Martin Sattler 1, Ravi Salgia 1, Gautam Shrikhande 1, Shalini Verma 1, Jung-Lim Choi 2, Larry R Rohrschneider 2 and James D Gri n 1

Size: px

Start display at page:

Download "Martin Sattler 1, Ravi Salgia 1, Gautam Shrikhande 1, Shalini Verma 1, Jung-Lim Choi 2, Larry R Rohrschneider 2 and James D Gri n 1"

Justina Davidson
6 years ago
Views:

1 Oncogene (1997) 15, 2379 ± Stockton Press All rights reserved 95 ± 9232/97 $12. The phosphatidylinositol polyphosphate 5-phosphatase and the protein tyrosine phosphatase SHP-2 form a complex in hematopoietic cells which can be regulated by BCR/ABL and growth factors Martin Sattler 1, Ravi Salgia 1, Gautam Shrikhande 1, Shalini Verma 1, Jung-Lim Choi 2, Larry R Rohrschneider 2 and James D Gri n 1 1 Division of Hematologic Malignancies, Department of Adult Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, Massachusetts 2115; 2 Division of Basic Science, Fred Hutchinson Cancer Research Center, 1124 Columbia St, B2-152, Seattle, Washington 98125, USA We report here that interleukin-3 (IL-3) and erythropoietin (EPO) induce formation of a complex composed of two SH2-containing phosphatases, the tyrosine phosphatase SHP-2 and the SH2 containing inositol 5- phosphatase (). Both SHP-2 and are known to be involved in growth factor signal transduction, but their potential interaction in the same pathway is novel. has previously been shown to associate with SHC, and potentially to be involved in regulating apoptosis. In contrast, in some model systems, SHP-2 has been demonstrated to positively regulate cell growth. Both phosphatases in the complex were tyrosine phosphorylated, and the amount of coprecipitating with SHP-2 was inversely related to the amount of coprecipitating with SHC. In hematopoietic cells transformed by the BCR/ABL oncogene, this phosphatase complex was found to be constitutively present with both components heavily tyrosine phosphorylated. Also, other proteins were detected in the complex, including BCR/ABL itself and c-cbl. However, transformation by BCR/ABL was associated with a reduced protein expression, which could further a ect the accumulation of various inositol polyphosphates in these leukemic cells. These data suggest that the function of and SHP-2 in normal cells are linked and that BCR/ABL alters the function of this signaling complex. Keywords: BCR/ABL; ; SHP-2; CBL Introduction The hematopoietic inositol-5 phosphatase has been recently cloned (Damen et al., 1996; Lioubin et al., 1996) and shown to be transiently tyrosine phosphorylated in response to growth factor receptor activated tyrosine kinases or constitutively phosphorylated by BCR/ABL (Damen et al., 1996; Matsuguchi et al., 1994). has speci c inositol-5-phosphatase activity for phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P 3 ] and inositol 1,3,4,5-tetraphosphate [Ins(1,2,4,5)P 4 ] and thus generates PtdIns(3,4)P 2 and Ins(1,3,4)P 3 respectively (Damen et al., 1996). In normal cells, the enzyme activity of has not been shown to change after receptor activation, Correspondence: JD Gri n Received 3 April 1997; revised 14 July 1997; accepted 14 July 1997 suggesting that interaction with other signaling proteins might be important to mediate biological e ects (Damen et al., 1996). has been shown to interact through its SH2 domain with the adapter protein SHC (Liu et al., 1997a), which in turn binds through its protein tyrosine binding (PTB) domain to phosphorylated NPXY motifs in (Kavanaugh et al., 1996; Lioubin et al., 1996). Another prominent binding protein is the adapter protein GRB2 that competes with for SH2 binding to SHC (Liu et al., 1994, 1997a) or binds to a C-terminal proline rich region in through its SH3 domains (Damen et al., 1996). Relocation of to the cell membrane may be an important regulatory mechanism, and thus transient interaction of with signaling complexes associated with transmembrane growth factor receptors may be quite important. The biological e ects of and its substrates or products are just beginning to be elucidated. However, recent studies suggest that negatively regulates growth and might have an important role in apoptosis (Lioubin et al., 1996; Liu et al., 1997a; Ono et al., 1996). Other molecules which bind to through its SH2 domain are likely to be important in this process, as deleting the SH2 domain impairs apoptotic activity of and prevents its tyrosine phosphorylation (Liu et al., 1997a). SHP-2 is a tyrosine phosphatase which, like, is involved in growth factor and oncogene-mediated signaling. SHP-2 and the related tyrosine phosphatase SHP-1 belong to a class of phosphatases containing two adjacent SH2 domains which are important for linking these phosphatases to activated receptors such as the PDGF or EGF receptor (Feng et al., 1993; Lechleider et al., 1993; Vogel et al., 1993). In PDGF signaling, SHP-2 binds directly to the receptor and recruits GRB2 and SOS to a phosphorylation site in SHP-2, thus providing a potential pathway which contributes to activation of RAS (Bazenet et al., 1996; Klingho er et al., 1996). Similarly, in Xenopus laevis, SHP-2 appears to positively regulate early development and is required for MAP kinase activation (Tang et al., 1995). Also, Corksrew (Crw) an SHP- 2 homolog in Drosophila melanogaster, is required for growth factor receptor mediated signaling and loss of function mutations in crw are lethal during development (Perkins et al., 1996). In this study, we found that and SHP-2 coimmunoprecipitate in hematopoietic cell lines. The amount of the complex and the amount of tyrosine

238 binds to the protein tyrosine phosphatase SHP-2 phosphorylation of and SHP-2 are increased by at least two di erent hematopoietic growth factors, interleukin-3 and erythropoietin, and regulated

Expression of tends to be reduced in cell lines transformed by BCR/ABL, but it is heavily tyrosine phosphorylated and forms a constitutive complex with SHP-2, which is also constitutively tyrosine

2 238 binds to the protein tyrosine phosphatase SHP-2 phosphorylation of and SHP-2 are increased by at least two di erent hematopoietic growth factors, interleukin-3 and erythropoietin, and regulated in a di erent way by BCR/ABL. Expression of tends to be reduced in cell lines transformed by BCR/ABL, but it is heavily tyrosine phosphorylated and forms a constitutive complex with SHP-2, which is also constitutively tyrosine phosphorylated. These data suggest that in normal cells the functions of both of these phosphatases are linked, and in BCR/ABLtransformed cells, the function of this complex of phosphatases may be permanently altered. a lysate IL-3 EP + + p Tyr Results IL-3 and EPO increase the association of with SHP-2 and SHC Hematopoietic growth factors including IL-3 and EPO induce tyrosine phosphorylation of cellular proteins (Figure 1a). Figure 1b (top panel) demonstrates that IL-3 and EPO induce tyrosine phosphorylation of the protein, which coprecipitated with tyrosine phosphoproteins of 52 and. Also, after EPO, but not IL-3 stimulation, a 4 phosphoprotein coprecipitated with. The 52 protein was identi ed as SHC and the proteins as SHP-2 (Figure 1b bottom panels). Inducible association between and SHC has been described before. Interestingly, there was constitutive association between and SHP-2 in unstimulated cells although the association between these two proteins increased after growth factor stimulation. These results were con rmed by immunoprecipitation with anti-shp-2 followed by immunoblotting with anti- using lysates of IL-3 or EPO stimulated cells (data not shown). In these experiments we did not detect coprecipitation of SHP- 2 with SHC. b IP: IL-3 EP + + forms complexes with both SHC and SHP-2 in pervanadate treated cells To investigate the potential interaction of SHP-2 with cellular proteins in a situation where tyrosine phosphatases are inactivated, cells were treated with pervanadate. Figure 2a demonstrates that pervanadate treatment leads to increased tyrosine phosphorylation of cellular proteins. Pervanadate increases the tyrosine phosphorylation of SHP-2 and increases its association with other signaling proteins (Figure 2b, right panel). Figure 2b (right panel) demonstrates that SHP-2 inducibly coprecipitates with at least three major tyrosine phosphorylated proteins of approximately, 1, and 65 after and min of pervanadate treatment. The band was identi ed as. In some experiments appeared as a doublet of and 14, which may represent isoforms of, as the antibody used in these experiments is known to recognize di erent isoforms. Interestingly, after pervanadate treatment and its isoforms also associated with SHP-2 (Figure 2b, bottom panels). itself was also found to be tyrosine phosphorylated after pervanadate treatment and to coprecipitate with tyrosine phos- 52 SHC Figure 1 IL-3 and EPO increase the association of with SHP-2 and SHC. or /EPOR cells were left untreated or treated for 7.5 min with IL-3 (1 ng/ml) or EPO (1 U/ml) respectively. (a) Protein tyrosine phosphorylation was detected in cell lysates ( cells) by immunoblotting (WB) with the monoclonal anti-phosphotyrosine antibody 4G1 (). The blot was stripped and reprobed with anti-shp-2 antibodies. (b) was immunoprecipitated (IP) from cell lysates (61 6 cells). Protein tyrosine phosphorylation was detected in the immunoprecipitations by immunoblotting with the monoclonal anti-phosphotyrosine antibody 4G1 (). The blots were stripped and reprobed with anti- and anti-shc antibodies

phorylated proteins of, 65, and 52 (Figure 2b, top left panel). Interestingly, in contrast to growth factor stimulation, the amount of the /SHP-2 complex decreased after pervanadate stimulation.

In control experiments the quality of the antiserum to speci cally recognize in SHC immunoprecipitates of cells was tested.

SHC and SHP-2 were not found to coprecipitate, suggesting that they are likely to be in di erent complexes (Figure 2b, bottom right panel).

3 phorylated proteins of, 65, and 52 (Figure 2b, top left panel). Interestingly, in contrast to growth factor stimulation, the amount of the /SHP-2 complex decreased after pervanadate stimulation. This decrease correlated with the increased interaction of with SHC (Figure 2b, bottom left panel). In control experiments the quality of the antiserum to speci cally recognize in SHC immunoprecipitates of cells was tested. SHC was found to be tyrosine phosphorylated after pervanadate treatment and to coprecipitate with. Also, preimmune rabbit serum did not precipitate, SHP-2 or SHC proteins (Figure 2c). SHC and SHP-2 were not found to coprecipitate, suggesting that they are likely to be in di erent complexes (Figure 2b, bottom right panel). These data suggest that inhibition of tyrosine phosphatases by pervanadate, which would include inhibition of SHP-2, induces tyrosine phosphorylation of, SHC and SHP-2 and also alters the composition of -containing complexes in Ba/F3 cells. binds to the protein tyrosine phosphatase SHP-2 and SHP-2 are associated in cells transformed by BCR/ABL BCR/ABL transforms cells and causes IL-3 independence. Transformation of these cells requires the ABL tyrosine kinase and leads to tyrosine phosphorylation of cellular proteins (Figure 3a, top panel). Interestingly, the protein expression of is drastically reduced in the BCR/ABL transfected and 32Dcl3 cells compared to the parent cell lines. In contrast, the expression of SHP-2 did not change in the BCR/ABL expressing cell lines (Figure 3a, bottom panels). Also, the protein expression of other proteins including c-cbl, the 85 subunit of phosphatidylinositol-3 kinase, CRK, CRKL, VAV, paxillin, SHC or SHP-1 did not change upon BCR/ABL transformation (data not shown). In BCR/ABL transformed cells, SHP-2, and SHC are tyrosine phosphorylated., ABL and SHP-2 immunoprecipitations in /p21 cells show coprecipitation of common phosphotyrosine proteins 2381 a lysate c IP: 85 NRS SHC b IP: 52/46 SHC 52 SHC Figure 2, SHP-2, and SHC form a stable complex in pervanadate treated cells. cells were left untreated or treated for the indicated times with mm pervanadate. (a) Protein tyrosine phosphorylation was detected in cell lysates ( cells) by immunoblotting (WB) with the monoclonal anti-phosphotyrosine antibody 4G1 (). (b) and SHP- 2 proteins were immunoprecipitated (IP) from cell lysates (61 6 cells). (c) As a control SHC antibody and normal rabbit serum (NRS) were used for immunoprecipitations. b and c Protein tyrosine phosphorylation was detected in the immunoprecipitations by immunoblotting (WB) with the monoclonal antiphosphotyrosine antibody 4G1 (). The blots were stripped and reprobed with anti-shp-2, anti-, or anti-shc antibodies

2382 b a 21 1 binds to the protein tyrosine phosphatase SHP-2 IP: /p21 /p21 lysate IP: ABL /p21 32Dcl3 32D/p21 IP: /p21 BCR/ABL CBL Figure 3 and SHP-2 are associated with BCR/ABL in /p21 cells.

(b) Lysates of 61 6 starved or /p21 cells were used for immunoprecipitation (IP) with anti-, anti-abl and anti- SHP-2 antibodies.

4 2382 b a 21 1 binds to the protein tyrosine phosphatase SHP-2 IP: /p21 /p21 lysate IP: ABL /p21 32Dcl3 32D/p21 IP: /p21 BCR/ABL CBL Figure 3 and SHP-2 are associated with BCR/ABL in /p21 cells. (a) Protein tyrosine phosphorylation was detected in cell lysates ( cells) of, /p21, 32Dcl3, and 32D/p21 cells by immunoblotting (WB) with the monoclonal anti-phosphotyrosine antibody 4G1 (). (b) Lysates of 61 6 starved or /p21 cells were used for immunoprecipitation (IP) with anti-, anti-abl and anti- SHP-2 antibodies. Protein tyrosine phosphorylation was detected in the immunoprecipitations by immunoblotting (WB) with the monoclonal anti-phosphotyrosine antibody 4G1 (). Coprecipitating proteins were detected by immunoprecipitation with anti-, anti-abl, anti-cbl or anti-shp-2 antibodies with an apparent molecular mass of 21,, 1, 55 and 5 (Figure 3b, top panel). Again, and SHP-2 were found to coprecipitate in cells. This complex was not increased in /p21 cells but coprecipitation was still detected despite the reduced expression of (Figure 3b, bottom panels). However, coprecipitation of a tyrosine phosphorylated band in immunoprecipitations could only be observed after long exposure of the immunoblot. The sustained interaction between and SHP-2 is likely to be in part due to increased SHP-2 phosphorylation, since the SH2 domain of ABL and can precipitate SHP-2 in BCR/ABL transformed but not in normal cells (data not shown). In all three immunoprecipitations the 21 protein was identi- ed as BCR/ABL and the 1 protein as c-cbl (Figure 3b, bottom panels). We also found a small amount of coprecipitating with SHP-2 using lysates of the Philadelphia chromosome positive cell lines BV173 and K562 (data not shown). These data suggest that in BCR/ABL transformed cells, in contrast to IL-3 and EPO stimulated cells, the formation of the /SHP-2 complex is small and possibly insignificant. Discussion In this report, we demonstrate by coimmunoprecipitation the presence of protein complexes that contain SHP-2 and in untransformed hematopoietic cell lines. This interaction is increased considerably following stimulation with IL-3 and EPO, which also induce tyrosine phosphorylation of both phosphatases. In BCR/ABL transformed cells, this complex is altered and the phosphatase complex coprecipitates with BCR/ ABL itself, and with another signaling protein previously linked to BCR/ABL transformation, c- CBL. Since both SHP-2 and are believed to have important signaling roles related to proliferation and viability, these results overall suggest that the functions of these two phosphatases are linked. The nature of the interaction of and SHP-2 may be quite complex. In untransformed cell lines, a small amount of SHP-2 was found to be preassociated with when neither protein was signi cantly tyrosine phosphorylated. After growth factor stimulation, both phosphatases in this complex are tyrosine phosphorylated, and it is likely that the overall increase in the complex is mediated through SH2 binding. In fact, during preparation of this manuscript, Liu et al. reported that after IL-3 stimulation the SH2 domain can inducibly bind directly to a phosphotyrosine residue in SHC or SHP-2 (Liu et al., 1997b). In pervanadate treated and growth factor stimulated cells we were unable to detect coprecipitation of SHC and SHP-2 suggesting that can bind to either SHC or SHP-2, but not to both at the same time. Also, increased binding of to SHC correlated with decreased binding to SHP-2, suggesting that SHC and SHP-2 may in fact compete for binding to. In BCR/ABL expressing cells the interactions are more complex and other proteins are contained with this phosphatase complex, such as c-cbl and BCR/ ABL. Since each of the proteins in the phosphatase

5 complex are tyrosine phosphorylated, additional interactions with and SHP-2 could be directed through multiple SH2 domain interactions and involve one or more adapter proteins. Onc consequence of the interaction between and SHP-2 is likely to be altered enzyme activity of either or both enzymes. This is of special interest since has previously been reported to be important in regulating apoptosis (Lioubin et al., 1996; Liu et al., 1997a; Ono et al., 1996). In primary hematopoetic cells, growth factors such as IL-3 and EPO are required to maintain viability, and one of the prominent biological e ects of BCR/ABL in primary CML cells is inhibition of apoptosis (Bedi et al., 1994; Sattler and Salgia, 1997). It will be of considerable interest to examine the role of in the altered regulation of viability that is characteristic of chronic myeloid leukemia cells. It is possible that one function of SHP-2 in this complex is to dephosphorylate, possibly reducing its accumulation at the cell membrane. If so, SHP-2 would then reduce the activity of an enzyme which may negatively regulate apoptosis. An alternative model would be that the enzymatic activity of is directly regulated by one or more tyrosine phosphorylation events. Finally, it is also possible that is not a substrate for SHP-2, but that they function in the same signaling pathway. In any event, the nding that these two phosphatases exist in a complex suggests that their functions are interrelated in a manner which will be of considerable interest to decipher. The role of this phosphatase complex in BCR/ABL transformation is also of interest. Constitutive activation of the Pl3K pathway by BCR/ABL has drawn special attention, since there is increasing evidence that Pl3K is important in transformation by BCR/ABL. Inhibition of Pl3K by the inhibitor wortmannin, a speci c inhibitor of the p11 subunit of Pl3K, inhibits proliferation of BCR/ABL transformed cells (Skorski et al., 1995). We recently described a complex containing Pl3K, c-cbl, and BCR/ABL (Sattler and Salgia, 1997; Sattler et al., 1996). Constitutive binding of Pl3K to tyrosine phosphorylated c-cbl is a possible mechanism of Pl3K activation and might contribute to BCR/ABLmediated transformation. The major in vivo substrates of Pl3K are PtdIns(4)P or PtdIns(4.5)P 2, which are converted by Pl3K to PtdIns(3.4)P 2 or PtdIns(3,4,5)P 3 (Divecha and Irvine, 1995). PtdIns(3,4)P 2 or PtdIns(3,4,5)P 3 are elevated in BCR/ABL transformed cells or after growth factor stimulation and one or both could have multiple activities related to viability, motility and other biological events. One possibility is that the activity is reduced in cells transformed by BCR/ABL, either by phosphorylation, sequestration with BCR/ABL, or reduced expression. This would lead to increased levels of 5-phosphate lipids, and possibly to increased viability and other e ects. It is intriguing that we observed reduced expression of in BCR/ABL transformed cell lines (compared to their untransformed counterparts). At the present binds to the protein tyrosine phosphatase SHP-2 time, we do not know if this is due to reduced protein production or accelerated destruction or both. This would suggest, at least in cell lines, that transformation by BCR/ABL is facilitated by having reduced activity, thus favoring a model in which increased activity leads to decreased viability. It will be of interest to determine the cellular localization of in BCR/ABL transformed cells and to directly look for deregulation of or SHP- 2 activity in these cells. Materials and methods Cells The murine IL-3-dependent pre B-cell line and the BCR/ABL transfected cell line /p21 were cultured as described (Sattler et al., 1995; Sattler et al., 1996). The human erythropoietin receptor expressing cell line /EPOR (obtained from Dr AD d'andrea, Dana-Farber Cancer Institute) were cultured under the same conditions as the parent cell line. Similarly, the murine IL-3-dependent myeloid cell line 32Dcl3 and one derivative, the BCR/ABL transfected 32Dcl3 cell line 32D/ p21 were cultured as described (Salgia et al., 1996). Stimulation of cells and preparation of cellular lysates Growth factor-deprived cells or cells transfected with the receptor for EPO were left untreated or stimulated with IL-3 (Upstate Biotechnology Inc., Lake Placid, NY) or EPO (OrthoBiotech, Raritan, NJ), respectively in RPMI 164 at a concentration of cells/ml. In some experiments cells were treated with mm pervanadate generated by oxidizing a mm vanadate solution with 5 mm peroxide. All cell lines were washed in Dulbecco's PBS at 48C before preparation of lysates as described (Sattler et al., 1995). Immunoprecipitation and Western blotting Immunoprecipitation and immunoblotting using a chemiluminescence technique was performed as described (Sattler et al., 1995). Polyclonal rabbit antisera against SHP-2 (Santa Cruz Biotechnology, Santa Cruz, CA), SHC (Transduction Laboratories, Lexington, KY), CBL (Santa Cruz Biotechnology) and two polyclonal rabbit antisera to (Lioubin et al., 1996) (#534: prepared to amino acid 67 ± 868 of the murine sequence; #5369: prepared to amino acid 889 ± 146 of the murine sequence) were used. Detection of tyrosine phosphorylated proteins in Western blots utilized monoclonal antibody 4G1 (obtained from Dr B Druker, Oregon Health Science University, Portland, OR). Also, the murine monoclonal anti-c-abl antibody Ab-3 (Calbiochem-Novatech, Cambridge, MA) was used for immunoprecipitations and Western blotting. Acknowledgements This work was supported by JoseÂ Carreras International Leukemia Foundation fellowship FIJC-95/INT (to MS), National Institute of Health Grants CA173 (to RS), and DK5654 (to JDG) 2383

6 2384 References binds to the protein tyrosine phosphatase SHP-2 Bazenet CE, Gelderloos JA and Kazlauskas A. (1996). Mol. Cell. Biol., 16, 6926 ± Bedi A, Zehnbauer BA, Barber JP, Sharkis SJ and Jones RJ. (1994). Blood, 83, 38 ± 44. Damen JE, Liu L, Rosten P, Humphries RK, Je erson AB, Majerus PW and Krystal G. (1996). Proc. Natl. Acad. Sci. USA, 93, 1689 ± Divecha N and Irvine RF. (1995). Cell, 8, 269 ± 278. Feng GS, Hui CC and Pawson T. (1993). Science, 259, 17 ± Kavanaugh WM, Pot DA, Chin SM, Deuter-Reinhard M, Je erson AB, Norris FA, Masiarz FR, Cousens LS, Majerus PW and Williams LT. (1996). Curr. Biol., 6, 438 ± 445. Klingho er RA, Duckworth B, Valius M, Cantley L and Kazlauskas A. (1996). Mol. Cell. Biol., 16, 595 ± Lechleider RJ, Sugimoto S, Bennett AM, Kashishian AS, Cooper JA, Shoelson SE, Walsh CT and Neel BG. (1993). J. Biol. Chem., 268, ± Lioubin MN, Algate PA, Tsai S, Carlberg K, Aebersold A and Rohrschneider LR. (1996). Genes Dev., 1, 184 ± 195. Liu L, Damen JE, Cutler RL and Krystal G. (1994). Mol. Cell. Biol., 14, 6926 ± Liu L, Damen JE, Hughes MR, Babic I, Jirik FR and Krystal G. (1997a). J. Biol. Chem., 2, 8983 ± Liu L, Damen JE, Ware MD and Krystal G. (1997b). J. Biol. Chem., 2, 1998 ± 111. Matsuguchi T, Salgia R, Hallek M, Eder M, Druker B, Ernst TJ and Gri n JD. (1994). J. Biol. Chem., 269, 516 ± 521. Ono M, Bolland S, Tempst P and Ravetch JV. (1996). Nature, 383, 263 ± 266. Perkins LA, Johnson MR, Melnick MB and Perrimon N. (1996). Dev. Biol., 18, 63 ± 81. Salgia R, Sattler M, Pisick E, Li J-L and Gri n JD. (1996). Exp. Hematol., 24, 31 ± 313. Sattler M, Durstin MA, Frank DA, Okuda K, Kaushansky K, Salgia R and Gri n JD. (1995). Exp. Hematol., 23, 14 ± 148. Sattler M and Salgia R. (1997). Cytokine Growth Factor Rev., 8, 63 ± 79. Sattler M, Salgia R, Okuda K, Uemura N, Durstin MA, Pisick E, Xu G, Li J-L, Prasad KV and Gri n JD. (1996). Oncogene, 12, 839 ± 846. Skorski T, Kanakaraj P, Nieborowska-Skorska M, Ratajczak MZ, Wen SC, Zon G, Gewirtz AM, Perussia B and Calabretta B. (1995). Blood, 86, 6 ± 736. Tang TL, Freeman Jr R, O'Reilly AM, Neel BG and Sokol SY. (1995). Cell, 8, 473 ± 483. Vogel W, Lammers R, Huang J and Ullrich A. (1993). Science, 259, 1611 ± 1614.

Molecular Characterization of Specific Interactions between SHP-2 Phosphatase and JAK Tyrosine Kinases*

Molecular Characterization of Specific Interactions between SHP-2 Phosphatase and JAK Tyrosine Kinases* THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 272, No. 2, Issue of January 10, pp. 1032 1037, 1997 1997 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Molecular Characterization