these associations. The association of Syp with gp130 was reduced with the peptide YSTV and blocked with the YSTV-containing phosphopeptide. The YSTV peptide sequence was derived from gp130 and is similar to sequences known to bind Syp(11, 33) . This finding suggests that Syp is involved in the interaction with the YSTV site on gp130. The unphosphorylated YSTV peptide, however, does not compete away JAK2 association with Syp as much, suggesting that the Syp-JAK2 association is unique and may not require the gp130-Syp association. The induced association of JAK2 and Syp has not been previously presented, although JAK2 has several potential Syp binding sites(26, 33, 34) . The association of Syp in all pathways that activate JAK2 such as erythropoietin, growth hormone, prolactin, granulocyte colony-stimulating factor, IL-3, IL-5, and granulocyte-macrophage colony-stimulating factor will require further investigation(35) .
Experiments with GST-Syp fusion proteins indicate specific IL-11-induced associations between the C-terminal SH2 of Syp and tyrosine-phosphorylated proteins. There are reports of the C-terminal SH2 domain having a higher affinity to the PDGF receptor (36) and the N-terminal SH2 domain binding preferably to the erythropoietin receptor (37) . The differences seen in the tyrosine-phosphorylated protein bands precipitated between the N/C- and C-terminal GST-SH2-Syp precipitations may indicate a role for the N-terminal GST-SH2 domain of Syp of increasing the specificity and affinity of binding of Syp toward associated proteins. This synergistic role for the two Syp SH2 domains has been reported recently in response to PDGF(36) . Tyrosine-phosphorylated protein bands of the size of JAK2 (130 kDa) and gp130 (150 kDa) were seen precipitated with GST-Syp in addition to 90- and 200-kDa proteins of undetermined origin. The large protein may be the insulin receptor substrate-1 docking protein, which binds with Syp (21) .
The time course of tyrosine phosphorylation for Syp differs from the results found for A31 mouse fibroblasts induced by PDGF(16) . Tyrosine phosphorylation of Syp may correspond directly to activation (19) or protein targeting(38) , and either may be the case here. Since activated Syp has not been found to be phosphorylated during insulin receptor substrate-1 activation(21) , tyrosine phosphorylation may not be required for activation. Syp-gp130 or Syp-JAK2 interactions may lead to activation of Syp as does a portion of the PDGF receptor and Syp (39) .
The IL-11-inducible binding of Syp to both gp130 and JAK2 was shown by reciprocal immunoprecipitations. It was shown previously that the 61 amino acids proximal to the trans-membrane region on gp130 are required for the mitogenic signal through gp130(9) . The suggested Syp binding site on gp130 (15) is not included in this region (25) and therefore not required for the mitogenic signal. A possible function of Syp-gp130 binding is the localization of Syp to the membrane to allow easier association with JAK2 since IL-11 induces association of gp130 and JAK2 (38) . The necessary site for gp130 and JAK2 association with each other is within the membrane proximal region of gp130 that is required for the mitogenic response(9) . Further evidence of Syp being involved in the mitogenic response is a series of microinjection experiments by Xiao and others(41) , which showed that the functional inhibition of Syp also inhibited the mitogenic signal. Another potential mitogenic pathway for Syp is as an adaptor protein for the Grb2-Sos-Ras pathway in response to PDGF(24) . The induced tyrosine phosphorylation of Shc and other members of the Ras pathway has been shown previously in the gp130 signal transduction pathway(15) .
Incubation of cells and lysates with the unphosphorylated YSTV peptide appeared to significantly reduce the IL-11-induced association of Syp with gp130 but not reduce the JAK2 association with Syp. Phosphotyrosine-independent peptide binding has been reported previously at a lower affinity than the same tyrosine-phosphorylated peptide at sites of SH2 domain interactions(42, 43) . A similar interaction may be occurring here. The affinities of the activated gp130 and YSTV peptide may be similar enough for them to compete and reduce gp130-Syp association. The activated JAK2 affinity for Syp may be stronger and thus less sensitive to competition with the unphosphorylated YSTV peptide. An alternative explanation is that the YSTV peptide bound to gp130 and altered the gp130 configuration or blocked gp130 dimerization and thus reduced its association with Syp.
Binding to Syp is strongly enhanced in phosphopeptides containing
the protein motif Y*XXV(33) , of which JAK2 has two
possible sites (YNSV and Y
YKV) (26) and gp130 has one (YSTV). The presence of these sites and
the subsequent inhibition of Syp binding to gp130 and JAK2 by the
phosphopeptide implies competitive inhibition with the peptide and
suggests possible sites for binding of Syp to gp130 and JAK2. In
vivo peptide addition produced no significant effects, implying
that in vitro peptide interactions alone, such as competition,
suppress association of Syp and gp130 or JAK2.
In summary, the ubiquitous tyrosine phosphatase Syp associates with the signal transducer gp130 and cytoplasmic kinase JAK2. This association increases with IL-11 stimulation and is decreased by a tyrosine-phosphorylated peptide containing the sequence YSTV. A non-phosphorylated peptide, YSTV, affects gp130-Syp but does not affect JAK2-Syp-induced association. These results support a model in which Syp interacts with gp130 and JAK2 independently. Further experiments must be conducted to determine the nature of these interactions.
Addendum-While this work was in review, a paper supporting the importance of the gp130 YSTV sequence for Syp tyrosine phosphorylation was published by Stahl et al.(40) . In this paper, a point mutation (Y to F, of YSTV) of an over-expressing gp130-neurotrophin-3 fusion protein blocked the induced antiphosphotyrosine immunoprecipitation of Syp.