Tyrosine Phosphorylation of Shc in Response to B Cell Antigen Receptor Engagement Depends on the SHIP Inositol Phosphatase1

Robert J. Ingham,2* Hidetaka Okada,2† May Dang-Lawson,* Jason Dinglasan,* Peter van der Geer,‡ Tomohiro Kurosaki,§ and Michael R. Gold3*

Tyrosine phosphorylation of Shc in response to B cell Ag receptor (BCR) engagement creates binding sites for the Src homology 2 (SH2) domain of Grb2. This facilitates the recruitment of both Grb2 ⅐ Sos complexes and Grb2 ⅐ SHIP complexes to the plasma membrane where Sos can activate Ras and SH2 domain-containing inositol phosphatase (SHIP) can dephosphorylate phospha- tidylinositol 3,4,5-trisphosphate. Given the importance of Shc phosphorylation, we investigated the mechanism by which the BCR stimulates this response. We found that both the SH2 domain and phosphotyrosine-binding (PTB) domain of Shc are important for BCR-induced tyrosine phosphorylation of Shc and the subsequent binding of Grb2 to Shc. The unexpected finding that the PTB domain of Shc is required for Shc phosphorylation was investigated further. Because the major ligand for the Shc PTB domain is SHIP, we asked whether the interaction of Shc with SHIP was required for BCR-induced tyrosine phosphorylation of Shc. Using SHIP-deficient DT40 cells, we show that SHIP is necessary for the BCR to induce significant levels of Shc tyrosine phosphorylation. BCR-induced tyrosine phosphorylation of Shc could be restored in the these cells by expressing wild-type SHIP but not by expressing a mutant form of SHIP that cannot bind to Shc. This suggests that BCR-induced tyrosine phosphorylation of Shc may depend on the binding of SHIP to the Shc PTB domain. Thus, we have described a novel role for SHIP in BCR signaling, promoting the tyrosine phosphorylation of Shc. The Journal of Immunology, 1999, 163: 5891–5895.

lustering of the B cell Ag receptor (BCR)4 by multivalent binding sites for the SH2 domain of Grb2 (3), an adapter protein Ags or by anti-Ig Abs activates multiple tyrosine kinases that uses its SH3 domains to bind several key signaling enzymes C (1). A prominent substrate of these BCR-associated ty- including Sos and the SH2 domain-containing inositol phosphatase rosine kinases is Shc (2), an adapter protein that consists almost (SHIP). entirely of protein-protein interaction domains. Shc may play an By binding Sos and SHIP (2–6), Shc may be a critical component important role in BCR signaling by facilitating the formation of in several BCR signaling pathways. Sos activates Ras, a GTPase that signaling complexes. The Src homology 2 (SH2) and phosphoty- regulates a kinase cascade culminating in the activation of the extra- rosine-binding (PTB) domains of Shc can bind to phosphoty- cellular signal-regulated kinase (ERK) mitogen-activated protein ki- rosine-containing sequences in other proteins. Moreover, BCR- nase. SHIP dephosphorylates the phosphatidylinositol 3-kinase prod- induced phosphorylation of tyrosines 239 and 313 in Shc creates uct phosphatidylinositol 3,4,5-trisphosphate (PIP3) and may limit the magnitude or duration of BCR signaling events that are dependent upon PIP , such as activation of the Btk tyrosine kinase (7), activation *Department of Microbiology and Immunology, University of British Columbia, 3 Vancouver, British Columbia, Canada; †Department of Obstetrics and Gynecology, of the Akt serine/threonine kinase (8), and increases in intracellular Kansai Medical University, Moriguchi, Japan; ‡Department of Chemistry and Bio- calcium (9). The mechanism by which SHIP binds to Shc is complex. chemistry, University of California at San Diego, La Jolla, CA 92093; and §Depart- ment of Molecular Genetics, Institute for Liver Research, Kansai Medical University, First, the PTB domain of Shc must bind to tyrosine residues on SHIP Moriguchi, Japan that are phosphorylated after BCR ligation (10). Mutations that inac- Received for publication September 1, 1999. Accepted for publication September tivate the Shc PTB domain prevent Shc from binding SHIP (10). 16, 1999. Similarly, changing tyrosines 917 and 1020 of SHIP to phenylala- The costs of publication of this article were defrayed in part by the payment of page nines prevents the creation of the phosphotyrosine-containing se- charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. quences that the Shc PTB domain binds to and blocks the Shc/SHIP 1 This work was supported by a grant from the Medical Research Council of Canada interaction (10). Second, it has been reported that a functional SHIP (to M.R.G) and by grants to T.K. from the Ministry of Education, Science, Sports, and SH2 domain is required for association of SHIP with Shc (11). Fi- Culture of Japan, the Ciba-Geigy Foundation (Japan), the Naito Foundation, and the nally, Grb2 is required to stabilize Shc ⅐ SHIP complexes (6), presum- Toray Science Foundation. R.J.I is a Terry Fox Research Student of the National Cancer Institute of Canada supported by funds provided by the Terry Fox Run. M.R.G ably by simultaneously binding via its SH3 domains to SHIP and is a recipient of a Medical Research Council of Canada Scholarship. binding via its SH2 domain binding to phosphotyrosine residues on 2 R.J.I. and H.O. contributed equally to this work and should be considered co-first Shc. The importance of this Grb2-mediated interaction between Shc authors. and SHIP is highlighted by the fact that SHIP is unable to associate 3 Address correspondence and reprint requests to Dr. Michael R. Gold, Department of with Shc in Grb2-deficient B cells (6). Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Vancouver, British Columbia V6T 1Z3, Canada. E-mail address: Both Sos and SHIP are cytosolic enzymes whose substrates are [email protected] localized to the inner face of the plasma membrane. Sos activates 4 Abbreviations used in this paper: BCR, B cell Ag receptor; SH2, Src homology 2; Ras (12), which is tethered to the inner face of the plasma mem- SHIP, SH2 domain-containing inositol phosphatase; PTB, phosphotyrosine-binding; brane by a lipid anchor, while SHIP dephosphorylates PIP3,a PIP3, phosphatidylinositol 3,4,5-trisphosphate; anti-P-Tyr, anti-phosphotyrosine; ITAM, immunoreceptor tyrosine-based activation motif. plasma membrane phospholipid (13). Thus, both Sos and SHIP

Copyright © 1999 by The American Association of Immunologists 0022-1767/99/$02.00 5892 SHIP IS REQUIRED FOR PHOSPHORYLATION OF Shc BY THE BCR must be recruited to the plasma membrane to perform their func- procedure routinely results in Ͼ95% of the surviving puromycin-resistant tions. This may be accomplished, at least in part, by their Grb2- cells expressing the transferred gene (20). mediated binding to tyrosine-phosphorylated Shc. Although Shc is Expression of SHIP proteins in DT40 cells found in the cytoplasm of resting B cells, it is recruited to the membrane after BCR ligation (2). Recruitment of Shc to the A mutant form of SHIP that is unable to bind to the Shc PTB domain (SHIP plasma membrane may bring it in close proximity to BCR-asso- Y917F/Y1020F) was generated by PCR and cloned into the pApuro ex- pression vector (21). SHIP-deficient DT40 cells (9) were transfected by ciated tyrosine kinases. Tyrosine phosphorylation of this mem- electroporation with cDNA encoding either the wild-type murine SHIP or brane-associated Shc would create a binding site for the Grb2 SH2 SHIP Y917F/Y1020F. After selection with 0.5 ␮g/ml puromycin, SHIP domains and allow Shc to recruit Grb2-containing signaling com- expression was assessed by immunoblotting. plexes to the cell membrane. In B cells stimulated through the BCR, both Grb2 ⅐ Sos complexes and Grb2 ⅐ SHIP complexes bind Cell stimulation, immunoprecipitation, and immunoblotting to tyrosine-phosphorylated Shc (2, 4, 6), and Shc ⅐ Grb2 ⅐ Sos com- WEHI-231 cells were resuspended to 2.5 ϫ 107/ml in modified HEPES- plexes are found in the membrane-enriched particulate fraction of buffered saline (2) and stimulated with 100 ␮g/ml goat anti-mouse IgM 7 ␮ the cells (2). Abs. DT40 cells were resuspended to 10 /ml and stimulated with 4 g/ml of the M4 anti-chicken IgM mAb (22). Reactions were stopped by adding Because Shc must be tyrosine phosphorylated to bind Grb2 and cold PBS containing 1 mM Na3VO4. The cells were solubilized in Triton to recruit Grb2-associated signaling proteins, it is important to un- X-100 lysis buffer (14), and detergent-insoluble material was removed by derstand how the BCR induces phosphorylation of Shc. BCR-in- centrifugation. Immunoprecipitations, immunoblotting, and detection of duced tyrosine phosphorylation of Shc is likely to require the re- immunoreactive bands by enhanced chemiluminescence were performed as described (14, 17). Each experiment was performed at least twice with cruitment of Shc to regions of the plasma membrane where BCR- similar results. activated tyrosine kinases such as Syk are located. This may be mediated by the SH2 domain of Shc binding to phosphotyrosine- Results containing sequences on membrane-associated proteins. In the BCR-induced tyrosine phosphorylation of Shc depends on both RAMOS human B cell line, we have shown that Shc binds via its the SH2 and PTB domains of Shc SH2 domain to Gab1, a membrane-associated docking protein that is tyrosine phosphorylated in response to BCR ligation (14). Shc BCR ligation causes Shc to bind via its SH2 domain to membrane- may also bind via its SH2 domain to the ITAMs (immunoreceptor associated docking proteins such as Gab1 (14). These interactions tyrosine-based activation motifs) in the BCR Ig␣/␤ subunit. The may bring Shc in close proximity to BCR-associated tyrosine ki- Shc SH2 domain can bind in vitro to phosphorylated Ig␣ (15, 16), nases and allow these kinases to phosphorylate Shc. This model and there is some evidence that Shc can bind to Ig␣ in vivo suggests that Shc must have a functional SH2 domain to become (15, 17). tyrosine phosphorylated in response to BCR engagement. To test To test the hypothesis that the Shc SH2 domain is required for this hypothesis, we expressed in WEHI-231 B lymphoma cells a phosphorylation of Shc by the BCR, we expressed in B cells a mutant form of Shc in which the SH2 domain was rendered non- mutant Shc protein in which the SH2 domain had been inactivated functional by a point mutation (Shc R401M). Fig. 1A shows that by a point mutation. As expected, we found that BCR-induced the Shc R401M SH2-domain mutant exhibited decreased BCR- tyrosine phosphorylation of this SH2 domain mutant was signifi- induced tyrosine phosphorylation compared with the wild-type cantly lower than tyrosine phosphorylation of wild-type Shc. Sur- Shc protein. Taking into account that the Shc R401M protein was prisingly, a Shc protein in which the PTB domain was inactivated expressed at somewhat lower levels than the wild-type Shc protein by a point mutation exhibited an even greater reduction in BCR- (e.g., see Fig. 1B, lower panel), densitometry showed that the rel- induced tyrosine phosphorylation. Because SHIP is the major li- ative BCR-induced tyrosine phosphorylation of the Shc R401M gand for the Shc PTB domain, we investigated whether it plays a protein was ϳ50% less than that for the wild-type Shc protein role in phosphorylation of Shc by the BCR. We found that efficient (data not shown). Thus, the SH2 domain of Shc is important for BCR-induced tyrosine phosphorylation of Shc required the expres- Shc to be tyrosine phosphorylated after BCR ligation. sion of SHIP and correlated with the ability of SHIP to bind to the In addition to its SH2 domain, Shc also has a PTB domain that PTB domain of Shc. These data suggest a novel role for SHIP in can bind phosphotyrosine-containing sequences. To determine BCR signaling, promoting the tyrosine phosphorylation of Shc. whether the Shc PTB domain contributed to the ability of Shc to be phosphorylated by the BCR, we expressed in WEHI-231 cells a Shc protein in which the PTB domain was inactivated by a point Materials and Methods mutation (Shc R175M). Surprisingly, we found that this mutation Antibodies reduced BCR-induced tyrosine phosphorylation of Shc to an even Abs to Shc, SHIP, and Sos1, as well as to the 4G10 anti-phosphotyrosine greater extent than the SH2 domain mutation (Fig. 1A). Thus, the (anti-P-Tyr) mAb were from Upstate Biotechnology (Lake Placid, NY). PTB domain of Shc also appears to be important for phosphory- The M2 anti-FLAG mAb was from Babco (Berkeley, CA). The anti-Grb2 lation of Shc by the BCR. Ab was from Santa Cruz Biotechnology (Santa Cruz, CA). Binding of SHIP to Shc depends on both the SH2 and PTB Expression of Shc proteins in WEHI-231 cells domains of Shc cDNAs encoding wild-type human Shc or Shc proteins with a point mu- Although expressing the Shc R401M and Shc R175M proteins in tation that inactivates either the SH2 domain (R401M) or the PTB domain WEHI-231 cells had no effect on overall BCR-induced tyrosine (R175M) were generated by PCR, amino-terminally tagged with the FLAG epitope, and cloned into the pMSCVpac retroviral expression vector (18). phosphorylation of proteins in total cell lysates (data not shown), The protocol for producing retrovirus particles and infecting WEHI-231 Fig. 1A shows that mutating the SH2 and PTB domains of Shc cells has been described in detail (19, 20). Briefly, 48 h after transfecting reduced the ability of Shc to bind other tyrosine-phosphorylated BOSC 23 cells with 2 ␮g of DNA, the retrovirus-containing culture su- proteins. Association of Shc with an unidentified 70-kDa phos- pernatants were collected and 2 ml of the supernatant was used to infect 5 ϫ 105 WEHI-231 cells. At 48 h postinfection, 0.25 ␮g/ml puromycin was phoprotein that did not react with anti-Syk Abs (R. Ingham, un- added to the cells. After 5 days of selection, stable populations of WEHI- published observation) required a functional SH2 domain but was 231 cells expressing the transfected gene were used for experiments. This unaffected by inactivation of the PTB domain. In contrast, the The Journal of Immunology 5893

FIGURE 2. BCR-induced binding of Grb2 to Shc depends on both the SH2 and PTB domains of Shc. The BOSC 23 packaging cell line was transfected with the pMSCVpac vector or pMSCVpac encoding the indi- cated Shc proteins. The resulting retrovirus particles were used to infect WEHI-231 cells. The WEHI-231 cells were then stimulated with anti-IgM Abs for the indicated times. Cell lysates were immunoprecipitated with the M2 anti-FLAG mAb and the precipitated proteins were analyzed by im- munoblotting with an anti-Grb2 Ab (upper panel). The blot then was FIGURE 1. BCR-induced tyrosine phosphorylation of Shc depends on stripped and reprobed with the M2 anti-FLAG mAb (lower panel). Mo- both the SH2 and PTB domains of Shc. The BOSC 23 packaging cell line lecular mass standards (in kDa) are indicated to the left. was transfected with the pMSCVpac vector or pMSCVpac containing cDNA encoding FLAG-tagged wild-type Shc (wt Shc), a Shc protein in which the SH2 domain was inactivated (Shc R401M), or a Shc protein in which the PTB domain was inactivated (Shc R175M). The resulting retro- virus particles were used to infect WEHI-231 cells. The WEHI-231 cells primarily on phosphorylation of tyrosine 239 of murine Shc and, to were then stimulated with anti-IgM Abs for the indicated times. Cell ly- a lesser extent, on phosphorylation of tyrosine 313. We found that sates were immunoprecipitated with the M2 anti-FLAG mAb, and the pre- BCR-induced association of Grb2 with Shc was greatly decreased cipitated proteins were analyzed by immunoblotting with either the 4G10 by inactivating either the SH2 domain (Shc R401M mutant) or the anti-P-Tyr mAb (A, upper panel) or an anti-SHIP Ab (B, upper panel). The PTB domain (Shc R175M mutant) of Shc (Fig. 2). Thus, Shc re- positions of the tyrosine phosphorylated FLAG-tagged Shc proteins (P- FLAG-Shc) (A, upper panel) and SHIP (B, upper panel) are indicated by quires both a functional SH2 domain and a functional PTB domain arrows. The blots were then stripped and reprobed with either an anti-Shc in order for BCR-associated tyrosine kinases to phosphorylate it on Ab (A, lower panel) or an anti-FLAG mAb (B, lower panel). Note that the sites that are important for Grb2 binding. bands in the “vector” lanes of the anti-Shc reprobe (A, lower panel) are the heavy chain of the Ab used for immunoprecipitation. Molecular mass stan- BCR-induced tyrosine phosphorylation of Shc depends on SHIP dards (in kDa) are indicated to the left. The unexpected finding that the PTB domain of Shc is required for BCR-induced tyrosine phosphorylation of Shc was investigated further. Because the major ligand for the Shc PTB domain is SHIP binding of a 140-kDa phosphoprotein to Shc was completely ab- (10), we asked whether the interaction of Shc with SHIP was re- lated by mutating the Shc PTB domain and was greatly reduced by quired for BCR-induced Shc phosphorylation. Because SHIP can mutating the Shc SH2 domain. Because SHIP binds to the Shc associate with the Syk tyrosine kinase (5), it is possible that the PTB domain and has a molecular mass of 135–140 kDa, we asked binding of SHIP to the Shc PTB domain brings Syk close to Shc whether the binding of SHIP to Shc showed the same dependence and that this facilitates Shc phosphorylation. To address whether on both the Shc PTB domain and the Shc SH2 domain. Immuno- SHIP is required for phosphorylation of Shc by the BCR, we made blotting with anti-SHIP Abs showed that after BCR ligation SHIP use of DT40 chicken B cells in which the genes encoding SHIP bound strongly to wild-type Shc (Fig. 1B). As expected, SHIP did have been disrupted (9). Fig. 3A shows that tyrosine phosphory- not bind to the Shc R175M protein which has a nonfunctional PTB lation of the three isoforms of chicken Shc was reduced in SHIP- domain. Interestingly, very little SHIP bound to the Shc R401M deficient DT40 cells compared with wild-type DT40 cells. Ex- protein which has a nonfunctional SH2 domain. This is probably a pressing an exogenous wild-type SHIP in the SHIP-deficient DT40 consequence of the decreased tyrosine phosphorylation of the Shc B cells restored the ability of the BCR to induce tyrosine phos- R401M protein compared with wild-type Shc (see Fig. 1A). Ty- phorylation of Shc (Fig. 3B). Thus, BCR-induced tyrosine phos- rosine phosphorylation of Shc is required for Grb2 to bind to Shc phorylation of Shc depends on the expression of SHIP. and for Grb2 to stabilize the interaction between SHIP and Shc (6). We also expressed in WEHI-231 cells a mutant form of SHIP Indeed, the Shc R401M protein shows significantly reduced bind- (SHIP Y917F/Y1020F) that lacks the tyrosine residues which upon ing of Grb2 after BCR ligation (see below and Fig. 2). Thus, both phosphorylation mediate the binding of SHIP to the Shc PTB do- the PTB domain and the SH2 domain of Shc contribute to the main (10). Fig. 3C shows that this SHIP Y917F/Y1020F protein ability of Shc to bind SHIP. did indeed have a greatly decreased ability to bind to Shc after BCR ligation compared with wild-type SHIP. This correlated with BCR-induced binding of Grb2 to Shc depends on both the SH2 the inability of this SHIP Y917F/Y1020F protein to restore BCR- and PTB domains of Shc induced tyrosine phosphorylation of Shc (Fig. 3B). This is in con- Because mutating either the Shc SH2 domain or the Shc PTB trast to the wild-type SHIP which bound to Shc after BCR ligation domain reduced BCR-induced tyrosine phosphorylation of Shc, we and fully restored BCR-induced tyrosine phosphorylation of Shc in asked whether this correlated with a decreased ability of these the SHIP-deficient DT40 cells. The simplest interpretation of this mutant Shc proteins to bind Grb2. Harmer et al. (3) showed that data is that BCR-induced tyrosine phosphorylation of Shc depends the binding of Grb2 to Shc in anti-Ig-stimulated B cells depends on the binding of SHIP to the PTB domain of Shc. 5894 SHIP IS REQUIRED FOR PHOSPHORYLATION OF Shc BY THE BCR

FIGURE 3. BCR-induced tyrosine phosphorylation of Shc depends on the binding of SHIP to the PTB domain of Shc. A, Wild-type (wt) or SHIP- deficient (SHIPϪ/Ϫ) DT40 cells were stimulated with the M4 anti-IgM mAb for the indicated times. Cell lysates were immunoprecipitated with an anti-Shc Ab and the immunoprecipitates were divided into two equal fractions. One fraction was analyzed by immunoblotting with the anti-P-Tyr mAb (upper panel), whereas the other fraction was blotted with an anti-Shc Ab (lower panel). B and C, SHIP-deficient (SHIPϪ/Ϫ) DT40 cells expressing either a transfected wild-type SHIP protein (wt SHIP) or a transfected SHIP protein in which the tyrosine residues critical for binding the Shc PTB domain had been mutated (Y917F/Y1020F SHIP) were stimulated with the M4 anti-IgM Ab for the indicated times. Cell lysates were immunoprecipitated with an anti-Shc Ab, and the immunoprecipitates were divided into two equal fractions. The phosphorylation of Shc was analyzed in B. One fraction was analyzed by immunoblotting with the anti-P-Tyr mAb (upper panel), whereas the other fraction was blotted with an anti-Shc Ab (lower panel). The binding of SHIP to Shc was analyzed in C. One fraction was analyzed by immunoblotting with an anti-SHIP Ab (upper panel), whereas the other fraction was blotted with an anti-Shc Ab (lower panel). Molecular mass standards (in kDa) are indicated to the left of each panel.

Discussion during immunoprecipitation. An alternative model is that the Shc PTB domain binds to molecules other than SHIP and in this way In this report, we show for the first time that BCR-induced tyrosine brings Shc close to SHIP ⅐ Syk complexes. However, no tyrosine- phosphorylation of Shc strongly depends on 1) Shc having a func- phosphorylated proteins other than SHIP bound to the Shc R401M tional PTB domain and 2) the expression of SHIP, a protein that protein which has a functional PTB domain but an inactivated SH2 binds to the PTB domain of Shc after BCR ligation. We also domain (Fig. 1A). Moreover, in the context of this model, it is showed that the ability of SHIP to promote BCR-induced tyrosine not clear why mutating tyrosines 917 and 1020 in SHIP would phosphorylation of Shc correlated with its ability to bind to Shc. BCR-induced tyrosine phosphorylation of Shc could be restored in prevent SHIP from facilitating the tyrosine phosphorylation of Shc SHIP-deficient DT40 cells by expressing wild-type SHIP but not (Fig. 3C). by expressing the SHIP Y917F/Y1020F protein which cannot bind A distinct model that could account for the role of the Shc PTB to the Shc PTB domain. Although we cannot rule out that tyrosines domain and SHIP in regulating Shc phosphorylation is that the ⅐ 917 and 1020 of SHIP perform some other role in promoting Shc binding of Grb2 SHIP complexes to Shc protects Shc from de- phosphorylation, the simplest interpretation of our data is that phosphorylation by tyrosine phosphatases. In this model, the bind- SHIP must bind to the PTB domain of Shc to promote phosphor- ing of SHIP to Shc does not promote Shc tyrosine phosphorylation ylation of Shc. Regardless of the mechanism, we have clearly but instead contributes to the maintenance of Shc phosphorylation. Stable binding of SHIP to Shc requires two distinct interactions: 1) shown that in addition to dephosphorylating PIP3, SHIP has a sec- ond role in BCR signaling: promoting the tyrosine phosphorylation the binding of phosphotyrosine-containing sequences on SHIP to of Shc. the Shc PTB domain (10) and 2) the bridging of SHIP to Shc by One model that would explain the requirement for both the Shc Grb2 (6). The SH3 domains of Grb2 bind to proline-rich regions PTB domain and SHIP in promoting the tyrosine phosphorylation on SHIP, while the SH2 domain of Grb2 binds to phosphotyrosine- of Shc is that the binding of SHIP to the Shc PTB domain brings containing sequences on Shc. In the SHIP-deficient DT40 cells, a tyrosine kinase close to Shc. This kinase is likely to be Syk even if Shc were tyrosine phosphorylated, there would be no because efficient BCR-induced phosphorylation of Shc requires Grb2 ⅐ SHIP complexes to bind to the phosphotyrosines on Shc Syk (17, 23). Although SHIP has been shown to bind to both Shc and protect them from phosphatases. Similarly, the Shc R175M and Syk in anti-Ig-stimulated B cells, Shc ⅐ SHIP ⅐ Syk complexes protein in which the PTB domain has been inactivated would be un- have not been detected (5). It is possible that such ternary com- able to recruit and stably bind Grb2 ⅐ SHIP complexes. Moreover, the plexes are of low abundance, exist only transiently, or dissociate SHIP Y907F/Y1020F protein which does not bind efficiently to Shc The Journal of Immunology 5895 would also be unable to protect the phosphotyrosines on Shc from ing SHC, GRB-2, mSos1, and a 145-kDa tyrosine-phosphorylated protein. J. Im- phosphatases. If the Grb2 ⅐ SHIP complexes do indeed protect Shc munol. 153:623. 3. Harmer, S. L., and A. L. DeFranco. 1997. Shc contains two Grb2 binding sites from dephosphorylation, our results imply that other Grb2 complexes needed for efficient formation of complexes with Sos in B lymphocytes. Mol. (e.g., Grb2 ⅐ Sos complexes) as well as free Grb2 cannot perform the Cell. Biol. 17:4087. 4. Lankester, A. C., G. M. van Schijndel, P. M. Rood, A. J. Verhoeven, and same function, either because they are much less abundant than R. A. van Lier. 1994. B cell antigen receptor cross-linking induces tyrosine phos- Grb2 ⅐ SHIP complexes in B cells or because they cannot bind stably phorylation and membrane translocation of a multimeric Shc complex that is to phosphorylated Shc if SHIP is not also present in the complex. augmented by CD19 co-ligation. Eur. J. Immunol. 24:2818. 5. Crowley, M. T., S. L. Harmer, and A. L. DeFranco. 1996. Activation-induced The models in which SHIP either promotes or maintains BCR- association of a 145-kDa tyrosine-phosphorylated protein with Shc and Syk in B induced tyrosine phosphorylation of Shc are not mutually exclu- lymphocytes and macrophages. J. Biol. Chem. 271:1145. sive. SHIP could recruit a tyrosine kinase to Shc while the subse- 6. Harmer, S. L., and A. L. DeFranco. 1999. The Src homology domain 2-contain- ⅐ ing inositol phosphatase SHIP forms a ternary complex with Shc and Grb2 in quent binding of Grb2 SHIP complexes to Shc could protect the antigen receptor-stimulated B cells. J. Biol. Chem. 274:12183. phosphorylated tyrosine residues from dephosphorylation by phos- 7. Bolland, S., R. N. Pearse, T. Kurosaki, and J. V. Ravetch. 1998. SHIP modulates phatases. The interactions between SHIP, Syk, Shc, Grb2, and Sos immune receptor responses by regulating membrane association of Btk. Immunity 8:509. are very complex and make it difficult to design unequivocal ex- 8. Aman, M. J., T. D. Lamkin, H. Okada, T. Kurosaki, and K. S. Ravichandran. periments to determine whether the binding of SHIP to Shc pro- 1998. The inositol phosphatase SHIP inhibits Akt/PKB activation in B cells. motes the phosphorylation of Shc, protects phosphorylated Shc J. Biol. Chem. 273:33922. 9. Okada, H., S. Bolland, A. Hashimoto, M. Kurosaki, Y. Kabuyama, M. Iino, and from tyrosine phosphatases, or facilitates both of these processes. T. Kurosaki. 1998. Role of the inositol phosphatase SHIP in B cell receptor- Nevertheless, we have clearly shown for the first time that BCR- induced Ca2ϩ oscillatory response. J. Immunol. 161:5129. induced tyrosine phosphorylation of Shc depends on the PTB do- 10. Lamkin, T. D., S. F. Walk, L. Liu, J. E. Damen, G. Krystal, and K. S. Ravichandran. 1997. Shc interaction with Src homology 2 domain contain- main of Shc and on the expression of SHIP, a protein that binds to ing inositol phosphatase (SHIP) in vivo requires the Shc-phosphotyrosine binding the Shc PTB domain. domain and two specific phosphotyrosines on SHIP. J. Biol. Chem. 272:10396. In addition to its PTB domain, the SH2 domain of Shc also 11. Liu, L., J. E. Damen, M. R. Hughes, I. Babic, F. R. Jirik, and G. Krystal. 1997. The Src homology 2 (SH2) domain of SH2-containing inositol phosphatase contributes to the ability of Shc to be tyrosine phosphorylated after (SHIP) is essential for tyrosine phosphorylation of SHIP, its association with Shc, BCR ligation. One possibility is that the Shc SH2 domain is re- and its induction of apoptosis. J. Biol. Chem. 272:8983. ⅐ 12. Egan, S. E., B. W. Giddings, M. W. Brooks, L. Buday, A. M. Sizeland, and quired for co-localizing Shc with SHIP Syk complexes. This may R. A. Weinberg. 1993. Association of Sos Ras exchange protein with Grb2 is involve translocation of Shc from the cytosol to the cell membrane. implicated in tyrosine kinase signal transduction and transformation. Nature Syk binds to the phosphorylated BCR ITAMs in activated B cells 363:45. 13. Damen, J. E., L. Liu, P. Rosten, R. K. Humphries, A. B. Jefferson, P. W. Majerus, (24) and SHIP is found in the membrane fraction of anti- and G. Krystal. 1996. The 145-kDa protein induced to associate with Shc by Ig-stimulated B cells (2). The Shc SH2 domain could mediate multiple cytokines is an inositol tetraphosphate and phosphatidylinositol 3,4,5- membrane translocation of Shc by binding membrane-associated triphosphate 5-phosphatase. Proc. Natl. Acad. Sci. USA 93:1689. 14. Ingham, R. J., M. Holgado-Madruga, C. Siu, A. J. Wong, and M. R. Gold. 1998. proteins such as Gab1 which are tyrosine phosphorylated after The Gab1 protein is a docking site for multiple proteins involved in signaling by BCR ligation. However, we cannot rule out other mechanisms by the B cell antigen receptor. J. Biol. Chem. 273:30630. which the Shc SH2 domain contributes to making Shc a better 15. D’Ambrosio, D., K. L. Hippen, and J. C. Cambier. 1996. Distinct mechanisms mediate SHC association with the activated and resting B cell antigen receptor. substrate for BCR-activated tyrosine kinases. Eur. J. Immunol. 26:1960. 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Versatile ret- protein interactions that are required for BCR-induced roviral vectors for potential use in gene therapy. Gene. Ther. 1:136. 19. Pear, W. S., G. P. Nolan, M. L. Scott, and D. Baltimore. 1993. Production of high phosphorylation of Shc. titer helper-free retroviruses by transient transfection. Proc. Natl. Acad. Sci. USA 90:8392. Acknowledgments 20. Krebs, D. L., Y. Yang, M. Dang, J. Haussmann, and M. R. Gold. 1999. Rapid and efficient retrovirus-mediated gene transfer into B cell lines. Methods Cell Sci. We thank Mari Kurosaki for technical help, Dr. Robert Hawley for the 21:57. pMSCVpac retroviral expression vector, and Drs. Anthony DeFranco and 21. Takata, M., H. Sabe, A. Hata, T. Inazu, Y. Homma, T. Nukada, and T. Kurosaki. 1994. Tyrosine kinases Lyn and Syk regulate B cell receptor-coupled Ca2ϩ mo- Linda Matsuuchi for critically reading the manuscript. bilization through distinct pathways. EMBO J. 13:1341. 22. Chen, C. L., J. E. Lehmeyer, and M. D. Cooper. 1982. Evidence for an IgD References homologue on chicken lymphocytes. J. Immunol. 129:2580. 23. Richards, J. D., M. R. Gold, S. L. Hourihane, A. L. DeFranco, and L. Matsuuchi. 1. DeFranco, A. L. 1997. The complexity of signaling pathways activated by the 1996. Reconstitution of B cell antigen receptor-induced signaling events in a BCR. Curr. Opin. Immunol. 9:296. nonlymphoid cell line by expressing the Syk protein-tyrosine kinase. J. Biol. 2. Saxton, T. M., I. van Oostveen, D. Bowtell, R. Aebersold, and M. R. Gold. 1994. Chem. 271:6458. B cell antigen receptor cross-linking induces phosphorylation of the p21ras on- 24. Kurosaki, T. 1997. Molecular mechanisms in B cell antigen receptor signaling. coprotein activators SHC and mSos1 as well as assembly of complexes contain- Curr. Opin. Immunol. 9:307.