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To whom correspondence should be addressed: INSERM Unit 461, Faculté de Pharmacie Paris-XI, 5 rue Jean-Baptiste Clément, 92296 Chatenay-Malabry Cedex, France. Tel.: 33-1-46-83-55-08; Fax: 33-1-46-83-54-96;
* This research was supported in part by INSERM and by research grants from Association pour la Recherche Contre le Cancer Grant 6310 and La Ligue Nationale Contre le Cancer.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. ‡ Supported by a fellowship from La Ligue Nationale Contre le Cancer.
Interleukin (IL)-2 is a major cytokine that controls differentiation and proliferation of T lymphocytes. In this report we characterize an as yet unidentified 97-kDa protein that is a major tyrosine kinase substrate in IL-2-stimulated cells. pp97 was found to associate with the p85·p110 phosphatidylinositol 3-kinase complex, the Src homology 2 (SH2) domain-containing tyrosine phosphatase SHP-2, and the adaptor molecules CrkL and Grb2. We demonstrate that these interactions are directly mediated through the SH2 domains of CrkL, p85, and SHP-2 and through the SH3 domains of Grb2. pp97 was found to mediate the IL-2-induced interaction between p85 and both a phosphorylated and a non-phosphorylated form of SHP-2. In this study we show that pp97 behaves as a docking protein and associates with at least CrkL, p85, and SHP-2 in the same multimolecular complex. We thus characterized pp97 as a new tyrosine kinase substrate in human T lymphocytes which might play a central role in the regulation of several pathways activated by IL-2.
-2 is a major cytokine that controls the transition from G1 to the S phase of the cell cycle and thus the proliferation of antigen-activated T lymphocytes. The high affinity IL-2 receptor (IL-2R) is composed of three subunits, α, β, and γ, the latter being shared with the IL-4, -7, -9, and -15 receptors (
). Only the β and γ chains have been demonstrated to contain a long enough cytoplasmic region to transduce signals, the α subunit solely functioning as an affinity converter. Although the IL-2R does not contain intrinsic enzymatic activity, cell stimulation by IL-2 leads to an important and rapid increase of tyrosine phosphorylation mediated by non-receptor tyrosine kinases that are physically associated with the IL-2Rβ and -γ chains. It has been shown that the first steps of IL-2 signaling depend upon the activation of several tyrosine kinases of the Jak, Syk, and Src families (
). PI3K activated in response to IL-2 is a type I PI3K composed of two subunits, a p85 regulatory subunit (p85) and a p110 catalytic subunit. p85 contains one SH3 domain, two proline-rich motifs, and two SH2 domains (
). Through its multiple possibilities of protein-protein interactions, p85 is responsible for the recruitment of p110 to the membrane and its activation. The lipid kinase activity of PI3K leads to the phosphorylation of phosphatidylinositol on the D3 position of the inositol ring. PI3K was found to be critical for the transduction of anti-apoptotic and proliferative signals as it regulates Bcl-2 expression and c-Myc activation, possibly through a pathway involving Akt/PKB and p70S6 kinases (
). This type of association recruits PI3K to the membrane receptor complex, a required and sufficient event to mediate activation, as recently emphasized by the observation that direct targeting of p110 to the cell membrane results in constitutive activation of PI3K (
). However, the mechanism of activation of PI3K in response to IL-2 remains to be determined since p85 tyrosine phosphorylation is not seen in most cells, in particular in the Kit 225 cell line used in this study, and since the IL-2 receptor does not contain a YXXM motif suggesting the involvement of intermediary molecules.
) reported the tyrosine phosphorylation of the SH2 domain-containing tyrosine phosphatase SHP-2 in response to IL-2. SHP-2 (also called Syp, PTP1D, PTP2C, or SHPTP2) is a ubiquitously expressed 70–72-kDa cytosolic phosphatase that contains two SH2 domains in tandem (
). SHP-2 has been described to transduce positive signals by binding directly to various receptor protein tyrosine kinases such as PDGF-, EGF-, or insulin receptors or to adaptor molecules such as IRS-1 or Grb2 leading to activation of Ras (
). IL-2-induced activation of SHP-2 was shown to be a Jak-independent mechanism probably mediated through Src kinases or a new type of kinase and is strictly dependent upon the expression of the IL-2Rβ and -γ chains (
). CrkL is a member of the Crk family, a group of SH2 and SH3 domains containing adaptor proteins, comprising the two splice products Crk-I and Crk-II and the product of a separate but related gene, CrkL (
). During the course of this previous study we observed a p97–100-kDa protein associated with CrkL and phosphorylated on tyrosine residues in response to IL-2. In this report, we demonstrate that pp97 phosphoprotein participates in the formation of a multimolecular complex containing the signaling molecules CrkL, Grb2, SHP-2, and the PI3K regulatory p85 subunit. pp97 is one of the major early tyrosine kinase substrates in IL-2 signaling, and it directly associates with CrkL, p85, and SHP-2 through their respective SH2 domains. We also provide evidence that pp97 mediates the interaction between p85 and SHP-2 that occurs in response to IL-2. Given its characteristic features, as described here, it is possible that pp97 may be related to phosphoproteins that display similar molecular mass and migration properties and that have been recently described. Indeed, three independent reports have described 97- and 100-kDa phosphoproteins in Bcr-Abl-transformed cells or in response to M-CSF or IL-3 (
). Similar to the pp97 described in our study, these phosphoproteins directly associate with SHP-2 and p85. The apparently central role of pp97 in the formation of a large and unique complex containing CrkL, SHP-2, and PI3K suggests that pp97 might exert an important function at the crossroad of three major signaling pathways triggered by IL-2.
Binding of IL-2 to its high affinity receptor leads to a rapid and important increase in tyrosine kinase activity, responsible for the phosphorylation of various substrates which results in the activation of downstream effectors such as PI3K and SHP-2. In this report, we demonstrate that a common 97-kDa protein that is tyrosine-phosphorylated in IL-2-stimulated T lymphocytes coprecipitates with the PI3K p85·p110 complex, the tyrosine phosphatase SHP-2, and the SH2-SH3-containing adaptor molecules CrkL and Grb2. As evidenced by pull-down and far Western experiments, pp97 interacts directly with the SH2 domains of p85, SHP-2, and CrkL, suggesting that pp97 may be phosphorylated on multiple tyrosine residues located in the specific environments required for recognition by these SH2 domains. Additionally, pp97 was shown to bind to the SH3 domains of CrkL and Grb2, through interactions which seem to be mediated by different mechanisms as the CrkL-SH3-pp97 association can be disrupted by phenyl phosphate whereas the Grb2-SH3-pp97 interaction cannot. These results indicate that the CrkL SH3 domains do not directly recognize pp97 and that this interaction requires an intermediary molecule that itself associates with pp97 through a phosphotyrosine-dependent mechanism. On the other hand Grb2 interacts with pp97 independently of phosphotyrosine residues. This interaction may be direct, although the presence of an intermediary molecule between Grb2-SH3 domains and pp97 could not be excluded since far Western blotting with the Grb2-SH3 domains did not evidence a direct interaction with pp97. Furthermore, pp97 is only detected through its phosphorylation, and the lack of specific antibodies against pp97 does not allow investigations of a possible constitutive association of non-phosphorylated p97 with SH3 domains containing proteins.
Since the identity of pp97 has not yet been established, the question of its relationship with other known proteins can only be addressed indirectly. In particular, the recently described SIRP/SHPS SHP-2-associated transmembrane glycoproteins share similar molecular mass and are heavily phosphorylated in response to growth factor stimulation (
). In cell fractionation experiments, however, we found that pp97 is mainly observed in the cytosol and thus is unlikely to be a transmembrane protein (not shown). Furthermore, unlike the SIRP/SHPS-glycosylated transmembrane protein family, the migration profile of pp97 was not modified by treatment withN-glycosidase. Finally, pp97 is not recognized, in Kit 225 cells, by two different antibodies kindly provided by A. Ullrich against murine SIRP (not shown). Gab1, another adaptor protein involved in EGF and insulin signaling has also been recently identified that shares some properties with pp97 (
), Gab-1 migrates at a significantly higher apparent molecular mass, closer to 115 kDa, than pp97 does and appears poorly expressed in lymphoid tissues. Additional experiments clearly excluded the possibility that pp97 be one of the already known tyrosine kinase substrates involved in IL-2 signaling such as Vav or STAT5 (not shown).
Between 30 s and 2 min of IL-2 stimulation pp97 is maximally tyrosine-phosphorylated and is detected as a broad band of 90–95 kDa apparent molecular mass and undergoes a migration shift after 2 min. Taken together with its association with the SH2 domains described above, this characteristic migration pattern is reminiscent of similar molecular weight phosphoproteins recently described in other systems (
) characterized a similar p97 phosphoprotein in Bcr-Abl-transformed and in IL-3-stimulated murine hematopoietic cells. It is thus possible that the pp97 described here, in human IL-2-stimulated cells, may be related to these. However, an answer to this question will have to wait until pp97 is identified or specific antibodies obtained.
As shown in several figures in this report, the tyrosine phosphorylation of pp97, as seen in anti-phosphotyrosine Western blotting, is maximum around the 2-min time point, then declines progressively. In parallel experiments we have also observed that when cells were stimulated by IL-2 in the presence of the phosphatase inhibitor pervanadate, the phosphorylation of pp97 was greatly increased (not shown). Although we did not address this question specifically, it is tempting to speculate that SHP-2, which associates with pp97, might be responsible for its dephosphorylation. Another characteristic feature of pp97 is a migration shift detectable after 2 min of stimulation. This shift is unlikely to be dependent on tyrosine phosphorylation since it persists while the amount of phosphotyrosine residues in pp97 decreases. The modifications of pp97 migration profile may be due to additional post-translational modifications such as ubiquitination or serine phosphorylation that require further investigation.
We also provide evidence, by peptide competition assays and immunodepletion experiments, that a large proportion of pp97 is simultaneously associated with CrkL, p85, and SHP-2. Furthermore, p85 and SHP-2 were found to associate directly with pp97 through their SH2 domains, whereas the interaction between p85 and SHP-2 is likely to depend upon an intermediary molecule. The observation that this p85-SHP-2 interaction is nevertheless disrupted by a pYXXM peptide, and thus depends upon the p85 SH2, and is only observed in the presence of pp97, led us to propose a hypothetical model (Fig. 10) in which pp97 mediates the interaction between p85 and SHP-2. The tyrosine phosphorylation of SHP-2 has been shown recently, but it is the first time that SHP-2 is reported to interact with other signaling molecules in response to IL-2. The function of SHP-2 tyrosine phosphorylation is not yet understood and may lead to SHP-2 activation or may be a required event to mediate protein-protein interactions. Regarding IL-2 signaling, it is not yet known if SHP-2 phosphatase activity is required or if it functions solely as an adaptor molecule. We provide evidence that p85 interacts with both a phosphorylated and a non-phosphorylated form of SHP-2 through a YXXM motif-dependent mechanism. YXXM motifs are present in SHP-2, but there are no indications as to whether they are phosphorylated or not in response to IL-2, and we have found no evidence that such motifs, if phosphorylated, are involved in p85/SHP-2 association. Rather the model would be consistent with our data showing that p85 can interact with SHP-2 independently of its tyrosine phosphorylation. This model also depicts the direct association of pp97 with CrkL and accounts for its presumed role in the association of CrkL with SHP-2. Since we have observed in far Western experiments that CrkL-SH2 did not bind pp97 very efficiently, CrkL may primarily associate with SHP-2 via a very indirect mechanism involving p85 as well as pp97, and the size of this complex probably explains at least in part the low amount of SHP-2 detectable in CrkL immunoprecipitates. On the other hand pp97 interacts with Grb2, and the scheme shown in Fig. 10 only illustrates the Grb2-SH3-pp97 interaction, as supported by our pull-down experimental data. However, Grb2 may also participate in this complex in several other ways, recognizing p85 proline motifs or phosphorylated SHP-2 (
F. Gesbert, C. Guenzi, and J. Bertoglio, personal observations
Grb2/SHP-2 association has already been reported in response to PDGF and was shown to mediate the recruitment of Grb2 to the PDGF receptor leading to the activation of Ras and the mitogen-activated protein kinase pathway (
). The scheme shown in Fig. 10 is based upon pull-down and immunodepletion experiments performed at 5 min following IL-2 stimulation. It is possible, although this has not been tested, that the complex behaves in a dynamic way, with a stoichiometry that could vary with time. Such an hypothesis would be consistent with our observation that at a later stimulation time pp97 appears to be present in larger amounts on p85 and SHP-2 than on CrkL and Grb2 immunoprecipitates.
The multiple protein interaction domains present in this large complex provide several possible means by which it could be recruited to the cell membrane and the IL-2R. Although pp97 is largely a cytosolic protein, a significant proportion can be found in membrane fractions after IL-2 stimulation (not shown). It is of interest that PI3K activity has been reported to associate with IL-2 receptor in response to IL-2 despite the absence of YXXM motifs in either chains of the IL-2R (
). IRS-2, also called 4PS, was initially reported to be tyrosine-phosphorylated in response to IL-4 in hematopoietic cells. The tyrosine phosphorylation of these proteins has recently been described in response to stimulation of the cytokine receptors that share the common γ chain (i.e. IL-2, -4, -7, and -15) (
). Indeed, we observed a pp180 associated to p85 in response to IL-2 that may represent IRS-1 or IRS-2 (Fig. 1, lane 4 and Fig. 8A, lane 8). Additionally it has been demonstrated that IRS-2 may serve as an alternate pathway to activate PI3K in response to erythropoietin (
). Not withstanding the possible role of IRS-1 or IRS-2, it is hypothesized that pp97 may play a central role in the recruitment of PI3K to the IL-2 receptor or to the membrane. Inin vitro experiments, fusion proteins containing the p85-SH2 domains precipitated a large amount of IL-2Rβ. However, IL-2Rβ was not evidenced in p85 immunoprecipitates and pp97 was not detectable in IL-2Rβ immunoprecipitates (not shown). This may be related to relatively labile interactions due to indirect interactions or to a low stoichiometry of these complexes in vivo. Alternatively, PI3K may be recruited to the membrane, where its preferred substrate phosphatidylinositol 4,5-bisphosphate is located, via CrkL interactions with cytoskeletal proteins, which would be consistent with the rapid phosphorylation of the CrkL-associated pp97. Consistent with this is the recent observation that CrkL plays a central role in β1 integrin-mediated adhesion mechanisms and has been shown to tightly interact with focal adhesion-associated proteins (
Our data thus suggest that pp97 may represent a novel example of large docking proteins such as IRS-1, IRS-2, or Gab-1 and may play important functions in several of the signal transduction pathways activated by IL-2. The formal identification of pp97 or the generation of specific antibodies should help in understanding these functions. Furthermore, the identification of pp97 should help in providing new insights in the respective functions of CrkL and SHP-2 in IL-2 signaling.
We thank T. Hori for kindly providing the Kit 225 cell line. We also thank J. Bosq, J. C. Cambier, P.-O. Couraud, B. Druker, P. Ferrara, C. Garbay, A. Kazlauskas, F. Parker, and S. Roche for essential reagents used in this study. We also thank G. Bismuth, J.-L. Zugaza, M. Pallardy, and J. Pierre for critical reading of this manuscript.