©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Crk Interacts with Tyrosine-phosphorylated p116 upon T Cell Activation (*)

(Received for publication, October 6, 1994; and in revised form, November 22, 1994)

Sansana Sawasdikosol Kodimangalam S. Ravichandran Kyungah Kay Lee Jin-Hong Chang Steven J. Burakoff

From the Division of Pediatric Oncology, Dana-Farber Cancer Institute and Harvard Medical School, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Products of the crk oncogene are expressed in all tissues. Crk proteins are composed exclusively of Src homology 2 (SH2) and Src homology 3 (SH3) domains, and they have been implicated in intracellular signaling. For example, they participate as mediators of Ras activation during nerve growth factor stimulation of PC12 pheochromocytoma cells. We examined the role of Crk proteins during T cell receptor-mediated signaling and observed that Crk proteins specifically interact, via their SH2 domains, with a tyrosine-phosphorylated 116-kDa protein upon T cell activation. p116 may be related to the recently cloned fibroblast p130 and/or p120-Cbl. In addition, we observed that GST-Crk fusion proteins and Crk-L bind, most likely via their SH3 domain, to C3G, a Ras guanine nucleotide exchange factor. Thus, the interaction of Crk with p116 and C3G strongly implicates Crk as a mediator of T cell receptor signaling, possibly involved in Ras activation.


INTRODUCTION

The product of the v-crk oncogene was identified in the retroviral genome of avian sarcoma virus-infected fibroblasts(1, 2) . Three different Crk homologues, Crk I, Crk II, and Crk-L, have been identified in mammalian cells(3, 4, 5) . Crk I is composed of a SH2 (^1)domain and a SH3 domain(3, 4) , whereas Crk II and Crk-L are composed of one SH2 domain and two SH3 domains(5) . All three Crk proteins lack an apparent catalytic domain and belong to the recently described class of SH2/SH3-containing adapter proteins(6, 7) , which includes Grb2(8) , Shc(9) , Nck(10) , and the p85 subunit of phosphatidylinositol 3-kinase(11) . SH2 domains bind to specific phosphotyrosine-containing sequences, while SH3 domains bind to proline-rich sequences(12, 13) .

During v-Crk-mediated transformation of chicken embryonic fibroblasts, three major proteins of 130, 110, and 70 kDa are tyrosine-phosphorylated and interact with Crk(1) . While the identity of the 110-kDa species remains unknown, the 70- and 130-kDa protein have been identified as the cytoskeletal protein paxillin (14) and the recently cloned Crk-associated substrate (p130)(15) , respectively. It has been demonstrated that Crk, via its SH2 domain, interacts with both phosphorylated paxillin and p130in vivo(15, 16) . In addition, Crk, via its SH3 domain, interacts with two Ras guanine nucleotide exchange factors, C3G (17, 18) and mSOS(17, 19) . The ability of Crk to simultaneously interact with the transformation-related proteins (paxillin and p130) via its SH2 domain and with guanine nucleotide exchange factors via its SH3 domains suggests that Crk may play a role in regulating the status of Ras activation. This postulation is supported by the finding that expression of Crk proteins carrying a point mutation in their SH3 domain (which do not interact with C3G and mSOS) resulted in diminished Ras activation following nerve growth factor stimulation of PC12 pheochromocytoma cells(17) . Furthermore, overexpression of Crk in PC12 cells leads to more rapid cellular differentiation upon nerve growth factor or basic fibroblast growth factor stimulation(20) .

We have examined the role of Crk in T cell activation and report here that, upon T cell activation, Crk specifically associates via its SH2 domain with a 116-kDa phosphorylated protein. Either CD4 or CD8 co-receptor cross-linking to TCR augmented the level of tyrosine phosphorylation of p116 resulting in its enhanced interaction with Crk. We observed that glutathione S-transferase (GST)-Crk fusion proteins can associate with C3G in T cell lysates. Furthermore, in vivo, Crk-L and perhaps Crk II form a complex with C3G. Thus, Crk, through its interaction with other proteins via its SH2 and SH3 domains, may function as an adapter protein in TCR signaling and Ras activation.


MATERIALS AND METHODS

Cells

BYDP, a L3T4 Lyt2/3 murine T cell hybridoma expressing comparable amount of retrovirus transduced human CD4 and human CD8alpha genes(21) , was grown in RPMI 1640 complete medium (RPMI 1640 supplemented with 5% fetal bovine serum, 5% iron-supplemented bovine calf serum, 2 mML-glutamine, 100 units of penicillin/streptomycin, and 2 times 10M beta-mercaptoethanol). Eighteen h prior to activation, BYDP was placed in serum-free medium (same as complete RPMI 1640 medium, except that 10% serum was replaced by 0.5% bovine serum albumin). Human peripheral mononuclear cells were isolated from the whole blood by Ficoll separation, followed by monocyte/macrophage depletion via plastic adherence. The remaining cells were enriched for T cells by passage through a nylon wool column. Using the anti-CD3 antibody (OKT3), a sample of enriched T cells were analyzed by flow cytometry to ensure that they comprised at least 80% T cells. Human peripheral T cells were also cultured in serum-free medium for 18 h before activation. The v-Src-transformed fibroblast cell line IV5 was a gift from Dr. S. J. Parsons(22) .

T Cell Activation

BYDP (1.5 times 10^7 cells/sample) were washed once and incubated with 1 µg of anti-murine TCR (F23.1) and/or anti-human CD4 (OKT4D) or anti-human CD8 (OKT8) as described previously(21) . After 10 min on ice with the stimulating antibodies, 7.5 µg of rabbit anti-mouse antibody was added for cross-linking, and incubated for an additional 10 min on ice. The cells were then warmed to 37 °C for the indicated times. For human peripheral blood T cell stimulation, 3 times 10^7 cells/sample were stimulated with anti-CD3 antibody (OKT3) as described above.

Immunoprecipitations and Immunoblotting

Cells were lysed in a buffer containing either 1% Nonidet P-40 or 1% Brij-96 and 50 mM Tris, pH 7.6, 150 mM NaCl, 1 mM Na(3)VO(4), 10 mM NaF, 10 mM sodium pyrophosphate, 10 µg/ml each aprotinin and leupeptin, and 2 mM phenylmethylsulfonyl fluoride. The lysates were precleared with protein A-Sepharose; subsequently, proteins were immunoprecipitated with either 2 µg of anti-Crk antibody or an equal amount of normal rabbit Ig by incubation at 4 °C for 2 h. The beads were washed extensively with 0.1% Nonidet P-40 or 0.1% Brij-96 in immunoprecipitation wash buffer (150 mM NaCl, 20 mM HEPES, pH 7.4, 10% glycerol, 1 mM Na(3)VO(4), 5 mM NaF, 10 µg/ml each of aprotinin and leupeptin), and the bead-bound proteins were separated by SDS-polyacrylamide gel electrophoresis. The proteins were transferred to a nitrocellulose membrane, immunoblotted with the indicated antibodies, and developed by the enhanced chemiluminescence (ECL) system (Amersham Corp.). As for precipitation with GST-Crk or GST-Crk SH2 fusion proteins, 2-5 µg of each fusion protein was incubated with T cell lysates. The precipitation and wash conditions were identical to those described above.

Reagents

All GST-Crk constructs were a gift from Dr. B. J. Mayer. The GST-Shc SH2 fusion protein was a gift from Dr. T. Pawson. The anti-Crk monoclonal antibody (mAb) and horseradish peroxidase-coupled anti-phosphotyrosine antibody (RC20H) were purchased from Transduction Laboratories (Lexington, KY). The anti-Crk II and anti-Crk-L polyclonal antibodies were purchased from Santa Cruz Biotechnologies (Santa Cruz, CA). The 4F4 anti-p130 Src substrate (p130) mAb was a gift from Dr. J. T. Parsons(23) , and the anti-C3G anti-serum was a gift from Dr. M. Matsuda(18) . The anti-phosphotyrosine mAb (4G10) was purchased from Upstate Biotechnology, Inc. (Lake Placid, NY). The anti-c-Cbl antibody was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA).


RESULTS AND DISCUSSION

The role of the Crk adapter protein in TCR-mediated signaling was investigated using a murine T cell hybridoma (BYDP). T cells were activated by antibody-mediated cross-linking of the TCR alone or by cross-linking the TCR with either the CD4 or CD8 co-receptors. Crk proteins were immunoprecipitated using an anti-Crk I mAb and immunoblotted with anti-phosphotyrosine antibody (4G10). Although in some transformed cells, Crk and Crk-L are tyrosine-phosphorylated (24, 25, 26) , upon T cell activation Crk did not appear to be tyrosine-phosphorylated; however, a 116-kDa tyrosine-phosphorylated protein co-precipitated with Crk only upon activation (Fig. 1A, lane4). p116 did not associate with other SH2/SH3-containing adapter proteins, Nck (Fig. 1A, lanes 5 and 6) or Shc (data not shown and (27) ). Addition of the Crk immunizing peptide during Crk immunoprecipitation resulted in the loss of p116 association (data not shown). Subsequent stripping and reprobing of the nitrocellulose membrane with the anti-Crk antibody indicated that the same level of Crk was precipitated in all lanes (Fig. 1B). The anti-Crk mAb used in this study, although raised against Crk I, cross-reacts with all three isoforms of Crk (Fig. 1B and data not shown). When Crk II or Crk-L specific antibodies were used to precipitate individual Crk isoforms, it was revealed that the p116 protein associates with all three isoforms of Crk upon TCR-mediated activation (data not shown).


Figure 1: Crk associates with p116 upon T cell activation. A, BYDP cells were activated by antibody-mediated cross-linking, as indicated, for 1 min at 37 °C. Activated (+) or non-activated(-) cells were lysed with 1% Nonidet P-40, and proteins were immunoprecipitated with anti-Crk, anti-Nck, or control Ig antibodies and immunoblotted with anti-phosphotyrosine (Anti-Ptyr) antibody RC20H. B, immunoprecipitates were also immunoblotted with anti-Crk mAb (lanes3, 4, 9, 10, 13, and 14). The arrows identify different Crk isoforms. Molecular size markers are shown on the left of all figures.



It has been reported that cross-linking CD4 or CD8 with the TCR leads to enhanced tyrosine phosphorylation of intracellular proteins. Consistent with this observation, cross-linking of CD4 or CD8 co-receptors with the TCR resulted in enhanced association of Crk with p116, due to enhanced phosphorylation of p116 (Fig. 1A, lanes10 and 14). Since both CD4 and CD8 associate with a Src family tyrosine kinase p56 (Lck), Lck may be involved in p116 phosphorylation. It has been shown that phosphorylated p116 also interacts with the SH2 domain of another Src family tyrosine kinase, p59 (Fyn)(28, 29) , which, in turn, associates with the TCR / and CD3 and chains(30, 31) . Since p116 has been reported to interact with the Fyn SH2 domain (30, 31) and with the SH3 domains of both Fyn and Lck(32) , both of these kinases may play a role, either directly or indirectly, in p116 phosphorylation.

The kinetics of the Crk-p116 association was assessed in a time course of T cell activation. T cells were activated by TCR cross-linking for different times, and Crk proteins were then immunoprecipitated with anti-Crk mAb and immunoblotted with anti-phosphotyrosine antibody for the presence of tyrosine-phosphorylated p116. Phosphorylated p116 associated with Crk as early as 15 s after activation (Fig. 2, lane3). The level of p116 in Crk immunoprecipitates was maximal by 3 min after activation, began to decline by 10 min, and diminished to near-basal levels by 60 min (Fig. 2, lanes 5-7).


Figure 2: Kinetics of Crk-p116 association. BYDP T cells were activated by TCR cross-linking for the indicated times and lysed with 1% Nonidet P-40, and proteins were immunoprecipitated with anti-Crk mAb (lanes3-14) and immunoblotted with anti-phosphotyrosine (Anti-Ptyr) antibody (RC20H).



To determine if Crk-p116 association also occurs in non-transformed T cells, lysates from human peripheral blood T cells were immunoprecipitated with anti-Crk mAb and immunoblotted with anti-phosphotyrosine antibody. As with the T cell hybridoma, phosphorylated p116 associated with Crk only upon TCR-mediated activation (Fig. 3, lane 4). There were trace amounts of p116 associated with Crk in lysates of non-stimulated T cells (Fig. 3, lane 3). This minimal phosphorylation of p116 in resting T cells may reflect heterogeneity in human peripheral T cells or minimal activation that could occur from the purification procedure.


Figure 3: Association between Crk and p116 in activated human peripheral blood T cells. Purified human T cells were stimulated by TCR cross-linking with OKT3 for 1 min at 37 °C. Cells were lysed in 1% Nonidet P-40, immunoprecipitated with anti-Crk mAb, and analyzed by anti-phosphotyrosine (Anti-Ptyr) immunoblotting with RC20H.



To determine which domain of Crk interacts with p116, GST fusion proteins, encoding either full-length Crk or only the Crk SH2 domain (bound to glutathione-agarose beads), were used to probe activated T cell lysates. Anti-phosphotyrosine blotting revealed that both GST-Crk I and GST-Crk II precipitated p116 from activated lysates but not from non-activated lysates (Fig. 4, lanes 3-6). Moreover, GST-Crk SH2 alone precipitated equivalent amounts of p116 compared to GST-Crk I and GST-Crk II, indicating that the SH2 domain of Crk is responsible for its association with p116 (Fig. 4, lane 8). No p116 was precipitated from activated lysates by either GST alone or GST-Shc SH2 fusion protein (Fig. 4, lanes 1, 2, 9, and 10).


Figure 4: Crk SH2 domain interacts with p116 from activated T cell lysates. GST-Crk I (lanes 3 and 4), GST-Crk II (lanes5 and 6), GST-Crk SH2 (lanes7 and 8), and GST-Shc SH2 (lanes9 and 10) bound to glutathione-agarose beads were added to lysates from non-activated or BYDP T cells activated by TCR cross-linking for 1 min at 37 °C. The phosphoproteins bound to the beads were analyzed by anti-phosphotyrosine (Anti-Ptyr) immunoblotting.



We and others have observed that the tyrosine-phosphorylated p116 detected in activated T cells may be related to the p130 observed in v-Src-transformed fibroblasts (data not shown and (28) and (29) ). Peptide maps of p116 and p130 revealed a similar pattern indicating that p116 in T cells may be an isoform of the p130 found in fibroblasts (data not shown and (28) and (29) ). p130 from fibroblasts has been recently cloned (18) and was found to contain nine tyrosine-containing motifs that represent the predicted optimal Crk SH2 binding sequence (YDXP, where X is any amino acid)(13) . To determine whether the p116 co-precipitated with Crk from activated T cells might represent the T cell isoform of p130, Crk immunoprecipitates were immunoblotted with 4F4, a mAb that recognizes both p130 and p116. The 4F4 mAb immunoprecipitated tyrosine-phosphorylated p116 from activated T cell lysates and phosphorylated p130 from Src-transformed fibroblasts (Fig. 5, left panel). p116 was detected by 4F4 immunoblotting in Crk immunoprecipitates from activated T cell lysates (Fig. 5, right panel) but not in control Ig or Nck immunoprecipitates (data not shown). These data suggest that p116 may also contain YDXP motifs similar to p130 and that tyrosine phosphorylation of this motif following T cell activation leads to its interaction with the Crk SH2 domain. It should be noted, however, that it has been recently shown that a significant portion of the tyrosine-phosphorylated proteins in the 116-kDa region of T cell lysates can be immunoprecipitated by an antibody to c-Cbl(33) . Cbl structure contains a YDXP Crk binding motif(34) , and it may be a component of the p116 complex that binds to Crk.


Figure 5: Crk-associated p116 may be related to p130. Leftpanel, lysates from BYDP T cells activated by TCR cross-linking for 1 min at 37 °C or Src-transformed fibroblasts (IV5) were immunoprecipitated with anti-p130 mAb (4F4) and immunoblotted with anti-phosphotyrosine (Anti-Ptyr) antibody. Right panel, Crk was immunoprecipitated with the Crk mAb from BYDP cells activated for 1 min by TCR cross-linking. Precipitates were immunoblotted with 4F4.



It has been reported that Crk, via its SH3 domain, is constitutively associated with a novel Ras GTP/GDP exchange factor, C3G in PC12 pheochromocytoma cells(17) . To determine whether Crk can interact with C3G in T cells, two approaches were taken. First, GST-Crk fusion proteins were incubated with activated and non-activated T cell lysates and immunoblotted with anti-C3G antibody. C3G was precipitated by full-length GST-Crk I and GST-Crk II from both activated and non-activated lysates (Fig. 6, lanes2, 3, 6, and 7). Irrespective of the activation status, GST-Crk SH2 did not associate with C3G (Fig. 6, lanes4 and 8). Second, Crk immunoprecipitates were immunoblotted for C3G (Fig. 7). C3G was detected when Crk-L immunoprecipitates were immunoblotted for C3G. However, only small amounts of C3G were found in Crk II immunoprecipitates and none were found in Crk I immunoprecipitates (Fig. 7). These data would suggest that there is an association of Crk-L with C3G in vivo. Because Crk I antibody interfered with Crk-L binding to C3G, we cannot rule out an interaction of Crk I and Crk II with C3G (data not shown).


Figure 6: C3G associates with GST-Crk fusion proteins. BYDP T cells were activated by TCR and CD4 cross-linking for 1 min at 37 °C and lysed with 1% Brij-96 lysis buffer. GST-Crk I and GST-Crk II fusion proteins were incubated with non-activated and activated BYDP lysates. Precipitates were analyzed by anti-C3G immunoblotting. As a control, C3G was immunoprecipitated from non-activated T cell lysates with anti-C3G antibody and immunoblotted with the anti-C3G antibody. The arrow indicates C3G.




Figure 7: C3G associates with Crk-L in vivo. BYDP T cells were activated by TCR and CD4 cross-linking for 1 min at 37 °C and lysed with 1% Brij-96 lysis buffer. Crk was immunoprecipated with Crk I, Crk II, or Crk-L specific mAbs from non-activated and activated lysates. Precipitates were analyzed by anti-C3G immunoblotting.



Taken together, these data suggest that, in T cells, Crk interacts with p116 via its SH2 domain and possibly with a mediator of Ras activation, C3G via its SH3 domain. Since phosphorylated p130 in fibroblasts has been localized to the plasma membrane (15) and T cell p116 has been shown to interact with TCR-associated Fyn(28, 29) , the Crk-p116 inter-action may serve to shuttle C3G to the membrane. This, in turn, may contribute to TCR-mediated, tyrosine kinase-dependent Ras activation. In T cells, three other proteins: Shc and p36 (via their interaction with Grb2 and mSOS)(27, 35) , and Vav (through its intrinsic nucleotide exchange activity)(36) , are also implicated in Ras activation. Since the Crk SH3 domain can also interact with mSOS(17, 19) , Crk might also be able to activate the Ras pathway via mSOS. Whether these are distinct pathways of Ras activation or whether they represent pathways employed under different stimulation conditions remains to be determined. While the precise role of Crk in TCR-mediated signaling remains to be established, our data strongly suggest an important role for Crk in T cell activation.


FOOTNOTES

*
This study was supported by National Institutes of Health Grant AI17258 (to S. J. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

(^1)
The abbreviations used are: SH2, Src homology 2; SH3, Src homology 3; GST, glutathione S-transferase; TCR, T cell receptor; mAb, monoclonal antibody.


ACKNOWLEDGEMENTS

We are very grateful to Dr. M. Matsuda for the anti-C3G antibody, Dr. B. J. Mayer for the GST-Crk constructs, Dr. T. Pawson for GST-Shc SH2 fusion protein, Dr. S. J. Parsons for the IV5 Src-transformed fibroblast cell line, and to Dr. J. T. Parsons for the 4F4 mAb. We thank L. Samelson and H. Meissner for helpful discussions.


REFERENCES

  1. Mayer, B. J., Hamaguchi, M., and Hanafusa, H. (1988) Nature 332, 272-275 [CrossRef][Medline] [Order article via Infotrieve]
  2. Tsuchie, H., Chang, C. H., Yoshida, M., and Vogt, P. K. (1989) Oncogene 4, 1281-1284 [Medline] [Order article via Infotrieve]
  3. Matsuda, M., Tanaka, S., Nagata, S., Kokima, A., Kurata, T., and Shibuya, M. (1992) Mol. Cell. Biol. 12, 3482-3489 [Abstract]
  4. Reichman, C. T., Mayer, B. J., Keshav, S., and Hanafusa, H. (1992) Cell Growth Diff. 3, 451-460 [Abstract]
  5. Hoeve, J., Morris, C., Heisterkamp, N., and Groffen, J. (1993) Oncogene 8, 2469-2474 [Medline] [Order article via Infotrieve]
  6. Cantley, L. C., Auger, K. R., Carpenter, C., Duckworth, B., Graziani, A., Kapeller, R., and Soltoff, S. (1991) Cell 64, 281-302 [Medline] [Order article via Infotrieve]
  7. Pawson, T., and Gish, G. D. (1992) Cell 71, 359-362 [Medline] [Order article via Infotrieve]
  8. Lowenstein, E. J., Daly, R. J., Batzer, A. G., Li, W., Margolis, B., Lammers, R., Ullrich, A., Skolnik, E. Y., Bar-Sagi, D., and Schlessinger, J. (1994) Cell 70, 431-442
  9. Pelicci, G., Lanfrancone, L., Grignani, F., McGlade, J., Cavallo, F., Forni, G., Nicoletti, I., Pawson, T., and Pelicci, P. G. (1992) Cell 70, 93-104 [Medline] [Order article via Infotrieve]
  10. Lehmann, J. M., Riethmuller, G., and Johnson, J. P. (1990) Nucleic Acids Res. 18, 1048 [Medline] [Order article via Infotrieve]
  11. Otsu, M., Hiles, I., Gout, I., Fry, M. J., Ruiz-Larrea, F., Panayotou, G., Thompson, A., Dhand, R., Hsuan, J., Totty, N., Smith, A. D., Morgan, S. J., Courtneidge, S. A., Parker, P. J., and Waterfield, M. D. (1991) Cell 65, 91-104 [Medline] [Order article via Infotrieve]
  12. Cicchetti, P., Mayer, B. J., Thiel, G., and Baltimore, D. (1992) Science 257, 803-806 [Medline] [Order article via Infotrieve]
  13. Songyang, Z., Shoelson, S. E., Chaudhri, M., Gish, G. D., Pawson, T., Haser, W., King, F., Roberts, T., Ratnofsky, S., Lechleider, R. J., Neel, B. G., Birge, R., Fajardo, J. E., Chou, M. M., Hanafusa, H., Schaffhausen, B., and Cantley, L. C. (1993) Cell 72, 767-778 [Medline] [Order article via Infotrieve]
  14. Birge, R. B., Fajardo, E. E., Reichman, C., Shoelson, S. E., Songyang, Z., Cantley, L. C., and Hanafusa, H. (1993) Mol. Cell. Biol. 13, 4648-4656 [Abstract]
  15. Sakai, R., Iwamatsu, A., Hirano, N., Ogawa, S., Tanaka, T., Mano, H., Yazaki, Y., and Hirai, H. (1994) EMBO J. 13, 3748-3756 [Abstract]
  16. Matsuda, M., Mayer, B. J., and Hanafusa, H. (1994) Mol. Cell. Biol. 11, 1697-1613
  17. Matsuda, M., Hashimoto, Y., Muroya, K., Hasegawa, H., Kurata, T., Tanaka, S., Nakamura, S., and Hattori, S. (1994) Mol. Cell. Biol. 14, 5495-5500 [Abstract]
  18. Tanaka, S., Morishita, T., Hashimoto, Y., Hattori, S., Nakamura, S., Shibuya, M., Matuoka, K., Takenawa, T., Kurata, T., Nagashima, K., and Matsuda, M. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 3443-3447 [Abstract]
  19. Gout, I., Dhand, R., Hiles, I. D., Fry, M. J., Panayotou, G., Das, P., Truong, O., Totty, N. F., Hsuan, J., Booker, G. W., Campbell, I. D., and Waterfield, M. D. (1993) Cell 75, 25-36 [Medline] [Order article via Infotrieve]
  20. Hempstead, B. L., Birge, R. B., Fajardo, J. E., Glassman, R., Mahadeo, D., Kraemer, R., and Hanafusa, H. (1994) Mol. Cell. Biol. 14, 1964-1971 [Abstract]
  21. Ravichandran, K. S., and Burakoff, S. J. (1994) J. Exp. Med. 179, 727-732 [Abstract]
  22. Luttrell, D. K., Luttrell, L. M., and Parsons, S. J. (1988) Mol. Cell. Biol. 8, 497-501 [Medline] [Order article via Infotrieve]
  23. Kanner, S. B., Reynolds, A. B., Vines, R. R., and Parsons, J. T. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 3328-3332 [Abstract]
  24. Feller, S. M., Knudsen, B., and Hanafusa, H. (1994) EMBO J. 13, 2341-2351 [Abstract]
  25. Ren, R., Ye, Z., and Baltimore, D. (1994) Genes & Dev. 8, 783-795
  26. Hoeve, J. T., Arlinghaus, R. B., Guo, J. Q., Heisterkamp, N., and Groffen, J. (1994) Blood 84, 1731-1736 [Abstract/Free Full Text]
  27. Ravichandran, K. S., Lee, K. K., Songyang, Z., Cantley, L. C., Burn, P., and Burakoff, S. J. (1993) Science 262, 902-905 [Medline] [Order article via Infotrieve]
  28. Tsygankov, A. Y., Spana, C., Rowley, R. B., Penhallow, R. C., Burkhardt, A. L., and Bolen, J. B. (1994) J. Biol. Chem. 269, 7792-7800 [Abstract/Free Full Text]
  29. DA Silva, A. J., Janssen, O., and Rudd, C. E. (1992) J. Exp. Med. 178, 2107-2113 [Abstract]
  30. Samelson, L. E., Phillips, A. F., Luong, E. T., and Klausner, R. D. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4358-4362 [Abstract]
  31. Timson Gauen, L. K., Kong, A. N., Samelson, L. E., and Shaw, A. S. (1992) Mol. Cell. Biol. 12, 5438-5446 [Abstract]
  32. Reedquist, K. A., Fukazawa, T., Druker, B., Panchamoorthy, G., Shoelson, S. E., and Band, H. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 4135-4139 [Abstract]
  33. Donovan, J. A., Wange, R. L., Langdon, W. Y., and Samelson, L. E. (1994) J. Biol. Chem. 269, 22921-22924 [Abstract/Free Full Text]
  34. Blake, T. J., Shapiro, M., Morse, H. C., III, and Langdon, W. Y. (1991) Oncogene 6, 653-657 [Medline] [Order article via Infotrieve]
  35. Buday, L., Egan, S. E., Viciana, P. R., Cantrell, D. A., and Downward, J. (1994) J. Biol. Chem. 269, 9019-9023 [Abstract/Free Full Text]
  36. Gulbins, E., Coggeshall, K. M., Baier, G., Katzav, S., Burn, P., and Altman, A. (1993) Science 260, 822-825 [Medline] [Order article via Infotrieve]

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