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To whom correspondence should be addressed: Dept. of Microbiology and Immunology, Faculty of Health Sciences, Ben Gurion University of the Negev, P. O. Box 653, Beer Sheva 84105, Israel. Tel.: 972-7-647-7267; Fax: 972-7-647-7626; E-mail: [email protected]
Department of Microbiology and Immunology, Faculty of Health Sciences, and the Cancer Research Center, Ben Gurion University of the Negev, Beer Sheva 84105, Israel
* The work reported herein was supported in part by grants from the Israel Science Foundation, the Israel Academy of Sciences and Humanities, the United States-Israel Binational Science Foundation, the Chief Scientist's office, Israel Ministry of Health, the Israel Cancer Association (ICA) through the ICA friends in Brazil, and the Israel Cancer Research Fund.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.
The Crk adapter proteins are assumed to play a role in T lymphocyte activation because of their induced association with tyrosine-phosphorylated proteins, such as ZAP-70 and Cbl, and with the phosphatidylinositol 3kinase regulatory subunit, p85, following engagement of the T cell antigen receptor. Although the exact mechanism of interaction between these molecules has not been fully defined, it has been generally accepted that Crk, ZAP-70, and p85 interact with tyrosine-phosphorylated Cbl, which serves as a major scaffold protein in activated T lymphocytes. Our present results demonstrate a cell activation-dependent reciprocal co-immunoprecipitation of CrkII and p85 from lysates of Jurkat T cells and a direct binding of CrkII to p85 in an overlay assay. The use of bead-immobilized GST fusion proteins indicated a complex mechanism of interaction between CrkII and p85 involving two distinct and mutually independent regions in each molecule. A relatively high affinity binding of the CrkII-SH3(N) domain to p85 and the p85-proline-B cell receptor-proline (PBP) region to CrkII was observed in lysates of either resting or activated T cells. Direct physical interaction between the CrkII-SH3(N) and the p85-PBP domain was demonstrated using recombinant fusion proteins and was further substantiated by binding competition studies. In addition, immobilized fusion proteins possessing the CrkII-SH2 and p85-SH3 domains were found to pull down p85 and CrkII, respectively, but only from lysates of activated T cells. Nevertheless, the GST-CrkII-SH2 fusion protein was unable to mediate direct association with p85 from lysates of either resting or activated T cells. Our results support a model in which T cell activation dependent conformational changes in CrkII and/or p85 promote an initial direct or indirect low affinity interaction between the two molecules, which is then stabilized by a secondary high affinity interaction mediated by direct binding of the CrkII-SH3(N) to the p85-PBP domain.
T cell antigen receptor
polyacrylamide gel electrophoresis
peripheral blood lymphocytes
protein tyrosine kinase
SH2 and SH3
Src homology 2 and 3, respectively
Physiological activation of T lymphocytes is initiated by T cell antigen receptor (TCR)1interaction with a major histocompatibility complex-bound peptide antigen on the surface of antigen-presenting cells. Engagement of the TCR triggers multiple intracellular biochemical events that operate in a sequence and transduce the extracellular signal into different subcellular compartments. This results in the induction of transcription of selected genes and translation of their corresponding mRNA, reorganization of cytoskeletal elements, modulation of expression of cell surface receptors, and secretion of lymphokines and other soluble immune mediators. These responses may lead to T cell proliferation, apoptosis, anergy, or differentiation into distinct types of effector cells. Determination of the specific differentiation pathway of engaged T cells is dependent upon the structure of the presented peptide antigen, and the nature of simultaneously engaged cell surface receptors.
Individual components of the TCR-linked signaling pathways are physically separated in resting cells, and they reassemble into functional complexes upon receptor engagement (
). Because this process occurs in a temporally and spatially regulated manner, it ensures an efficient signaling, which, under appropriate conditions that follow a productive T cell interaction, lead to cell activation and differentiation.
Adapter molecules such as Grb2, Shc, and Crk possess multiple protein-protein interaction domains, which allows them to play a critical role in the assembly of multimolecular activation complexes (
). Many of these adapter proteins are involved in the regulation of cell growth and differentiation by coupling proximal biochemical events, initiated by cell surface receptor engagement, with distal signal transducing pathways.
The Crk adaptor protein was originally identified as a product of the v-crk oncogene in the avian retrovirus CT10 (
). Although adapter proteins such as Grb2 and Shc were found to play a positive role in the regulation of T cell activation, other adapter proteins, including Crk and Cbl, were predominantly implicated in the negative regulation of TCR-linked signaling pathways. It is possible therefore that Crk and Cbl regulate the termination of TCR-linked activation signals or are involved in biochemical processes promoting T cell suppression and induction of immune anergy (
). This model is further supported by the findings that TCR ligation and the subsequent tyrosine phosphorylation of Cbl promotes Grb2 dissociation from Cbl and the recruitment of other effector molecules, including phosphatidylinositol 3-kinase (PI3K) (
The Cbl-associated PI3K may also be involved in the negative regulation of T cell responsiveness, because overexpression of a mutated, constitutively active form of PI3K resulted in a reduced TCR-dependent activation of the NF-AT transcription factor (
). We now demonstrate that a direct physical interaction exists between Crk and the PI3K regulatory subunit, p85, in activated T cells and further characterizes the mechanism of this interaction.
Crk proteins were shown to be involved in multiple signaling pathways linked to different cell surface receptors. Nevertheless, their mechanism of action in T lymphocytes following engagement of the TCR has not been studied thoroughly. In the present work we found that T cell activation results in CrkII association with the regulatory subunit of PI3K, as demonstrated by reciprocal co-immunoprecipitation studies. CrkII association with p85 reached its maximal level within 2 min of TCR cross-linking, and after about 10 min it started to decline gradually. Furthermore, T cell activation resulted in tyrosine phosphorylation of CrkII and, to a lesser degree, p85. However, maximal p85-CrkII interaction preceded the peak of phosphorylation of both proteins, suggesting that p85-CrkII interaction may be independent of the tyrosine phosphorylation state of the two proteins.
Studies on the regulation of Crk in activated cells combined with previous results on the Crk regulation in stimulated cells (
) support a model in which Crk phosphorylation induces the formation of an intramolecular SH2-phosphotyrosine interaction, preventing Crk from association with other effector molecules. It is possible therefore that Crk proteins in activated T cells exist in at least two major distinct forms: one that is tyrosine-phosphorylated and uncomplexed and a second that is not phosphorylated and can interact with effector molecules such as p85 and Cbl. Initial attempts to reevaluate this model in vivo in activated T cells revealed that only nonphosphorylated Crk proteins co-immunoprecipitated with anti-Cbl Abs.
S. Gelkop, Y. Babichev, and N. Isakov, unpublished data.
Because a significant fraction of the Crk proteins in the cells were tyrosine-phosphorylated but were excluded from the Cbl-Crk complex, the results suggest that the above mentioned model may also be applied to the regulation of Crk in activated T cells.
To test whether Crk association with p85 is mediated via a linker protein (such as Cbl) as suggested by some studies (
) or via a direct interaction, we performed an overlay assay on membrane-blotted p85 using soluble GST-Crk fusion proteins. We found a direct binding of CrkII to p85, which, in contrast to the results of the immunoprecipitation, was independent of the activation state of the cells. Furthermore, direct binding to p85 appeared to be mediated by a non-SH2-containing region in CrkII. A pull-down assay using bead-immobilized fusion proteins indicated that the p85-binding region corresponds to the CrkII-SH3(N) and also demonstrated a lower binding affinity of the CrkII-SH2 to p85 in activated but not in resting T cells.
The observation that CrkII binding to p85 is mediated by a direct physical interaction is similar to the findings obtained with CrkL, a protein that is highly homologous to and a close relative of CrkII. In these studies, CrkL was found to mediate direct binding (via its SH3(N) domain) to p85 in steel factor-responsive MO7e promegakaryoblastic cells (
). It is possible therefore that the CrkII and CrkL SH3(N) domains exhibit very similar binding specificity and therefore compete for binding to the same ligands.
Pull-down assays with GST-p85 fusion proteins revealed that two distinct regions mediate binding to CrkII. The p85-PBP region pulled down CrkII very efficiently from lysates of either resting or activated T cells, whereas p85-SH3 pulled down CrkII, almost exclusively, from lysates of activated cells. The observation that the p85-PBP domain can pull down CrkII but not the Cbl protein from a total cell lysate provides an additional support for the assumption that p85 can directly associate with CrkII in the absence of Cbl. This is further supported by the results in Fig. 9 showing the ability of p85 to co-immunoprecipitate with CrkII from a Cbl-depleted Jurkat cell lysate.
The involvement of p85-PBP and CrkII-SH3 in the reciprocal protein-protein interaction raised the possibility that these two regions bind to each other. This hypothesis was confirmed both by direct binding studies and by binding competition analysis. However, direct binding of p85-PBP to CrkII-SH3 was independent of the activation state of the T cells, in contrast to the results of the co-immunoprecipitation, which indicated that p85 association with CrkII occurs almost exclusively in activated T cells. Furthermore, although the GST-CrkII-SH2 was able to pull down p85 from a lysate of activated T cells, it was incapable of mediating direct binding. These results suggested the involvement of a third party molecule that functions as a link between p85 and CrkII.
An obvious candidate for mediating this linkage was the Cbl protein, which by itself is a major tyrosine-phosphorylated substrate in activated T cells and was shown to interact with multiple effector molecules, including Crk and p85, in an activation-dependent manner. We found that CrkII can associate directly with Cbl (via its SH2 domain) and that p85 can pull down Cbl from a lysate of activated T cells. In addition, Cbl associated with the p85-SH3 domain in lysates of both resting and activated T cells and with the p85-SH2 in a cell activation-dependent manner.
It is interesting to note that different regions of p85 pulled down distinct migratory forms of CrkII (Fig. 7, upper panel). For example, a fast migrating form of CrkII was associated with the p85-SH3 domain, whereas slower migrating forms of CrkII interacted with the p85-PBP region. The results suggest that post-translational modifications of CrkII, which are known to affect the protein migration rate on SDS-PAGE, also determine the ability of CrkII to interact with other molecules. Thus, the fastest migrating form of CrkII appears to be capable of interacting with the p85-SH3 (but not with the p85-PBP region), possibly via an indirect mechanism mediated by the simultaneous interaction of CrkII and p85 with tyrosine-phosphorylated Cbl. In contrast, the slow migrating forms of CrkII can bind to the p85-PBP region, apparently in a Cbl-independent manner. It is possible, however, that binding of the different forms of CrkII to isolated regions of recombinant p85 fusion proteins are not necessarily representative of all in vivo interactions between the endogenous CrkII and p85 native proteins.
Based on the present results we suggest that T cell activation that results in Cbl tyrosine phosphorylation at multiple sites creates two adjacent low affinity binding sites for the SH2 domains of Crk (possibly 774YDVP or 700YMTP; see Refs.
)) and a Cbl-derived proline-rich motif (possibly 494PPVPPRLDLL). The bimodal interaction of the two distinct p85 domains with Cbl may possibly induce a conformational change in the intermediate region of p85, the PBP proline-rich region, and position it adjacent to the Cbl-bound CrkII to facilitate its interaction with the CrkII-SH3(N) domain. Because the predicted consensus region for the Crk-SH3(N) corresponds to PPXLPXK (
Another proline-rich region that may be relevant to the interaction of CrkII with p85 is located within the CrkII-SH2 domain (residues 63–105) but is dispensable for interaction with phosphopeptides. Binding affinity of this proline-rich region to the Abl-SH3 was found to significantly increase under cell activation conditions when CrkII undergoes phosphorylation on Tyr221 and forms an intramolecular association with the SH2 domain (
). As a result, folding of CrkII induces a new conformation in which the proline-rich region becomes more accessible for interaction with SH3 peptides. Because a sequence in this region (69PPVPPS) is likely to functions as a putative ligand for the p85-SH3, the data of Anafiet al. (
) provide a good explanation for the SH3-mediated cell activation dependent interactions observed in our system.
The CrkII-SH3C appears to take no part in the formation of the Crk-Cbl-p85 trimolecular complex, suggesting that CrkI, which is devoid of a SH3C domain, can also be found in this complex. This assumption was confirmed in additional studies in which Crk-specific Abs (raised against the Crk amino terminus) detected a 28-kDa protein band that co-immunoprecipitated with Cbl. In contrast to CrkII, this protein band did not react with a CrkII-SH3C-specific Ab (not shown).
) have indicated that Crk association with tyrosine-phosphorylated Cbl is mediated by the Crk-SH2 domain and that the Crk-SH3(N) associates with the Rap1 guanine nucleotide exchange protein, C3G. These results raised the possibility that the Cbl-bound Crk protein can associate simultaneously with the C3G protein. It is possible therefore that the Cbl-bound Crk could associate via its SH3(N) with either p85 or C3G and that each of the two complexes could operate in a distinct signaling pathway. However, Crk is the only molecule in the Crk-Cbl-C3G complex that interacts simultaneously with two other partners, whereas each of the three partners in the Crk-Cbl-p85 complex can mediate at least two simultaneous interactions with other partners. Furthermore, immunoprecipitation of each of the three partners in the Crk-Cbl-p85 complex pulled down the other two partners (Figs. 1, 2, and 4and Ref.
). It is possible therefore that T cell activation and tyrosine phosphorylation of Cbl, which result in Cbl interaction with Crk and p85, will lead to a predominant interaction of the Crk-SH3 domain with p85. Interaction of C3G with Cbl-bound Crk will therefore occur only under the conditions (or at subcellular locations) when p85 is absent.
We thank Drs. J. Bertoglio, J. Bolen, B. Druker, M. Matsuda, and A. Weiss for gifts of reagents.