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*
Department of Microbiology and Immunology, University of British Columbia, Vancouver, British Columbia, Canada;
Department of Obstetrics and Gynecology, Kansai Medical University, Moriguchi, Japan;
Department of Chemistry and Biochemistry, University of California at San Diego, La Jolla, CA 92093; and
§
Department of Molecular Genetics, Institute for Liver Research, Kansai Medical University, Moriguchi, Japan
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Abstract |
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 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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By binding Sos and SHIP (2, 3, 4, 5, 6), Shc may be a critical component in several BCR signaling pathways. Sos activates Ras, a GTPase that regulates a kinase cascade culminating in the activation of the extracellular signal-regulated kinase (ERK) mitogen-activated protein kinase. SHIP dephosphorylates the phosphatidylinositol 3-kinase product phosphatidylinositol 3,4,5-trisphosphate (PIP3) and may limit the magnitude or duration of BCR signaling events that are dependent upon PIP3, such as activation of the Btk tyrosine kinase (7), activation of the Akt serine/threonine kinase (8), and increases in intracellular calcium (9). The mechanism by which SHIP binds to Shc is complex. First, the PTB domain of Shc must bind to tyrosine residues on SHIP that are phosphorylated after BCR ligation (10). Mutations that inactivate the Shc PTB domain prevent Shc from binding SHIP (10). Similarly, changing tyrosines 917 and 1020 of SHIP to phenylalanines prevents the creation of the phosphotyrosine-containing sequences that the Shc PTB domain binds to and blocks the Shc/SHIP interaction (10). Second, it has been reported that a functional SHIP SH2 domain is required for association of SHIP with Shc (11). Finally, Grb2 is required to stabilize Shc · SHIP complexes (6), presumably by simultaneously binding via its SH3 domains to SHIP and binding via its SH2 domain binding to phosphotyrosine residues on Shc. The importance of this Grb2-mediated interaction between Shc and SHIP is highlighted by the fact that SHIP is unable to associate with Shc in Grb2-deficient B cells (6).
Both Sos and SHIP are cytosolic enzymes whose substrates are localized to the inner face of the plasma membrane. Sos activates Ras (12), which is tethered to the inner face of the plasma membrane by a lipid anchor, while SHIP dephosphorylates PIP3, a plasma membrane phospholipid (13). Thus, both Sos and SHIP must be recruited to the plasma membrane to perform their functions. This may be accomplished, at least in part, by their Grb2-mediated binding to tyrosine-phosphorylated Shc. Although Shc is found in the cytoplasm of resting B cells, it is recruited to the membrane after BCR ligation (2). Recruitment of Shc to the plasma membrane may bring it in close proximity to BCR-associated tyrosine kinases. Tyrosine phosphorylation of this membrane-associated Shc would create a binding site for the Grb2 SH2 domains and allow Shc to recruit Grb2-containing signaling complexes to the cell membrane. In B cells stimulated through the BCR, both Grb2 · Sos complexes and Grb2 · SHIP complexes bind to tyrosine-phosphorylated Shc (2, 4, 6), and Shc · Grb2 · Sos complexes are found in the membrane-enriched particulate fraction of the cells (2).
Because Shc must be tyrosine phosphorylated to bind Grb2 and to recruit
Grb2-associated signaling proteins, it is important to understand how
the BCR induces phosphorylation of Shc. BCR-induced tyrosine
phosphorylation of Shc is likely to require the recruitment of Shc to
regions of the plasma membrane where BCR-activated tyrosine kinases
such as Syk are located. This may be mediated by the SH2 domain of Shc
binding to phosphotyrosine-containing sequences on membrane-associated
proteins. In the RAMOS human B cell line, we have shown that Shc binds
via its SH2 domain to Gab1, a membrane-associated docking protein that
is tyrosine phosphorylated in response to BCR ligation
(14). Shc may also bind via its SH2 domain to the ITAMs
(immunoreceptor tyrosine-based activation motifs) in the BCR Ig
/ß
subunit. The Shc SH2 domain can bind in vitro to phosphorylated Ig
(15, 16), and there is some evidence that Shc can bind to
Ig in vivo (15, 17).
To test the hypothesis that the Shc SH2 domain is required for phosphorylation of Shc by the BCR, we expressed in B cells a mutant Shc protein in which the SH2 domain had been inactivated by a point mutation. As expected, we found that BCR-induced tyrosine phosphorylation of this SH2 domain mutant was significantly lower than tyrosine phosphorylation of wild-type Shc. Surprisingly, a Shc protein in which the PTB domain was inactivated by a point mutation exhibited an even greater reduction in BCR-induced tyrosine phosphorylation. Because SHIP is the major ligand for the Shc PTB domain, we investigated whether it plays a role in phosphorylation of Shc by the BCR. We found that efficient BCR-induced tyrosine phosphorylation of Shc required the expression of SHIP and correlated with the ability of SHIP to bind to the PTB domain of Shc. These data suggest a novel role for SHIP in BCR signaling, promoting the tyrosine phosphorylation of Shc.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Abs to Shc, SHIP, and Sos1, as well as to the 4G10 anti-phosphotyrosine (anti-P-Tyr) mAb were from Upstate Biotechnology (Lake Placid, NY). The M2 anti-FLAG mAb was from Babco (Berkeley, CA). The anti-Grb2 Ab was from Santa Cruz Biotechnology (Santa Cruz, CA).
Expression of Shc proteins in WEHI-231 cells
cDNAs encoding wild-type human Shc or Shc proteins with a point mutation that inactivates either the SH2 domain (R401M) or the PTB domain (R175M) were generated by PCR, amino-terminally tagged with the FLAG epitope, and cloned into the pMSCVpac retroviral expression vector (18). The protocol for producing retrovirus particles and infecting WEHI-231 cells has been described in detail (19, 20). Briefly, 48 h after transfecting BOSC 23 cells with 2 µg of DNA, the retrovirus-containing culture supernatants were collected and 2 ml of the supernatant was used to infect 5 x 105 WEHI-231 cells. At 48 h postinfection, 0.25 µg/ml puromycin was added to the cells. After 5 days of selection, stable populations of WEHI-231 cells expressing the transfected gene were used for experiments. This procedure routinely results in >95% of the surviving puromycin-resistant cells expressing the transferred gene (20).
Expression of SHIP proteins in DT40 cells
A mutant form of SHIP that is unable to bind to the Shc PTB domain (SHIP Y917F/Y1020F) was generated by PCR and cloned into the pApuro expression vector (21). SHIP-deficient DT40 cells (9) were transfected by electroporation with cDNA encoding either the wild-type murine SHIP or SHIP Y917F/Y1020F. After selection with 0.5 µg/ml puromycin, SHIP expression was assessed by immunoblotting.
Cell stimulation, immunoprecipitation, and immunoblotting
WEHI-231 cells were resuspended to 2.5 x 107/ml in modified HEPES-buffered saline (2) and stimulated with 100 µg/ml goat anti-mouse IgM Abs. DT40 cells were resuspended to 107/ml and stimulated with 4 µg/ml of the M4 anti-chicken IgM mAb (22). Reactions were stopped by adding cold PBS containing 1 mM Na3VO4. The cells were solubilized in Triton X-100 lysis buffer (14), and detergent-insoluble material was removed by centrifugation. Immunoprecipitations, immunoblotting, and detection of immunoreactive bands by enhanced chemiluminescence were performed as described (14, 17). Each experiment was performed at least twice with similar results.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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BCR ligation causes Shc to bind via its SH2 domain to
membrane-associated docking proteins such as Gab1 (14).
These interactions may bring Shc in close proximity to BCR-associated
tyrosine kinases and allow these kinases to phosphorylate Shc. This
model suggests that Shc must have a functional SH2 domain to become
tyrosine phosphorylated in response to BCR engagement. To test this
hypothesis, we expressed in WEHI-231 B lymphoma cells a mutant form of
Shc in which the SH2 domain was rendered nonfunctional by a point
mutation (Shc R401M). Fig. 1
A
shows that the Shc R401M SH2-domain mutant exhibited decreased
BCR-induced tyrosine phosphorylation compared with the wild-type Shc
protein. Taking into account that the Shc R401M protein was expressed
at somewhat lower levels than the wild-type Shc protein (e.g., see Fig. 1B, lower panel), densitometry showed that the
relative BCR-induced tyrosine phosphorylation of the Shc R401M protein
was 
50% less than that for the wild-type Shc protein (data not
shown). Thus, the SH2 domain of Shc is important for Shc to be tyrosine
phosphorylated after BCR ligation.
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A). Thus, the PTB domain of Shc also appears
to be important for phosphorylation of Shc by the BCR. Binding of SHIP to Shc depends on both the SH2 and PTB domains of Shc
Although expressing the Shc R401M and Shc R175M proteins in
WEHI-231 cells had no effect on overall BCR-induced tyrosine
phosphorylation of proteins in total cell lysates (data not shown),
Fig. 1A shows that mutating the SH2 and PTB domains of Shc
reduced the ability of Shc to bind other tyrosine-phosphorylated
proteins. Association of Shc with an unidentified 70-kDa phosphoprotein
that did not react with anti-Syk Abs (R. Ingham, unpublished
observation) required a functional SH2 domain but was unaffected by
inactivation of the PTB domain. In contrast, the binding of a 140-kDa
phosphoprotein to Shc was completely ablated by mutating the Shc PTB
domain and was greatly reduced by mutating the Shc SH2 domain. Because
SHIP binds to the Shc PTB domain and has a molecular mass of 135–140
kDa, we asked whether the binding of SHIP to Shc showed the same
dependence on both the Shc PTB domain and the Shc SH2 domain.
Immunoblotting with anti-SHIP Abs showed that after BCR ligation
SHIP bound strongly to wild-type Shc (Fig. 1B). As expected,
SHIP did not bind to the Shc R175M protein which has a nonfunctional
PTB domain. Interestingly, very little SHIP bound to the Shc R401M
protein which has a nonfunctional SH2 domain. This is probably a
consequence of the decreased tyrosine phosphorylation of the Shc R401M
protein compared with wild-type Shc (see Fig. 1A). Tyrosine
phosphorylation of Shc is required for Grb2 to bind to Shc and for Grb2
to stabilize the interaction between SHIP and Shc (6).
Indeed, the Shc R401M protein shows significantly reduced binding of
Grb2 after BCR ligation (see below and Fig. 2). Thus, both the PTB domain and the SH2
domain of Shc contribute to the ability of Shc to bind SHIP.
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Because mutating either the Shc SH2 domain or the Shc PTB domain
reduced BCR-induced tyrosine phosphorylation of Shc, we asked whether
this correlated with a decreased ability of these mutant Shc proteins
to bind Grb2. Harmer et al. (3) showed that the binding of
Grb2 to Shc in anti-Ig-stimulated B cells depends primarily on
phosphorylation of tyrosine 239 of murine Shc and, to a lesser extent,
on phosphorylation of tyrosine 313. We found that BCR-induced
association of Grb2 with Shc was greatly decreased by inactivating
either the SH2 domain (Shc R401M mutant) or the PTB domain (Shc R175M
mutant) of Shc (Fig. 2). Thus, Shc requires both a functional SH2
domain and a functional PTB domain in order for BCR-associated tyrosine
kinases to phosphorylate it on sites that are important for Grb2
binding.
BCR-induced tyrosine phosphorylation of Shc depends on SHIP
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
(10), we asked whether the interaction of Shc with SHIP
was required for BCR-induced Shc phosphorylation. Because SHIP can
associate with the Syk tyrosine kinase (5), it is possible
that the binding of SHIP to the Shc PTB domain brings Syk close to Shc
and that this facilitates Shc phosphorylation. To address whether SHIP
is required for phosphorylation of Shc by the BCR, we made use of DT40
chicken B cells in which the genes encoding SHIP have been disrupted
(9). Fig. 3A
shows that tyrosine phosphorylation of the three isoforms of chicken
Shc was reduced in SHIP-deficient DT40 cells compared with wild-type
DT40 cells. Expressing an exogenous wild-type SHIP in the
SHIP-deficient DT40 B cells restored the ability of the BCR to induce
tyrosine phosphorylation of Shc (Fig. 3B). Thus, BCR-induced
tyrosine phosphorylation of Shc depends on the expression of
SHIP.
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C shows that this SHIP Y917F/Y1020F
protein did indeed have a greatly decreased ability to bind to Shc
after BCR ligation compared with wild-type SHIP. This correlated with
the inability of this SHIP Y917F/Y1020F protein to restore BCR-induced
tyrosine phosphorylation of Shc (Fig. 3B). This is in
contrast to the wild-type SHIP which bound to Shc after BCR ligation
and fully restored BCR-induced tyrosine phosphorylation of Shc in the
SHIP-deficient DT40 cells. The simplest interpretation of this data is
that BCR-induced tyrosine phosphorylation of Shc depends on the binding
of SHIP to the PTB domain of Shc. |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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One model that would explain the requirement for both the Shc PTB
domain and SHIP in promoting the tyrosine phosphorylation of Shc is
that the binding of SHIP to the Shc PTB domain brings a tyrosine kinase
close to Shc. This kinase is likely to be Syk because efficient
BCR-induced phosphorylation of Shc requires Syk (17, 23).
Although SHIP has been shown to bind to both Shc and Syk in
anti-Ig-stimulated B cells, Shc · SHIP · Syk complexes have
not been detected (5). It is possible that such ternary
complexes are of low abundance, exist only transiently, or dissociate
during immunoprecipitation. An alternative model is that the Shc PTB
domain binds to molecules other than SHIP and in this way brings Shc
close to SHIP · Syk complexes. However, no tyrosine-phosphorylated
proteins other than SHIP bound to the Shc R401M protein which has a
functional PTB domain but an inactivated SH2 domain (Fig. 1A). Moreover, in the context of this model, it is not
clear why mutating tyrosines 917 and 1020 in SHIP would prevent SHIP
from facilitating the tyrosine phosphorylation of Shc (Fig. 3C).
A distinct model that could account for the role of the Shc PTB domain and SHIP in regulating Shc phosphorylation is that the binding of Grb2 · SHIP complexes to Shc protects Shc from dephosphorylation by tyrosine phosphatases. In this model, the binding of SHIP to Shc does not promote Shc tyrosine phosphorylation but instead contributes to the maintenance of Shc phosphorylation. Stable binding of SHIP to Shc requires two distinct interactions: 1) the binding of phosphotyrosine-containing sequences on SHIP to the Shc PTB domain (10) and 2) the bridging of SHIP to Shc by Grb2 (6). The SH3 domains of Grb2 bind to proline-rich regions on SHIP, while the SH2 domain of Grb2 binds to phosphotyrosine-containing sequences on Shc. In the SHIP-deficient DT40 cells, even if Shc were tyrosine phosphorylated, there would be no Grb2 · SHIP complexes to bind to the phosphotyrosines on Shc and protect them from phosphatases. Similarly, the Shc R175M protein in which the PTB domain has been inactivated would be unable to recruit and stably bind Grb2 · SHIP complexes. Moreover, the SHIP Y907F/Y1020F protein which does not bind efficiently to Shc would also be unable to protect the phosphotyrosines on Shc from phosphatases. If the Grb2 · SHIP complexes do indeed protect Shc from dephosphorylation, our results imply that other Grb2 complexes (e.g., Grb2 · Sos complexes) as well as free Grb2 cannot perform the same function, either because they are much less abundant than Grb2 · SHIP complexes in B cells or because they cannot bind stably to phosphorylated Shc if SHIP is not also present in the complex.
The models in which SHIP either promotes or maintains BCR-induced tyrosine phosphorylation of Shc are not mutually exclusive. SHIP could recruit a tyrosine kinase to Shc while the subsequent binding of Grb2 · SHIP complexes to Shc could protect the phosphorylated tyrosine residues from dephosphorylation by phosphatases. The interactions between SHIP, Syk, Shc, Grb2, and Sos are very complex and make it difficult to design unequivocal experiments to determine whether the binding of SHIP to Shc promotes the phosphorylation of Shc, protects phosphorylated Shc from tyrosine phosphatases, or facilitates both of these processes. Nevertheless, we have clearly shown for the first time that BCR-induced tyrosine phosphorylation of Shc depends on the PTB domain of Shc and on the expression of SHIP, a protein that binds to the Shc PTB domain.
In addition to its PTB domain, the SH2 domain of Shc also contributes to the ability of Shc to be tyrosine phosphorylated after BCR ligation. One possibility is that the Shc SH2 domain is required for co-localizing Shc with SHIP · Syk complexes. This may involve translocation of Shc from the cytosol to the cell membrane. Syk binds to the phosphorylated BCR ITAMs in activated B cells (24) and SHIP is found in the membrane fraction of anti-Ig-stimulated B cells (2). The Shc SH2 domain could mediate membrane translocation of Shc by binding membrane-associated proteins such as Gab1 which are tyrosine phosphorylated after BCR ligation. However, we cannot rule out other mechanisms by which the Shc SH2 domain contributes to making Shc a better substrate for BCR-activated tyrosine kinases.
In summary, we have shown that BCR-induced tyrosine phosphorylation of Shc depends on both the SH2 domain and the PTB domain of Shc, and that the SHIP inositol phosphatase plays an important role in this process. Further work is required to determine which domains of Shc are required for its membrane translocation and to determine the precise nature of all the protein-protein interactions that are required for BCR-induced phosphorylation of Shc.
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Acknowledgments |
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Footnotes |
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2 R.J.I. and H.O. contributed equally to this work and should be considered co-first authors.
3 Address correspondence and reprint requests to Dr. Michael R. Gold, Department of Microbiology and Immunology, University of British Columbia, 6174 University Boulevard, Vancouver, British Columbia V6T 1Z3, Canada. E-mail address:
4 Abbreviations used in this paper: BCR, B cell Ag receptor; SH2, Src homology 2; SHIP, SH2 domain-containing inositol phosphatase; PTB, phosphotyrosine-binding; PIP3, phosphatidylinositol 3,4,5-trisphosphate; anti-P-Tyr, anti-phosphotyrosine; ITAM, immunoreceptor tyrosine-based activation motif.
Received for publication September 1, 1999. Accepted for publication September 16, 1999.
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References |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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