Advertisement
JBC

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rui, L.
Right arrow Articles by Carter-Su, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rui, L.
Right arrow Articles by Carter-Su, C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

J Biol Chem, Vol. 273, Issue 33, 21239-21245, August 14, 1998


Platelet-derived Growth Factor (PDGF) Stimulates the Association of SH2-Bbeta with PDGF Receptor and Phosphorylation of SH2-Bbeta *

Liangyou RuiDagger and Christin Carter-Su§

From the Department of Physiology, University of Michigan Medical School, Ann Arbor, Michigan 48109-0622

    ABSTRACT
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We recently identified SH2-Bbeta as a JAK2-binding protein and substrate involved in the signaling of receptors for growth hormone and interferon-gamma . In this work, we report that SH2-Bbeta also functions as a signaling molecule for platelet-derived growth factor (PDGF). SH2-Bbeta fused to glutathione S-transferase (GST) bound PDGF receptor (PDGFR) from PDGF-treated but not control cells. GST fusion protein containing only the SH2 domain of SH2-Bbeta also bound PDGFR from PDGF-treated cells. An Arg to Glu mutation within the FLVRQS motif in the SH2 domain of SH2-Bbeta inhibited GST-SH2-Bbeta binding to tyrosyl-phosphorylated PDGFR. The N-terminal truncated SH2-Bbeta containing the entire SH2 domain interacted directly with tyrosyl-phosphorylated PDGFR from PDGF-treated cells but not unphosphorylated PDGFR from control cells in a Far Western assay. These results suggest that the SH2 domain of SH2-Bbeta is necessary and sufficient to mediate the interaction between SH2-Bbeta and PDGFR. PDGF stimulated coimmunoprecipitation of endogenous SH2-Bbeta with endogenous PDGFR in both 3T3-F442A and NIH3T3 cells. PDGF stimulated the rapid and transient phosphorylation of SH2-Bbeta on tyrosines and most likely on serines and/or threonines. Similarly, epidermal growth factor stimulated the phosphorylation of SH2-Bbeta ; however, phosphorylation appears to be predominantly on serines and/or threonines. In response to PDGF, SH2-Bbeta associated with multiple tyrosyl-phosphorylated proteins, at least one of which (designated p84) does not bind to PDGFR. Taken together, these data strongly argue that, in response to PDGF, SH2-Bbeta directly interacts with PDGFR and is phosphorylated on tyrosine and most likely on serines and/or threonines, and acts as a signaling protein for PDGFR.

    INTRODUCTION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

We recently identified the SH21 domain-containing molecule, SH2-Bbeta , as a substrate of JAK2 involved in signaling by growth hormone (GH) and interferon-gamma (1). Receptors for GH and interferon-gamma are members of the cytokine receptor family and are known to bind JAK tyrosine kinases (JAK2 for GH and both JAK1 and JAK2 for interferon-gamma ). After GH binding, JAK2 is activated and tyrosyl-phosphorylates its associated GH receptor as well as JAK2 itself (2, 3). As a consequence of JAK2 autophosphorylation, SH2-Bbeta is recruited into receptor/JAK2 complexes at least in part via the direct interaction of the SH2 domain of SH2-Bbeta with phosphotyrosine containing motif(s) in JAK2 (1). GH promotes not only the association of SH2-Bbeta with tyrosyl-phosphorylated JAK2, but also the tyrosyl phosphorylation of SH2-Bbeta (1). SH2-Bbeta also appears to be phosphorylated on serine(s) and/or threonine(s), even in the absence of ligand stimulation (1). These findings suggested that SH2-Bbeta , which contains multiple potential sites for protein-protein interaction in addition to its SH2 domain (9 tyrosines, a pleckstrin homology (PH) domain, and multiple proline-rich motifs) (Fig. 1A), serves as an adapter protein and recruits additional signaling molecules into cytokine receptor-JAK2 complexes (1).

Many signaling molecules are shared by cytokine receptors and receptor tyrosine kinases, particularly those signaling molecules containing SH2 domains. For example, Shc, Grb2, and phosphatidylinositol 3'-kinase are reported to play an important role in the biological actions of GH (2, 4) and platelet-derived growth factor (PDGF) (5, 6). In support of SH2-Bbeta serving as a signaling molecule for receptor tyrosine kinase(s), SH2-Bbeta was found to interact with receptors for insulin and insulin-like growth factor-1 (1, 7, 8). In this work, we demonstrate that PDGF stimulates association of SH2-Bbeta with PDGF receptor (PDGFR), and phosphorylation of SH2-Bbeta . We also show that SH2-Bbeta associates, not via the PDGFR, with a tyrosyl-phosphorylated, 84-kDa protein. These results provide strong evidence that SH2-Bbeta is a previously unknown signaling molecule for PDGF.

    EXPERIMENTAL PROCEDURES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

Cells and Reagents-- The stock of 3T3-F442A fibroblasts was kindly provided by H. Green (Harvard University, Cambridge, MA). Recombinant human GH was a gift of Eli Lilly and Co. Recombinant human PDGF-BB was from Life Technologies, Inc. Recombinant human PDGF-AA was from Collaborative Biomedical Products. Glutathione-agarose beads were from Sigma. Recombinant protein A-agarose was from Repligen. Alkaline phosphatase, aprotinin, leupeptin, and Triton X-100 were from Boehringer Mannheim. Protein phosphatase 2A (PP2A) was from Upstate Biotechnology, Inc. Enhanced chemiluminescence (ECL) detection system was from Amersham Corp. Antibodies to rat SH2-Bbeta (alpha SH2-B) were raised against GST-SH2-Bbeta c as described previously (1), and used at a dilution of 1:100 for immunoprecipitation and 1:15,000 for immunoblotting. Anti-JAK2 antiserum (alpha JAK2) was raised in rabbits against a synthetic peptide corresponding to amino acids 758-776 of murine JAK2 (9) and was used at a dilution of 1:500 for immunoprecipitation and 1:15,000 for immunoblotting. Monoclonal anti-phosphotyrosine antibody 4G10 (alpha PY) and polyclonal antibody against human PDGF receptor (alpha PDGFR, recognizing both alpha  and beta subunits) were from Upstate Biotechnology, Inc. and used at a dilution of 1:7500 and 1:1000 for immunoblotting, respectively. alpha PDGFR was used at a dilution of 1:100 for immunoprecipitation.

Methods-- 3T3-F442A fibroblasts were treated for 10 min with 25 ng/ml PDGF-BB, vehicle, or other ligands as indicated. For GST fusion protein pull-down assays, whole cell lysates were precipitated with GST fusion proteins immobilized on glutathione-agarose beads and subsequently immunoblotted with alpha PDGFR or alpha PY as described previously (1). GST fusion proteins containing SH2-Bbeta or mutant SH2-Bbeta were prepared as described previously (1). Arg within the FLVRQS motif in the SH2 domain of SH2-Bbeta was mutated to Glu using a site-directed mutagenesis kit (Stratagene), and the mutation was confirmed by DNA sequencing. The mutant SH2-Bbeta (SH2-Bbeta (R-E)) was subcloned into pGEX-KG to generate a GST fusion protein. For immunoprecipitations, cell lysates were incubated with the indicated antibody on ice for 2 h. The immune complexes were collected on protein A-agarose (50 µl) during a 1-h incubation at 4 °C. In some experiments, alpha SH2-B immunoprecipitates were dephosphorylated by alkaline phosphatase or PP2A as described previously (1). The immunoprecipitates were immunoblotted with the indicated antibody. Some membranes were stripped by incubation at 55 °C for 30-60 min in stripping buffer (100 mM beta -mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl, pH 6.7) and reprobed with a different antibody. For Far Western blotting, PDGFR was immunoprecipitated with alpha PDGFR from solubilized 3T3-F442A fibroblasts, subjected to SDS-PAGE, and transferred onto nitrocellulose. The nitrocellulose was incubated with GST-SH2-Bbeta c (1.5 µg/ml) at 4 °C overnight. After extensive washing, the membrane was immunoblotted with alpha SH2-B. The blot was stripped and reprobed with alpha PDGFR and alpha PY sequentially.

    RESULTS
Top
Abstract
Introduction
Procedures
Results
Discussion
References

SH2-Bbeta Binds to PDGFR Only from PDGF-treated Cells-- To determine whether SH2-Bbeta serves as a signaling molecule for PDGF, we first tested whether SH2-Bbeta binds to PDGFR. 3T3-F442A fibroblasts, which express endogenous PDGFR (10), were deprived of serum overnight and treated with 25 ng/ml PDGF-BB or vehicle for 10 min. Cell lysates were incubated with immobilized GST or GST fusion protein containing full-length SH2-Bbeta , N-terminally truncated SH2-Bbeta (SH2-Bbeta c), or the SH2 domain of SH2-Bbeta (Fig. 1A), and immunoblotted with alpha PDGFR. GST-SH2-Bbeta bound to PDGFR in a ligand-dependent manner (Fig. 1B, lanes 1 and 2). GST-SH2-Bbeta c (Fig. 1B, lanes 3 and 4) and GST-SH2 domain of SH2-Bbeta (Fig. 1B, lanes 5 and 6) also bound PDGFR from PDGF- but not vehicle-treated cells. Reprobing with alpha PY showed that PDGFR that is associated with SH2-Bbeta or truncated SH2-Bbeta is tyrosyl-phosphorylated (data not shown). In contrast, GST alone did not bind PDGFR (Fig. 1B, lane 7). These results suggest that SH2-Bbeta interacts with activated, tyrosyl-phosphorylated PDGFR, and that the SH2 domain of SH2-Bbeta may mediate the interaction between SH2-Bbeta and PDGFR.


View larger version (40K):
[in this window]
[in a new window]
  		Fig. 1.   GST-SH2-Bbeta binds to PDGFR from PDGF-treated cells. A, schematic representation of full-length and truncated SH2-Bbeta . Tyrosines and the SH2 and PH domains are designated. The C-terminal gray region designates the amino acids that are not shared with SH2-Balpha . B, 3T3-F442A cells were treated for 10 min with vehicle (lanes 1, 3, and 5) or 25 ng/ml PDGF-BB (lanes 2, 4, 6, and 7). Whole cell lysates were incubated with the indicated GST fusion protein immobilized on glutathione-agarose beads. Bound proteins were separated by SDS-PAGE and immunoblotted with alpha PDGFR. The amount of GST-SH2-Bbeta was less than one-third the amount of the other GST fusion proteins.

SH2-Bbeta Binds Directly to Tyrosyl-phosphorylated PDGFR through Its SH2 Domain-- To investigate whether SH2-Bbeta binds PDGFR directly or indirectly through some intermediate molecule, the ability of GST-SH2-Bbeta c to bind PDGFR was determined by Far Western blotting. 3T3-F442A cells were treated with 25 ng/ml PDGF-BB or vehicle. PDGFR was immunoprecipitated with alpha PDGFR, resolved by SDS-PAGE, and transferred to nitrocellulose. The nitrocellulose was incubated first with GST-SH2-Bbeta c, and then with alpha SH2-B. GST-SH2-Bbeta c bound directly to PDGFR from PDGF-treated (Fig. 2A, lane 2) but not vehicle-treated cells (Fig. 2A, lane 1), although an equal amount of PDGFR was present (Fig. 2A, lanes 3 and 4). Reprobing the same blot with alpha PY confirmed the PDGF-dependent tyrosyl phosphorylation of PDGFR (Fig. 2A, lanes 5 and 6). These results indicate that SH2-Bbeta c binds directly to tyrosyl-phosphorylated, activated PDGFR.


View larger version (33K):
[in this window]
[in a new window]
  		Fig. 2.   SH2-Bbeta binds directly to tyrosyl-phosphorylated PDGFR via the SH2 domain of SH2-Bbeta . A, 3T3-F442A cells were treated for 10 min with 25 ng/ml PDGF-BB (lanes 2, 4, and 6) or vehicle (lanes 1, 3, and 5). Solubilized proteins were immunoprecipitated with alpha PDGFR. The immunoprecipitated proteins were separated by SDS-PAGE and transferred onto nitrocellulose. The nitrocellulose was incubated with GST-SH2-Bbeta c at 4 °C overnight. After extensive washing, the nitrocellulose was immunoblotted with alpha SH2-B (lanes 1 and 2). The same blot was sequentially reprobed with alpha PDGFR (lanes 3 and 4) and alpha PY (lanes 5 and 6). B, 3T3-F442A (lanes 1-3) and NIH3T3 (lanes 4-6) cells were treated for 10 min with vehicle (lanes 1 and 4) or 25 ng/ml PDGF-BB (lanes 2, 3, 5, and 6). Whole cell lysates were incubated with the indicated GST fusion protein immobilized on glutathione-agarose beads. Bound proteins were separated by SDS-PAGE and immunoblotted with alpha PY. The migration of molecular weight standards (×10-3) (panel A) and PDGFR (panels A and B) is indicated.

To demonstrate the crucial role of the SH2 domain of SH2-Bbeta in mediating the interaction of SH2-Bbeta with PDGFR, we examined the ability of PDGFR to bind to GST fusion protein containing a mutant SH2-Bbeta in which Glu replaced the Arg (amino acid 555) within the FLVRQS motif in the SH2 domain. This positively charged Arg within the highly conserved FLVRQS motif in SH2 domains has been shown to interact with the negatively charged phosphate on the phosphotyrosine of its binding partner (11-13). Mutation of this critical Arg has been shown to abolish the binding ability of the SH2 domain to its corresponding phosphotyrosine motif for several molecules (14, 15). GST fusion protein containing this mutant SH2-Bbeta ((GST-SH2-Bbeta (R-E)) immobilized on glutathione-agarose beads was incubated with cell lysates of either 3T3-F442A or NIH3T3 cells. The bound proteins were separated by SDS-PAGE and immunoblotted with alpha PY. GST-SH2-Bbeta bound to tyrosyl-phosphorylated PDGFR from PDGF-treated NIH3T3 cells (Fig. 2B, lane 5) as well as 3T3-F442A cells (Fig. 2B, lane 2), indicating that the interaction of SH2-Bbeta with PDGFR is not cell-type-specific. Mutation of Arg to Glu within the SH2 domain of SH2-Bbeta inhibited binding of SH2-Bbeta to tyrosyl-phosphorylated PDGFR from either 3T3-F442A (Fig. 2B, lane 3) or NIH3T3 (Fig. 2B, lane 6) cells. These results suggest that the SH2 domain of SH2-Bbeta mediates the interaction between SH2-Bbeta and PDGFR.

PDGF-BB Stimulates the Association of Endogenous SH2-Bbeta with PDGFR-- To examine whether endogenous SH2-Bbeta associates with endogenous PDGFR in mammalian cells, 3T3-F442A fibroblasts, shown previously to express endogenous SH2-Bbeta (1), were treated with PDGF-BB or vehicle. Solubilized proteins were immunoprecipitated with alpha SH2-B and immunoblotted with alpha PDGFR. SH2-Bbeta was observed to coimmunoprecipitate with PDGFR in PDGF-stimulated (Fig. 3, lane 2) but not control cells (Fig. 3, lane 1), consistent with the findings (shown in Figs. 1B and 2) that SH2-Bbeta binds only to tyrosyl-phosphorylated, activated PDGFR. Pre-immune serum was unable to precipitate PDGFR from PDGF-treated cells (Fig. 3, lanes 3 and 6). Reprobing the same blot with alpha PY confirmed that PDGFR associated with SH2-Bbeta is tyrosyl-phosphorylated (Fig. 3, lane 5). Similarly, SH2-Bbeta was detected in alpha PDGFR immunoprecipitates only when cells were stimulated with PDGF (data not shown). SH2-Bbeta also coimmunoprecipitated with PDGFR in NIH3T3 cells in response to PDGF-BB (Fig. 3, lanes 7 and 8), further demonstrating that the association of SH2-Bbeta with PDGFR is not cell-type-specific. Taken together, the results of Figs. 1-3 suggest that PDGF stimulates the recruitment of SH2-Bbeta to PDGFR presumably via a direct interaction of the SH2 domain of SH2-Bbeta with phosphotyrosine-containing motif(s) in the activated PDGFR.


View larger version (20K):
[in this window]
[in a new window]
  		Fig. 3.   PDGF stimulates association of SH2-Bbeta with PDGFR. 3T3-F442A (lanes 1-6) and NIH3T3 (lanes 7 and 8) cells were treated for 10 min with 25 ng/ml PDGF-BB (lanes 2, 3, 5, 6, and 8) or vehicle (lanes 1, 4, and 7). Solubilized proteins were immunoprecipitated with alpha SH2-B (lanes 1, 2, 4, 5, 7, and 8) or preimmune serum (PI, lanes 3 and 6), and subsequently immunoblotted with alpha PDGFR (lanes 1-3) or alpha PY (lanes 7 and 8). The blot corresponding to lanes 1-3 was stripped and reprobed with alpha PY (lanes 4-6).

PDGF-BB Promotes Tyrosyl Phosphorylation of SH2-Bbeta -- SH2-Bbeta was previously shown to be tyrosyl-phosphorylated in response to GH and interferon-gamma (1). To test whether PDGF is able to stimulate tyrosyl phosphorylation of SH2-Bbeta , 3T3-F442A cells were treated with PDGF-BB, and cell lysates were immunoprecipitated with alpha SH2-B and immunoblotted with alpha PY. PDGF-BB stimulated tyrosyl phosphorylation of SH2-Bbeta (Fig. 4, upper panel; Fig. 5B, lane 4; Fig. 6, lane 4; Fig. 7, lane 3). Based upon the migration of proteins immunoblotted with alpha SH2-B (Fig. 4, lower panel; Fig. 5B, lane 9; Fig. 7, lane 8), SH2-Bbeta migrates as a diffuse band indicated by the bracket. For reasons discussed below, the tight band, designated p84 in Figs. 4, 5B, 6, and 7, is believed to be a tyrosyl-phosphorylated protein that coimmunoprecipitates with SH2-Bbeta , and not a form of SH2-Bbeta . As predicted from Figs. 1-3, tyrosyl-phosphorylated PDGFR coimmunoprecipitated with SH2-Bbeta from PDGF-BB-treated cells (Fig. 4, lanes 2-7, upper panel; Fig. 5B, lanes 4 and 5; Fig. 6, lane 4; Fig. 7, lanes 2 and 3) but not from control (Fig. 4, lane 1; Fig. 5B, lanes 1-3; Fig. 6, lane 3; Fig. 7, lane 1) or GH- or EGF-treated cells (Fig. 7, lanes 4 and 5). When the blots were reprobed with alpha SH2-B, PDGF-BB was observed to cause a significant upward shift in the mobility of SH2-Bbeta (Fig. 4, lower panel; Fig. 5A, lane 2; Fig. 5B, lane 9; Fig. 7, lane 8), consistent with SH2-Bbeta being phosphorylated in response to PDGF. The PDGF-BB-induced tyrosyl phosphorylation and shift in mobility of SH2-Bbeta were rapid (within 1 min), transient (Fig. 4), and dose-dependent (data not shown), indicating that phosphorylation of SH2-Bbeta is a tightly regulated process. Interestingly, the greatest shift in SH2-Bbeta mobility was observed after 5 min of 25 ng/ml PDGF (Fig. 4, lower panel), while the tyrosyl phosphorylation of SH2-Bbeta was not maximal until 15 min (Fig. 4, upper panel). Similarly, the mobility shift of SH2-Bbeta was the greatest at a dose of 5 ng/ml for 15 min, but the tyrosyl phosphorylation was not maximal until 25 ng/ml (data not shown). Because the multiple SH2-Bbeta bands in control and GH-treated 3T3-F442A cells have been shown to be differentially phosphorylated forms of SH2-Bbeta , this discrepancy between mobility shift and alpha PY signal suggests that in addition to stimulating tyrosyl phosphorylation, PDGF-BB also promotes the phosphorylation of SH2-Bbeta on serine(s) and/or threonine(s). Curiously, the degree of tyrosyl phosphorylation of SH2-Bbeta measured using alpha PY Western blotting is less than that of the PDGFR, which coimmunoprecipitates with SH2-Bbeta (Figs. 4 and 5B). Although the reason for the lower signal is not known, it may reflect: 1) fewer phosphorylated tyrosines in SH2-Bbeta compared with PDGFR (reported to be phosphorylated on at least 10 tyrosines (5, 16); 2) tyrosyl phosphorylation of only a subset of that SH2-Bbeta that binds to PDGFR; or 3) poorer recognition by the 4G10 antibody of phosphorylated tyrosines in SH2-Bbeta compared with those in PDGFR.


View larger version (65K):
[in this window]
[in a new window]
  		Fig. 4.   Time course of PDGF-stimulated phosphorylation of SH2-Bbeta . 3T3-F442A cells were treated with 25 ng/ml PDGF-BB for the indicated times. Proteins in the cell lysates were immunoprecipitated with alpha SH2-B and immunoblotted with alpha PY (upper panel). The same blots were reprobed without stripping with alpha SH2-B (lower panel).


View larger version (40K):
[in this window]
[in a new window]
  		Fig. 5.   PDGF stimulates phosphorylation of SH2-Bbeta at multiple sites. A, 3T3-F442A cells were treated for 10 min with 25 ng/ml PDGF-BB (lanes 2-4) or with vehicle (lane 1), and solubilized proteins were immunoprecipitated with alpha SH2-B. The immunoprecipitates were incubated at 37 °C for 60 min in dephosphorylation buffer with (lanes 3 and 4) or without (lanes 1 and 2) alkaline phosphatase (AP), and with (lane 4) or without (lanes 1-3) Na3VO4. The reaction mixtures were subjected to SDS-PAGE and immunoblotted with alpha SH2-B. B, 3T3-F442A cells were treated for 10 min with 25 ng/ml PDGF-BB or vehicle as indicated, and solubilized proteins were immunoprecipitated with alpha SH2-B. alpha SH2-B immunoprecipitates were incubated with (lanes 2, 3, 5, 7, 8, and 10-12) or without (lanes 1, 4, 6, and 9) PP2A in the presence (lane 12) or absence (lanes 1-11) of okadaic acid. The reaction mixtures were subjected to SDS-PAGE and immunoblotted with alpha PY (lanes 1-5) or alpha SH2-B (lanes 11 and 12). The blot corresponding to lanes 1-5 was stripped and reprobed with alpha SH2-B (lanes 6-10). The migration of molecular weight standards (×10-3), p84, PDGFR, and SH2-Bbeta is indicated.


View larger version (41K):
[in this window]
[in a new window]
  		Fig. 6.   SH2-Bbeta associates with multiple tyrosyl-phosphorylated proteins in PDGF-treated cells. 3T3-F442A cells were treated for 10 min with 25 ng/ml PDGF-BB (lanes 2, 4, and 5) or vehicle (lanes 1 and 3). Solubilized proteins were immunoprecipitated with alpha PDGFR (lanes 1 and 2) or alpha SH2-B (lanes 3-5). For lane 5, alpha SH2-B immunoprecipitates were boiled for 5 min in lysis buffer containing 1% SDS, and then diluted 5 times with lysis buffer and re-immunoprecipitated with alpha SH2-B. The blots were immunoblotted with alpha PY. The migration of molecular weight standards (×10-3), p84, PDGFR, and SH2-Bbeta is indicated.


View larger version (40K):
[in this window]
[in a new window]
  		Fig. 7.   SH2-Bbeta is phosphorylated in response to PDGF-AA, PDGF-BB, GH, and EGF. 3T3-F442A cells were treated for 10 min with 25 ng/ml PDGF-AA (lanes 2 and 7), 25 ng/ml PDGF-BB (lanes 3 and 8), 500 ng/ml GH (lanes 4 and 9) or 100 ng/ml EGF (lanes 5 and 10). Whole cell lysates were immunoprecipitated with alpha SH2-B and immunoblotted with alpha PY (lanes 1-5). The same blot was stripped and reprobed with alpha SH2-B (lanes 6-10). The migration of molecular weight standards (×10-3), p84, PDGFR, EGFR, and SH2-Bbeta is indicated.

PDGF-BB Promotes Phosphorylation of SH2-Bbeta at Multiple Sites-- To provide evidence that the multiple proteins recognized by alpha SH2-B in PDGF-treated cells reflect different phosphorylation states of SH2-Bbeta , alpha SH2-B immunoprecipitates were treated with alkaline phosphatase in the presence or absence of sodium vanadate, an inhibitor of alkaline phosphatase. As discussed above, PDGF-BB treatment decreased the migration of SH2-Bbeta (Fig. 5A, lane 2). Alkaline phosphatase treatment reduced the multiple forms of SH2-Bbeta observed in PDGF-BB-treated cells (Fig. 5A, lane 2) to a faster migrating form (Fig. 5A, lane 3). Simultaneously, the intensity of the faster migrating form of SH2-Bbeta increased significantly (Fig. 5A, lane 3), indicating a shift of SH2-Bbeta from slower to faster migrating forms. Sodium vanadate significantly reduced the effect of alkaline phosphatase on the PDGF-BB-induced mobility shift of SH2-Bbeta (Fig. 5A, lane 4), indicating that the change of migration of SH2-Bbeta by alkaline phosphatase is due to dephosphorylation.

To provide evidence for the multiple bands in the presence of PDGF being due at least in part to serine and/or threonine phosphorylation, cell lysates from PDGF-BB-treated 3T3-F442A cells were incubated with alpha SH2-B. alpha SH2-B immunoprecipitates were treated with PP2A (a serine/threonine-specific phosphatase) and immunoblotted with alpha PY (Fig. 5B, lanes 1-5) and subsequently with alpha SH2-B (Fig. 5B, lanes 6-12). Consistent with our previous report (1), PP2A treatment of proteins from control cells reduced the multiple forms of SH2-Bbeta to a single faster migrating form (Fig. 5B, lanes 6-8). Interestingly, in PDGF-BB-stimulated cells, PP2A treatment reduced the broad, diffuse bands of SH2-Bbeta (Fig. 5B, lane 9) to two distinct, faster migrating protein bands (Fig. 5B, lanes 10 and 11). This condensation of bands is consistent with SH2-Bbeta being phosphorylated on multiple serines/threonines in the presence of PDGF. The two bands observed with PP2A treatment aligned exactly with the two major tyrosyl-phosphorylated SH2-Bbeta bands observed in the corresponding alpha PY blot (Fig. 5B, lane 5), suggesting that PDGF stimulates the phosphorylation of at least 2 tyrosines within SH2-Bbeta . Okadaic acid, a potent inhibitor of PP2A, completely abrogated the effect of PP2A on the PDGF-induced mobility shift of SH2-Bbeta (Fig. 5B, lane 12). Although PP2A increased substantially the migration of tyrosyl-phosphorylated SH2-Bbeta (Fig. 5B, lane 5), it did not affect the degree of tyrosyl phosphorylation of either SH2-Bbeta or SH2-Bbeta -associated PDGFR (Fig. 5B, lanes 4 and 5), indicating that it is specific for serines/threonines as expected.

Multiple Tyrosyl-phosphorylated Proteins Associate with SH2-Bbeta in Response to PDGF-- A tight, tyrosyl-phosphorylated protein band designated p84 coimmunoprecipitated with SH2-Bbeta (Figs. 4, 5B, 6, and 7). Interestingly, p84 aligns with neither of the two forms of SH2-Bbeta after dephosphorylation by PP2A (Fig. 5B, lanes 5, 10, and 11), suggesting that p84 is an SH2-Bbeta -interacting protein and not a form of SH2-Bbeta itself. PDGF stimulation increased the amount of tyrosyl-phosphorylated p84 associated with SH2-Bbeta (Fig. 4, upper panel; Fig. 6, lane 4). p84 does not appear to associate with SH2-Bbeta via PDGFR because alpha PDGFR did not immunoprecipitate p84 (Fig. 6, lane 2). Furthermore, when alpha SH2-B immunoprecipitates were first dissociated by boiling in SDS-containing buffer, and then re-immunoprecipitated with alpha SH2-B, p84, like PDGFR, did not coimmunoprecipitate with SH2-Bbeta (Fig. 6, lane 5). These data indicate that alpha SH2-B does not cross-react with either PDGFR or p84, and that both PDGFR and p84 associate with SH2-Bbeta in cells stimulated with PDGF. In addition to PDGFR, SH2-Bbeta , and p84, multiple other tyrosyl-phosphorylated proteins were present in alpha SH2-B immunoprecipitates when cells were treated with PDGF-BB (Fig. 4, lanes 2-6, upper panel; Fig. 6, lane 4). Because tyrosyl-phosphorylated proteins of similar size are also precipitated by alpha PDGFR and PDGFR coimmunoprecipitates with SH2-Bbeta , it is not clear whether these other phosphoproteins associate with SH2-Bbeta directly or indirectly through their interaction with SH2-Bbeta -bound PDGFR. It is also unclear whether PDGF stimulates the association of p84 and/or these other phosphoproteins with SH2-Bbeta or SH2-Bbeta constitutively associates with these proteins and PDGF stimulates their tyrosyl phosphorylation.

PDGF-AA and Epidermal Growth Factor (EGF) Stimulate Phosphorylation and a Shift in Mobility of SH2-Bbeta -- As PDGF-BB is able to activate both alpha  and beta  subunits of PDGFR, it is not clear which subunits in 3T3-F442A fibroblasts recruit and phosphorylate SH2-Bbeta in response to PDGF-BB. To begin to dissect which subunit of PDGFR utilizes SH2-Bbeta , 3T3-F442A cells were treated with PDGF-AA (which activates only the alpha  subunit of PDGFR; Ref. 17), and solubilized proteins were immunoprecipitated with alpha SH2-B and immunoblotted with alpha PY. The extent of ligand-induced tyrosyl phosphorylation of SH2-Bbeta and the multiple other proteins including PDGFR that coimmunoprecipitate with SH2-Bbeta was similar between cells stimulated with PDGF-AA and PDGF-BB (Fig. 7, lanes 2 and 3), with PDGF-AA being a little less effective than PDGF-BB at the same dosage. PDGF-AA, like PDGF-BB, caused a decrease in SH2-Bbeta mobility (Fig. 7, lanes 7 and 8). These data suggest that the alpha  subunit of PDGFR recruits SH2-Bbeta as a signaling protein in 3T3-F442A cells. Whether SH2-Bbeta also associates with PDGFRbeta remains to be determined.

To investigate whether other receptor tyrosine kinases regulate SH2-Bbeta , 3T3-F442A cells, which are also responsive to EGF (10), were stimulated with 100 ng/ml EGF for 10 min, and alpha SH2-B immunoprecipitates were immunoblotted with alpha PY. A tyrosyl-phosphorylated protein of a size appropriate for EGF receptor coimmunoprecipitated with SH2-Bbeta from EGF-treated (Fig. 7, lane 5) but not control (Fig. 7, lane 1) cells, suggesting that SH2-Bbeta is present in a complex with EGF receptor. Surprisingly, tyrosyl phosphorylation of SH2-Bbeta was not detectable using alpha PY (Fig. 7, lane 5), although a significantly reduced migration rate of SH2-Bbeta was observed in EGF-treated cells (Fig. 7, lane 10). Similarly, EGF stimulated a large shift in mobility of SH2-B in PC12 cells without detectable tyrosyl phosphorylation (data not shown). Incubation of alpha SH2-B immunoprecipitates with alkaline phosphatase completely abolished the EGF-induced mobility shift (data not shown). These data suggest that the EGF-induced mobility shift is most likely due to phosphorylation, and that the phosphorylation of SH2-Bbeta elicited by EGF occurs mainly on serines and/or threonines. The proposal that EGF, as well as PDGF-BB, stimulates phosphorylation of SH2-Bbeta on serines/threonines is further suggested by the finding that EGF and PDGF-BB stimulate tyrosyl phosphorylation of SH2-Bbeta to a much lesser extent than GH (Fig. 7, lanes 3-5) but cause a greater (PDGF-BB) or similar (EGF) shift in mobility of SH2-Bbeta (Fig. 7, lanes 8-10).

    DISCUSSION
Top
Abstract
Introduction
Procedures
Results
Discussion
References

In the current study, we show that SH2-Bbeta binds directly to tyrosyl-phosphorylated, but not unphosphorylated, PDGFR in both GST fusion protein pull-down and Far Western blotting assays. The SH2 domain of SH2-Bbeta is sufficient for binding to PDGFR in the GST fusion protein pull-down assay. Furthermore, when the conserved Arg was mutated to Glu within the FLVRQS motif in the SH2 domain of SH2-Bbeta , the ability of mutant SH2-Bbeta to bind PDGFR was dramatically inhibited. These results suggest that the SH2 domain of SH2-Bbeta is both necessary and sufficient for binding to tyrosyl-phosphorylated, activated PDGFR. The ligand-dependent interaction of SH2-Bbeta with PDGFR was further confirmed by the coimmunoprecipitation of endogenous SH2-Bbeta with endogenous PDGFR in both 3T3-F442A and NIH3T3 cells.

The finding that there is an upward shift in mobility of SH2-Bbeta upon PDGF-BB treatment that is abolished by alkaline phosphatase provides clear evidence that PDGF promotes phosphorylation of SH2-Bbeta . Similarly, blotting with alpha PY provides strong evidence that PDGF promotes tyrosyl phosphorylation of SH2-Bbeta . The fact that PP2A condenses the broad SH2-Bbeta band to two faster migrating bands suggests that at least two tyrosines are phosphorylated. Because SH2-Bbeta binds directly to activated PDGFR, it is logical to hypothesize that SH2-Bbeta is phosphorylated directly by PDGFR. In support of this, when coexpressed in COS cells, SH2-Bbeta is tyrosyl-phosphorylated by PDGFR beta  subunit.2

The fact that PP2A increases the migration of SH2-Bbeta in control and PDGF-treated cells suggests that SH2-Bbeta is phosphorylated on serines and/or threonines. In support of PDGF and/or EGF stimulating the serine/threonine phosphorylation of SH2-Bbeta , there is a discrepancy between changes in SH2-Bbeta mobility and amount of alpha PY binding to SH2-Bbeta . A maximal decrease in mobility of SH2-Bbeta occurs at shorter times and at lower PDGF-BB concentrations than the maximal increase in tyrosyl phosphorylation as detected by alpha PY. In the extreme case of EGF, no signal is detectable by alpha PY but a significant decrease in SH2-Bbeta mobility is observed. In addition, PDGF-BB stimulates a greater decrease in SH2-Bbeta mobility than GH, but is much less effective than GH at stimulating tyrosyl phosphorylation of SH2-Bbeta . Although we favor the hypothesis that PDGF and EGF stimulate the serine/threonine phosphorylation of SH2-Bbeta , our data do not exclude the possibility that SH2-Bbeta is constitutively phosphorylated on serines/threonines and that EGF, PDGF, and GH stimulate the phosphorylation of different tyrosines on SH2-Bbeta . However, one would have to hypothesize that those tyrosines phosphorylated by EGF receptor are not recognized by the alpha PY used in this study, that those phosphorylated by PDGFR include some that are recognized by alpha PY and some that are not, and that those phosphorylated in response to GH bind alpha PY with high affinity. It would not be surprising for SH2-Bbeta to be phosphorylated on multiple serines/threonines because sequence analysis reveals that SH2-Bbeta has 82 serines and 26 threonines, including multiple potential phosphorylation sites for protein kinase C and a potential site (PLSP) for mitogen-activated protein kinases (e.g. ERK1/2). Protein kinase Cs and/or ERKs are potential candidates for PDGF-induced serine/threonine phosphorylation of SH2-Bbeta , because PDGF is reported to activate multiple isoforms of protein kinase C (18-22) and ERKs 1 and 2 (23).3

We observed a tyrosyl-phosphorylated protein with Mr ~ 84,000 (p84) coimmunoprecipitating with SH2-Bbeta in PDGF-stimulated cells. When the alpha SH2-B immunocomplex was dissociated by boiling in SDS-containing buffer, p84 was no longer immunoprecipitated by alpha SH2-B, suggesting that alpha SH2-B interacts with p84 indirectly through SH2-Bbeta rather than directly binding to p84. p84 does not coimmunoprecipitate with PDGFR, suggesting that the interaction of SH2-Bbeta with p84 is not mediated by PDGFR. The identity of p84 is not known. It is unlikely that p84 is the p85 subunit of phosphatidylinositol 3'-kinase because p84 is not recognized by anti-p85 in immunoblots (data not shown). Interestingly, when alpha SH2-B raised from a different rabbit (rabbit 2) was used to immunoprecipitate SH2-Bbeta , another tyrosyl-phosphorylated protein with Mr ~ 145,000 (p145) was observed in alpha SH2-B immunoprecipitates only from PDGF-stimulated cells (data not shown). We therefore believe that SH2-Bbeta interacts with multiple proteins besides PDGFR, as expected for an adapter protein involved in PDGFR signaling. SH2-Bbeta thereby may actively regulate PDGFR signaling by initiating some as yet unidentified pathways.

PDGF-induced phosphorylation of SH2-Bbeta may play a significant role in PDGFR signaling. The phosphorylated tyrosines in SH2-Bbeta may form docking sites for other signaling molecules which contain SH2 or phosphotyrosine binding domains, which may include p84 and p145 as discussed above. The significance of serine and/or threonine phosphorylation of SH2-Bbeta is unclear. Phosphoserine(s)/threonines in SH2-Bbeta could serve as a binding site for other signaling molecules such as 14-3-3 (24-29). Serine/threonine phosphorylation of SH2-Bbeta could also inhibit tyrosine phosphorylation of SH2-Bbeta , as reported for insulin receptor substrate-1 (30, 31), or affect the association of SH2-Bbeta with other signaling molecules, as reported for Sos association with Grb2 (11, 12, 32, 33).

Two isoforms of SH2-B, designated SH2-Balpha and SH2-Bbeta , have been described to date (1, 34). SH2-B, along with Lnk and APS, are proposed to form a new adapter family (35). Lnk, with an SH2 domain 68% identical to SH2-B, is expressed preferentially in lymphoid tissues and has been shown to bind to phosphatidylinositol 3'-kinase, Grb2, and phospholipase Cgamma (36). APS, with a PH domain 58% identical to that of SH2-B and an SH2 domain 80% identical to that of SH2-B, was cloned as a binding protein for the kinase domain of c-Kit receptor and is predicted to play a role in B cell antigen receptor activation (35). As the SH2 domain of SH2-Bbeta , which is highly conserved among SH2-Bbeta , Lnk and APS, mediates the interaction between SH2-Bbeta and PDGFR, we predict that Lnk, APS, SH2-Balpha , or their homologues also bind to activated PDGFR and serve as signaling molecules for PDGFR in those cells that express both PDGFR and the SH2-B-related proteins.

In summary, we have shown that in response to PDGF, SH2-Bbeta is recruited onto PDGFR complexes via direct interaction with PDGFR, and is tyrosyl-phosphorylated. SH2-Bbeta is also phosphorylated on serines/threonines. Serine/threonine phosphorylation of SH2-Bbeta appears to be increased by PDGF and EGF stimulation. The SH2 domain of SH2-Bbeta is required and sufficient for the interaction of SH2-Bbeta with tyrosyl-phosphorylated PDGFR. As a consequence of association of SH2-Bbeta with PDGFR, signaling molecules bound to SH2-Bbeta such as p84 are also recruited by PDGFR. We conclude that SH2-Bbeta is a previously unknown signaling molecule for PDGF signaling. It will be interesting to determine whether SH2-Bbeta mediates some of the actions of PDGF that cannot be accounted for by previously identified PDGF signaling molecules.

    ACKNOWLEDGEMENTS

We thank M. R. Stofega and Drs. J. B. Herrington, L. S. Argetsinger, and J. A. VanderKuur for their helpful suggestions. We thank P. Du for technical assistance and B. Hawkins for assistance with the manuscript. Oligonucleotide synthesis was performed by the Biomedical Research Core Facilities, University of Michigan, supported in part by grants to the Cancer Center, Michigan Diabetes Research and Training Center (P60-DK-20572), and UM-MAC (P60-AR20557).

    FOOTNOTES

* This work was supported by National Institutes of Health Grant DK 34171 (to C. C.-S.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger Recipient of a predoctoral fellowship and a Distinguished Research Partnership Fellowship from the Rackham School of Graduate Studies, University of Michigan.

§ To whom correspondence should be addressed: Dept. of Physiology, The University of Michigan Medical School, Ann Arbor, MI 48109-0622. Fax: 734-647-9523; E-mail: cartersu{at}umich.edu.

The abbreviations used are: SH, Src homology; PDGF, platelet-derived growth factor; GH, growth hormone; EGF, epidermal growth factor; PDGFR, platelet-derived growth factor receptor; PP2A, protein phosphatase 2A; GST, glutathione S-transferasePAGE, polyacrylamide gel electrophoresisERK, extracellular signal regulated kinase.

2 L. Rui, A. Kazlauskas, and C. Carter-Su, unpublished data.

3 L. Rui and C. Carter-Su, unpublished data.

    REFERENCES
Top
Abstract
Introduction
Procedures
Results
Discussion
References

  1. Rui, L., Mathews, L. S., Hotta, K., Gustafson, T. A., and Carter-Su, C. (1997) Mol. Cell. Biol. 17, 6633-6644[Abstract]
  2. Argetsinger, L. S., and Carter-Su, C. (1996) Physiol. Rev. 76, 1089-1107[Abstract/Free Full Text]
  3. Argetsinger, L. S., Campbell, G. S., Yang, X., Witthuhn, B. A., Silvennoinen, O., Ihle, J. N., and Carter-Su, C. (1993) Cell 74, 237-244[CrossRef][Medline] [Order article via Infotrieve]
  4. Carter-Su, C., Schwartz, J., and Smit, L. S. (1996) Annu. Rev. Physiol. 58, 187-207[CrossRef][Medline] [Order article via Infotrieve]
  5. Claesson-Welsh, L. (1994) J. Biol. Chem. 269, 32023-32026[Free Full Text]
  6. Claesson-Welsh, L. (1996) Int. J. Biochem. Cell Biol. 28, 373-385[CrossRef][Medline] [Order article via Infotrieve]
  7. Riedel, H., Wang, J., Hansen, H., and Yousaf, N. (1997) J. Biochem. (Tokyo) 122, 1105-1113[Abstract/Free Full Text]
  8. Wang, J., and Riedel, H. (1998) J. Biol. Chem. 273, 3136-3139[Abstract/Free Full Text]
  9. Silvennoinen, O., Witthuhn, B., Quelle, F. W., Cleveland, J. L., Yi, T., and Ihle, J. N. (1993) Proc. Natl. Acad. Sci. U. S. A. 90, 8429-8433[Abstract/Free Full Text]
  10. Campbell, G. S., Christian, L. J., and Carter-Su, C. (1993) J. Biol. Chem. 268, 7427-7434[Abstract/Free Full Text]
  11. Waksman, G., Kominos, D., Robertson, S. C., Pant, N., Baltimore, D., Birge, R. B., Cowburn, D., Hanafusa, H., Mayer, B. J., and Overduin, M. (1992) Nature 358, 646-653[CrossRef][Medline] [Order article via Infotrieve]
  12. Waksman, G., Shoelson, S. E., Pant, N., Cowburn, D., and Kuriyan, J. (1993) Cell 72, 779-790[CrossRef][Medline] [Order article via Infotrieve]
  13. Pascal, S. M., Singer, A. U., Gish, G., Yamazaki, T., Shoelson, S. E., Pawson, T., Kay, L. E., and Forman-Kay, J. D. (1994) Cell 77, 461-472[CrossRef][Medline] [Order article via Infotrieve]
  14. Mayer, B. J., Jackson, P. K., Van Etten, R. A., and Baltimore, D. (1992) Mol. Cell. Biol. 12, 609-618[Abstract/Free Full Text]
  15. Zhu, G., Decker, S. J., Mayer, B. J., and Saltiel, A. R. (1993) J. Biol. Chem. 268, 1775-1779[Abstract/Free Full Text]
  16. Ruusala, A., Sundberg, C., Arvidsson, A. K., Rupp-Thuresson, E., Heldin, C. H., and Claesson-Welsh, L. (1998) J. Cell Sci. 111, 111-120[Abstract]
  17. Heldin, C. H. (1992) EMBO J. 11, 4251-4259[Medline] [Order article via Infotrieve]
  18. Li, W., Yu, J. C., Michieli, P., Beeler, J. F., Ellmore, N., Heidaran, M. A., and Pierce, J. H. (1994) Mol. Cell. Biol. 14, 6727-6735[Abstract/Free Full Text]
  19. Ma, H., Matsunaga, H., Li, B., Marrero, M. B., and Ling, B. N. (1996) Am. J. Physiol. 271, F994-F1003[Abstract/Free Full Text]
  20. Akimoto, K., Takahashi, R., Moriya, S., Nishioka, N., Takayanagi, J., Kimura, K., Fukui, Y., Osada, S., Mizuno, K., Hirai, S., Kazlauskas, A., and Ohno, S. (1996) EMBO J. 15, 788-798[Medline] [Order article via Infotrieve]
  21. Inui, H., Kitami, Y., Tani, M., Kondo, T., and Inagami, T. (1994) J. Biol. Chem. 269, 30546-30552[Abstract/Free Full Text]
  22. Moriya, S., Kazlauskas, A., Akimoto, K., Hirai, S., Mizuno, K., Takenawa, T., Fukui, Y., Watanabe, Y., Ozaki, S., and Ohno, S. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 151-155[Abstract/Free Full Text]
  23. Lubinus, M., Meier, K. E., Smith, E. A., Gause, K. C., LeRoy, E. C., and Trojanowska, M. (1994) J. Biol. Chem. 269, 9822-9825[Abstract/Free Full Text]
  24. Ichimura, T., Uchiyama, J., Kunihiro, O., Ito, M., Horigome, T., Omata, S., Shinkai, F., Kaji, H., and Isobe, T. (1995) J. Biol. Chem. 270, 28515-28518[Abstract/Free Full Text]
  25. Ichimura, T., Ito, M., Itagaki, C., Takahashi, M., Horigome, T., Omata, S., Ohno, S., and Isobe, T. (1997) FEBS Lett. 413, 273-276[CrossRef][Medline] [Order article via Infotrieve]
  26. Furukawa, Y., Ikuta, N., Omata, S., Yamauchi, T., Isobe, T., and Ichimura, T. (1993) Biochem. Biophys. Res. Commun. 194, 144-149[CrossRef][Medline] [Order article via Infotrieve]
  27. Muslin, A. J., Tanner, J. W., Allen, P. M., and Shaw, A. S. (1996) Cell 84, 889-897[CrossRef][Medline] [Order article via Infotrieve]
  28. Zha, J., Harada, H., Yang, E., Jockel, J., and Korsmeyer, S. J. (1996) Cell 87, 619-628[CrossRef][Medline] [Order article via Infotrieve]
  29. Craparo, A., Freund, R., and Gustafson, T. A. (1997) J. Biol. Chem. 272, 11663-11669[Abstract/Free Full Text]
  30. Tanti, J.-F., Gremeaux, T., van Obberghen, E., and Le Marchand-Brustel, Y. (1994) J. Biol. Chem. 269, 6051-6057[Abstract/Free Full Text]
  31. Hotamisligil, G. S., Peraldi, P., Budavari, A., Ellis, R., White, M. F., and Spiegelman, B. M. (1996) Science 271, 665-668[Abstract]
  32. Cherniack, A. D., Klarlund, J. K., Conway, B. R., and Czech, M. P. (1995) J. Biol. Chem. 270, 1485-1488[Abstract/Free Full Text]
  33. Chen, H., Charlat, O., Tartaglia, L. A., Woolf, E. A., Weng, X., Ellis, S. J., Lakey, N. D., Culpepper, J., Moore, K. J., Breitbart, R. E., Duyk, G. M., Tepper, R. I., and Morgenstern, J. P. (1996) Cell 84, 491-495[CrossRef][Medline] [Order article via Infotrieve]
  34. Osborne, M. A., Dalton, S., and Kochan, J. P. (1995) Bio/Technology 13, 1474-1478[CrossRef][Medline] [Order article via Infotrieve]
  35. Yokouchi, M., Suzuki, R., Masuhara, M., Komiya, S., Inoue, A., and Yoshimura, A. (1997) Oncogene 15, 7-15[CrossRef][Medline] [Order article via Infotrieve]
  36. Huang, X., Li, Y., Tanaka, K., Moore, K. G., and Hayashi, J. I. (1995) Proc. Natl. Acad. Sci. U. S. A. 92, 11618-11622[Abstract/Free Full Text]

Copyright © 1998 by The American Society for Biochemistry and Molecular Biology, Inc.
Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 Mol. Endocrinol.Home page
L. Rider, J. Tao, S. Snyder, B. Brinley, J. Lu, and M. Diakonova
Adapter Protein SH2B1{beta} Cross-Links Actin Filaments and Regulates Actin Cytoskeleton
Mol. Endocrinol., July 1, 2009; 23(7): 1065 - 1076.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
T. J. Maures, L. Chen, and C. Carter-Su
Nucleocytoplasmic Shuttling of the Adapter Protein SH2B1{beta} (SH2-B{beta}) Is Required for Nerve Growth Factor (NGF)-Dependent Neurite Outgrowth and Enhancement of Expression of a Subset of NGF-Responsive Genes
Mol. Endocrinol., July 1, 2009; 23(7): 1077 - 1091.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Endocrinol.Home page
D. Yoshiga, N. Sato, T. Torisu, H. Mori, R. Yoshida, S. Nakamura, G. Takaesu, T. Kobayashi, and A. Yoshimura
Adaptor Protein SH2-B Linking Receptor-Tyrosine Kinase and Akt Promotes Adipocyte Differentiation by Regulating Peroxisome Proliferator-Activated Receptor {gamma} Messenger Ribonucleic Acid Levels
Mol. Endocrinol., May 1, 2007; 21(5): 1120 - 1131.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
A. M. Mazurkiewicz-Munoz, L. S. Argetsinger, J.-L. K. Kouadio, A. Stensballe, O. N. Jensen, J. M. Cline, and C. Carter-Su
Phosphorylation of JAK2 at Serine 523: a Negative Regulator of JAK2 That Is Stimulated by Growth Hormone and Epidermal Growth Factor.
Mol. Cell. Biol., June 1, 2006; 26(11): 4052 - 4062.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
M. Nishi, E. D. Werner, B.-C. Oh, J. D. Frantz, S. Dhe-Paganon, L. Hansen, J. Lee, and S. E. Shoelson
Kinase Activation through Dimerization by Human SH2-B
Mol. Cell. Biol., April 1, 2005; 25(7): 2607 - 2621.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
C. Duan, H. Yang, M. F. White, and L. Rui
Disruption of the SH2-B Gene Causes Age-Dependent Insulin Resistance and Glucose Intolerance
Mol. Cell. Biol., September 1, 2004; 24(17): 7435 - 7443.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
L. Chen and C. Carter-Su
Adapter Protein SH2-B{beta} Undergoes Nucleocytoplasmic Shuttling: Implications for Nerve Growth Factor Induction of Neuronal Differentiation
Mol. Cell. Biol., May 1, 2004; 24(9): 3633 - 3647.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
X. Wang, L. Chen, T. J. Maures, J. Herrington, and C. Carter-Su
SH2-B Is a Positive Regulator of Nerve Growth Factor-mediated Activation of the Akt/Forkhead Pathway in PC12 Cells
J. Biol. Chem., January 2, 2004; 279(1): 133 - 141.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
I. Nobuhisa, M. Takizawa, S. Takaki, H. Inoue, K. Okita, M. Ueno, K. Takatsu, and T. Taga
Regulation of Hematopoietic Development in the Aorta-Gonad-Mesonephros Region Mediated by Lnk Adaptor Protein
Mol. Cell. Biol., December 1, 2003; 23(23): 8486 - 8494.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. B. O'Brien, L. S. Argetsinger, M. Diakonova, and C. Carter-Su
YXXL Motifs in SH2-Bbeta Are Phosphorylated by JAK2, JAK1, and Platelet-derived Growth Factor Receptor and Are Required for Membrane Ruffling
J. Biol. Chem., March 28, 2003; 278(14): 11970 - 11978.
[Abstract] [Full Text] [PDF]

Home page
 JEMHome page
L. Velazquez, A. M. Cheng, H. E. Fleming, C. Furlonger, S. Vesely, A. Bernstein, C. J. Paige, and T. Pawson
Cytokine Signaling and Hematopoietic Homeostasis Are Disrupted in Lnk-deficient Mice
J. Exp. Med., June 17, 2002; 195(12): 1599 - 1611.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Kong, C. S. Wang, and D. J. Donoghue
Interaction of Fibroblast Growth Factor Receptor 3 and the Adapter Protein SH2-B. A ROLE IN STAT5 ACTIVATION
J. Biol. Chem., May 3, 2002; 277(18): 15962 - 15970.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
S. Ohtsuka, S. Takaki, M. Iseki, K. Miyoshi, N. Nakagata, Y. Kataoka, N. Yoshida, K. Takatsu, and A. Yoshimura
SH2-B Is Required for Both Male and Female Reproduction
Mol. Cell. Biol., May 1, 2002; 22(9): 3066 - 3077.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
M. Diakonova, D. R. Gunter, J. Herrington, and C. Carter-Su
SH2-Bbeta Is a Rac-binding Protein That Regulates Cell Motility
J. Biol. Chem., March 15, 2002; 277(12): 10669 - 10677.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
K. B. O'Brien, J. J. O'Shea, and C. Carter-Su
SH2-B Family Members Differentially Regulate JAK Family Tyrosine Kinases
J. Biol. Chem., March 1, 2002; 277(10): 8673 - 8681.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Cell. Biol.Home page
L. Rui, D. R. Gunter, J. Herrington, and C. Carter-Su
Differential Binding to and Regulation of JAK2 by the SH2 Domain and N-Terminal Region of SH2-Bbeta
Mol. Cell. Biol., May 1, 2000; 20(9): 3168 - 3177.
[Abstract] [Full Text]

Home page
 J. Biol. Chem.Home page
J. Herrington, M. Diakonova, L. Rui, D. R. Gunter, and C. Carter-Su
SH2-B Is Required for Growth Hormone-induced Actin Reorganization
J. Biol. Chem., April 21, 2000; 275(17): 13126 - 13133.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Rui, S. F. Archer, L. S. Argetsinger, and C. Carter-Su
Platelet-derived Growth Factor and Lysophosphatidic Acid Inhibit Growth Hormone Binding and Signaling via a Protein Kinase C-dependent Pathway
J. Biol. Chem., January 28, 2000; 275(4): 2885 - 2892.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Rui, J. Herrington, and C. Carter-Su
SH2-B, a Membrane-associated Adapter, Is Phosphorylated on Multiple Serines/Threonines in Response to Nerve Growth Factor by Kinases within the MEK/ERK Cascade
J. Biol. Chem., September 10, 1999; 274(37): 26485 - 26492.
[Abstract] [Full Text] [PDF]

Home page
 Proc. Natl. Acad. Sci. USAHome page
L. Rui and C. Carter-Su
Identification of SH2-Bbeta as a potent cytoplasmic activator of the tyrosine kinase Janus kinase 2
PNAS, June 22, 1999; 96(13): 7172 - 7177.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
S. A. Moodie, J. Alleman-Sposeto, and T. A. Gustafson
Identification of the APS Protein as a Novel Insulin Receptor Substrate
J. Biol. Chem., April 16, 1999; 274(16): 11186 - 11193.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
L. Rui, J. Herrington, and C. Carter-Su
SH2-B Is Required for Nerve Growth Factor-induced Neuronal Differentiation
J. Biol. Chem., April 9, 1999; 274(15): 10590 - 10594.
[Abstract] [Full Text] [PDF]

Home page
 J. Biol. Chem.Home page
N. Yousaf, Y. Deng, Y. Kang, and H. Riedel
Four PSM/SH2-B Alternative Splice Variants and Their Differential Roles in Mitogenesis
J. Biol. Chem., October 26, 2001; 276(44): 40940 - 40948.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Submit a Letter to Editor
Right arrow Alert me when this article is cited
Right arrow Alert me when eLetters are posted
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowRequest Permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rui, L.
Right arrow Articles by Carter-Su, C.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rui, L.
Right arrow Articles by Carter-Su, C.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
 All ASBMB Journals   Molecular and Cellular Proteomics 
 Journal of Lipid Research   ASBMB Today 
Copyright © 1998 by the American Society for Biochemistry and Molecular Biology.
Advertisement
spacer
Advertisement
Advertisement