scholarly journals Structural Determinants of SHP-2 Function and Specificity in Xenopus Mesoderm Induction

1998 ◽  
Vol 18 (1) ◽  
pp. 161-177 ◽  
Author(s):  
Alana M. O’Reilly ◽  
Benjamin G. Neel
Keyword(s):  
Tyrosine Phosphatase ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Mesoderm Induction ◽  
Structural Determinants ◽  
Tyrosine Kinase Signaling ◽  
Biologically Relevant ◽  
Sh2 Domains ◽  
Tyrosyl Phosphorylation

ABSTRACT SHP-2 is a positive component of many receptor tyrosine kinase signaling pathways. The related protein-tyrosine phosphatase (PTP) SHP-1 usually acts as a negative regulator. The precise domains utilized by SHP-2 to transmit positive signals in vivo and the basis for specificity between SHP-1 and SHP-2 are not clear. InXenopus, SHP-2 is required for mesoderm induction and completion of gastrulation. We investigated the effects of SHP-2 mutants and SHP-2/SHP-1 chimeras on basic fibroblast growth factor-induced mesoderm induction. Both SH2 domains and the PTP domain are required for normal SHP-2 function in this pathway. The N-terminal SH2 domain is absolutely required, whereas the C-terminal SH2 contributes to wild-type function. The C-terminal tyrosyl phosphorylation sites and proline-rich region are dispensable, arguing against adapter models of SHP-2 function. Although the SH2 domains contribute to SHP-2 specificity, studies of SHP chimeras reveal that substantial specificity resides in the PTP domain. Thus, PTP domains exhibit biologically relevant specificity in vivo, and noncatalytic and catalytic domains of PTPs contribute to specificity in a combinatorial fashion.

scholarly journals Regulation of colony-stimulating factor 1 receptor signaling by the SH2 domain-containing tyrosine phosphatase SHPTP1.

1996 ◽  
Vol 16 (7) ◽  
pp. 3685-3697 ◽  
Author(s):  
H E Chen ◽  
S Chang ◽  
T Trub ◽  
B G Neel
Keyword(s):  
Tyrosine Phosphatase ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Colony Stimulating Factor ◽  
Sh2 Domains ◽  
Associated Proteins ◽  
Primary Bone ◽  
Macrophage Colony ◽  
Stimulating Factor

SHPTP1 (PTP1C, HCP, SHP) is an SH2 domain-containing tyrosine phosphatase expressed predominantly in hematopoietic cells. A frameshift mutation in the SHPTP1 gene causes the motheaten (me/me) mouse. These mice are essentially SHPTP1 null and display multiple hematopoietic abnormalities, most prominently hyperproliferation and inappropriate activation of granulocytes and macrophages. The me/me phenotype suggests that SHPTP1 negatively regulates macrophage proliferative pathways. Using primary bone marrow-derived macrophages from me/me mice and normal littermates, we examined the role of SHPTP1 in regulating signaling by the major macrophage mitogen colony-stimulating factor 1 (CSF-1) (also known as macrophage colony-stimulating factor). Macrophages from me/me mice hyperproliferate in response to CSF-1. In the absence of SHPTP1, the CSF-1 receptor (CSF-1R) is hyperphosphorylated upon CSF-1 stimulation, suggesting that SHPTP1 dephosphorylates the CSF-1R. At least some CSF-1R-associated proteins also are hyperactivated. SHPTP1 is associated constitutively, via its SH2 domains, with an unidentified 130-kDa phosphotyrosyl protein (P130). P130 and SHPTP1 are further tyrosyl phosphorylated upon CSF-1 stimulation. Tyrosyl-phosphorylated SHPTP1 binds to Grb2 via the Grb2 SH2 domain. Moreover, in me/me macrophages, Grb2 is associated, via its SH3 domains, with several tyrosyl phosphoproteins. These proteins are hyperphosphorylated on tyrosyl residues in me/me macrophages, suggesting that Grb2 may recruit substrates for SHPTP1. Our results indicate that SHPTP1 is a critical negative regulator of CSF-1 signaling in vivo and suggest a potential new function for Grb2.

scholarly journals Direct association with and dephosphorylation of Jak2 kinase by the SH2-domain-containing protein tyrosine phosphatase SHP-1.

1996 ◽  
Vol 16 (12) ◽  
pp. 6985-6992 ◽  
Author(s):  
H Jiao ◽  
K Berrada ◽  
W Yang ◽  
M Tabrizi ◽  
L C Platanias ◽  
...  
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Tyrosine Kinases ◽  
Tyrosine Phosphatase ◽  
Binding Activity ◽  
Sh2 Domain ◽  
Erythropoietin Receptor ◽  
Protein Tyrosine ◽  
Sh2 Domains

SHP-1 is an SH2-containing cytoplasmic tyrosine phosphatase that is widely distributed in cells of the hematopoietic system. SHP-1 plays an important role in the signal transduction of many cytokine receptors, including the receptor for erythropoietin, by associating via its SH2 domains to the receptors and dephosphorylating key substrates. Recent studies have suggested that SHP-1 regulates the function of Jak family tyrosine kinases, as shown by its constitutive association with the Tyk2 kinase and the hyperphosphorylation of Jak kinases in the motheaten cells that lack functional SHP-1. We have examined the interactions of SHP-1 with two tyrosine kinases activated during engagement of the erythropoietin receptor, the Janus family kinase Jak-2 and the c-fps/fes kinase. Immunoblotting studies with extracts from mouse hematopoietic cells demonstrated that Jak2, but not c-fes, was present in anti-SHP-1 immunoprecipitates, suggesting that SHP-1 selectively associates with Jak2 in vivo. Consistent with this, when SHP-1 was coexpressed with these kinases in Cos-7 cells, it associated with and dephosphorylated Jak2 but not c-fes. Transient cotransfection of truncated forms of SHP-1 with Jak2 demonstrated that the SHP-1-Jak2 interaction is direct and is mediated by a novel binding activity present in the N terminus of SHP-1, independently of SH2 domain-phosphotyrosine interaction. Such SHP-1-Jak2 interaction resulted in induction of the enzymatic activity of the phosphatase in in vitro protein tyrosine phosphatase assays. Interestingly, association of the SH2n domain of SHP-1 with the tyrosine phosphorylated erythropoietin receptor modestly potentiated but was not essential for SHP-1-mediated dephosphorylation of Jak2 and had no effect on c-fes phosphorylation. These data indicate that the main mechanism for regulation of Jak2 phosphorylation by SHP-1 involves a direct, SH2-independent interaction with Jak2 and suggest the existence of similar mechanisms for other members of the Jak family of kinases. They also suggest that such interactions may provide one of the mechanisms that control SHP-1 substrate specificity.

scholarly journals Negative Regulation of Ros Receptor Tyrosine Kinase Signaling

2001 ◽  
Vol 152 (2) ◽  
pp. 325-334 ◽  
Author(s):  
Heike Keilhack ◽  
Marit Müller ◽  
Sylvia-Annette Böhmer ◽  
Carsten Frank ◽  
K. Michael Weidner ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Receptor Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Tyrosine Phosphatase ◽  
Sh2 Domain ◽  
Glutathione S Transferase ◽  
Tyrosine Kinase Signaling ◽  
Ros Signaling ◽  
Targeted Inactivation

Male “viable motheaten” (mev) mice, with a naturally occurring mutation in the gene of the SH2 domain protein tyrosine phosphatase SHP-1, are sterile. Known defects in sperm maturation in these mice correlate with an impaired differentiation of the epididymis, which has similarities to the phenotype of mice with a targeted inactivation of the Ros receptor tyrosine kinase. Ros and SHP-1 are coexpressed in epididymal epithelium, and elevated phosphorylation of Ros in the epididymis of mev mice suggests that Ros signaling is under control of SHP-1 in vivo. Phosphorylated Ros strongly and directly associates with SHP-1 in yeast two-hybrid, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Strong binding of SHP-1 to Ros is selective compared to six other receptor tyrosine kinases. The interaction is mediated by the SHP-1 NH2-terminal SH2 domain and Ros phosphotyrosine 2267. Overexpression of SHP-1 results in Ros dephosphorylation and effectively downregulates Ros-dependent proliferation and transformation. We propose that SHP-1 is an important downstream regulator of Ros signaling.

scholarly journals Coordinated Regulation of Insulin Signaling by the Protein Tyrosine Phosphatases PTP1B and TCPTP

2005 ◽  
Vol 25 (2) ◽  
pp. 819-829 ◽  
Author(s):  
Sandra Galic ◽  
Christine Hauser ◽  
Barbara B. Kahn ◽  
Fawaz G. Haj ◽  
Benjamin G. Neel ◽  
...  
Keyword(s):  
Insulin Signaling ◽  
Tyrosine Phosphatase ◽  
Protein Tyrosine Phosphatases ◽  
Negative Regulator ◽  
Mitogen Activated Protein Kinase ◽  
Protein Tyrosine ◽  
Akt Activation ◽  
Protein Levels ◽  
Mitogen Activated Protein

ABSTRACT The protein tyrosine phosphatase PTP1B is a negative regulator of insulin signaling and a therapeutic target for type 2 diabetes. Our previous studies have shown that the closely related tyrosine phosphatase TCPTP might also contribute to the regulation of insulin receptor (IR) signaling in vivo (S. Galic, M. Klingler-Hoffmann, M. T. Fodero-Tavoletti, M. A. Puryer, T. C. Meng, N. K. Tonks, and T. Tiganis, Mol. Cell. Biol. 23:2096-2108, 2003). Here we show that PTP1B and TCPTP function in a coordinated and temporally distinct manner to achieve an overall regulation of IR phosphorylation and signaling. Whereas insulin-induced phosphatidylinositol 3-kinase/Akt signaling was prolonged in both TCPTP−/− and PTP1B−/− immortalized mouse embryo fibroblasts (MEFs), mitogen-activated protein kinase ERK1/2 signaling was elevated only in PTP1B-null MEFs. By using phosphorylation-specific antibodies, we demonstrate that both IR β-subunit Y1162/Y1163 and Y972 phosphorylation are elevated in PTP1B−/− MEFs, whereas Y972 phosphorylation was elevated and Y1162/Y1163 phosphorylation was sustained in TCPTP−/− MEFs, indicating that PTP1B and TCPTP differentially contribute to the regulation of IR phosphorylation and signaling. Consistent with this, suppression of TCPTP protein levels by RNA interference in PTP1B−/− MEFs resulted in no change in ERK1/2 signaling but caused prolonged Akt activation and Y1162/Y1163 phosphorylation. These results demonstrate that PTP1B and TCPTP are not redundant in insulin signaling and that they act to control both common as well as distinct insulin signaling pathways in the same cell.

Binding of the Src SH2 domain to phosphopeptides is determined by residues in both the SH2 domain and the phosphopeptides

1993 ◽  
Vol 13 (12) ◽  
pp. 7278-7287
Author(s):  
K B Bibbins ◽  
H Boeuf ◽  
H E Varmus
Keyword(s):  
Intramolecular Interaction ◽  
Growth Factor Receptor ◽  
Sh2 Domain ◽  
Binding Properties ◽  
Vitro Assay ◽  
Sh2 Domains ◽  
Free Peptide ◽  
C Terminus

Src homology 2 (SH2) domains are found in a variety of signaling proteins and bind phosphotyrosine-containing peptide sequences. To explore the binding properties of the SH2 domain of the Src protein kinase, we used immobilized phosphopeptides to bind purified glutathione S-transferase-Src SH2 fusion proteins. With this assay, as well as a free-peptide competition assay, we have estimated the affinities of the Src SH2 domain for various phosphopeptides relative to a Src SH2-phosphopeptide interaction whose Kd has been determined previously (YEEI-P; Kd = 4 nM). Two Src-derived phosphopeptides, one containing the regulatory C-terminal Tyr-527 and another containing the autophosphorylation site Tyr-416, bind the Src SH2 domain in a specific though low-affinity manner (with about 10(4)-lower affinity than the YEEI-P peptide). A platelet-derived growth factor receptor (PDGF-R) phosphopeptide containing Tyr-857 does not bind appreciably to the Src SH2 domain, suggesting it is not the PDGF-R binding site for Src as previously reported. However, another PDGF-R-derived phosphopeptide containing Tyr-751 does bind the Src SH2 domain (with an affinity approximately 2 orders of magnitude lower than that of YEEI-P). All of the phosphopeptides which bind to the Src SH2 domain contain a glutamic acid at position -3 or -4 with respect to phosphotyrosine; changing this residue to alanine greatly diminishes binding. We have also tested Src SH2 mutants for their binding properties and have interpreted our results in light of the recent crystal structure solution for the Src SH2 domain. Mutations in various conserved and nonconserved residues (R155A, R155K, N198E, H201R, and H201L) cause slight reductions in binding, while two mutations cause severe reductions. The W148E mutant domain, which alters the invariant tryptophan that marks the N-terminal border of the SH2 domain, binds poorly to phosphopeptides. Inclusion of the SH3 domain in the fusion protein partially restores the binding by the W148E mutant. A change in the invariant arginine that coordinates twice with phosphotyrosine in the peptide (R175L) results in a nearly complete loss of binding. The R175L mutant does display high affinity for the PDGF-R peptide containing Tyr-751, via an interaction that is at least partly phosphotyrosine independent. We have used this interaction to show that the R175L mutation also disrupts the intramolecular interaction between the Src SH2 domain and the phosphorylated C terminus within the context of the entire Src protein; thus, the binding properties observed for mutant domains in an in vitro assay appear to mimic those that occur in vivo.

scholarly journals Regulation of Microtubule Nucleation in Mouse Bone Marrow-Derived Mast Cells by Protein Tyrosine Phosphatase SHP-1

Cells ◽  
2019 ◽  
Vol 8 (4) ◽  
pp. 345 ◽  
Author(s):  
Klebanovych ◽  
Sládková ◽  
Sulimenko ◽  
Vosecká ◽  
Čapek ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Mast Cells ◽  
Protein Tyrosine Phosphatase ◽  
Tyrosine Phosphatase ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Mast Cell Activation ◽  
Mouse Bone Marrow ◽  
Microtubule Nucleation ◽  
Protein Tyrosine

The antigen-mediated activation of mast cells initiates signaling events leading to their degranulation, to the release of inflammatory mediators, and to the synthesis of cytokines and chemokines. Although rapid and transient microtubule reorganization during activation has been described, the molecular mechanisms that control their rearrangement are largely unknown. Microtubule nucleation is mediated by γ-tubulin complexes. In this study, we report on the regulation of microtubule nucleation in bone marrow-derived mast cells (BMMCs) by Src homology 2 (SH2) domain-containing protein tyrosine phosphatase 1 (SHP-1; Ptpn6). Reciprocal immunoprecipitation experiments and pull-down assays revealed that SHP-1 is present in complexes containing γ-tubulin complex proteins and protein tyrosine kinase Syk. Microtubule regrowth experiments in cells with deleted SHP-1 showed a stimulation of microtubule nucleation, and phenotypic rescue experiments confirmed that SHP-1 represents a negative regulator of microtubule nucleation in BMMCs. Moreover, the inhibition of the SHP-1 activity by inhibitors TPI-1 and NSC87877 also augmented microtubule nucleation. The regulation was due to changes in γ-tubulin accumulation. Further experiments with antigen-activated cells showed that the deletion of SHP-1 stimulated the generation of microtubule protrusions, the activity of Syk kinase, and degranulation. Our data suggest a novel mechanism for the suppression of microtubule formation in the later stages of mast cell activation.

scholarly journals Tyrosine 425 within the activated erythropoietin receptor binds Syp, reduces the erythropoietin required for Syp tyrosine phosphorylation, and promotes mitogenesis

Blood ◽  
1996 ◽  
Vol 87 (11) ◽  
pp. 4495-4501 ◽  
Author(s):  
T Tauchi ◽  
JE Damen ◽  
K Toyama ◽  
GS Feng ◽  
HE Broxmeyer ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Intracellular Signaling ◽  
Tyrosine Phosphatase ◽  
Erythropoietin Receptor ◽  
Sh2 Domains ◽  
Proliferation And Differentiation ◽  
Stimulation Of

Erythropoietin (Epo), the primary in vivo stimulator of erythroid proliferation and differentiation, acts, in part, by altering the tyrosine phosphorylation levels of various intracellular signaling molecules. These phosphorylation levels are tightly regulated by both tyrosine kinases and tyrosine phosphatases. We have recently shown that the SH2 containing tyrosine phosphatase, Syp, binds directly to both the tyrosine phosphorylated form of the Epo receptor (EpoR) and to Grb2 after Epo stimulation of M07e cells engineered to express high levels of human EpoRs (T. Tauchi, et al: J Biol Chem 270:5631, 1995). To determine which tyrosine within the EpoR is responsible for binding Syp, we examined DA-3 cell lines expressing full-length mutant EpoRs bearing tyrosine to phenylalanine substitutions for each of the eight tyrosines within the intracellular domain of the EpoR. We found that: (1) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, coimmunoprecipitated with Syp; (2) all Epo-stimulated mutant EpoRs, except for the Y425F EpoR, bound to a GST-fusion protein containing both SH2 domains of Syp; (3) Jak2 could phosphorylate GST-Syp in vitro after Epo stimulation of wild-type (wt) EpoR expressing DA-3 cells; (4) Epo-stimulated tyrosine phosphorylation of Syp in vivo was markedly reduced in Y425F EpoR expressing DA-3 calls; and (5) DA-3 cells expressing the Y425F EpoR grow less well in response to Epo than wt EpoR expressing cells. These results suggest that Syp binds via its SH2 domains to phosphorylated Y425 within the EpoR and is then phosphorylated on tyrosine residues by Jak2. Moreover, Y425 in the EpoR reduces the Epo requirement for Syp tyrosine phosphorylation and promotes proliferation.

scholarly journals Mutagenic analysis of the roles of SH2 and SH3 domains in regulation of the Abl tyrosine kinase.

1994 ◽  
Vol 14 (5) ◽  
pp. 2883-2894 ◽  
Author(s):  
B J Mayer ◽  
D Baltimore
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Sh3 Domain ◽  
Sh2 Domain ◽  
Sh3 Domains ◽  
Sh2 Domains ◽  
Abl Tyrosine Kinase ◽  
Transforming Activity

We have used in vitro mutagenesis to examine in detail the roles of two modular protein domains, SH2 and SH3, in the regulation of the Abl tyrosine kinase. As previously shown, the SH3 domain suppresses an intrinsic transforming activity of the normally nontransforming c-Abl product in vivo. We show here that this inhibitory activity is extremely position sensitive, because mutants in which the position of the SH3 domain within the protein is subtly altered are fully transforming. In contrast to the case in vivo, the SH3 domain has no effect on the in vitro kinase activity of the purified protein. These results are consistent with a model in which the SH3 domain binds a cellular inhibitory factor, which in turn must physically interact with other parts of the kinase. Unlike the SH3 domain, the SH2 domain is required for transforming activity of activated Abl alleles. We demonstrate that SH2 domains from other proteins (Ras-GTPase-activating protein, Src, p85 phosphatidylinositol 3-kinase subunit, and Crk) can complement the absence of the Abl SH2 domain and that mutants with heterologous SH2 domains induce altered patterns of tyrosine-phosphorylated proteins in vivo. The positive function of the SH2 domain is relatively position independent, and the effect of multiple SH2 domains appears to be additive. These results suggest a novel mechanism for regulation of tyrosine kinases in which the SH2 domain binds to, and thereby enhances the phosphorylation of, a subset of proteins phosphorylated by the catalytic domain. Our data also suggest that the roles of the SH2 and SH3 domains in the regulation of Abl are different in several respects from the roles proposed for these domains in the closely related Src family of tyrosine kinases.

Adaptor Protein Lnk Binds to PDGFRA, PDGFRB and FIP1L1-PDGFRA, but Not to the TEL-PDGFRB Fusion Protein.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2213-2213
Author(s):  
Saskia Gueller ◽  
Sigal Gery ◽  
H. Phillip Koeffler
Keyword(s):  
Fusion Protein ◽  
Western Blot ◽  
Adaptor Protein ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Pleckstrin Homology Domain ◽  
Wild Type ◽  
Nih3t3 Cells ◽  
Sh2 Domains ◽  
Juxtamembrane Region

Abstract PDGFRA and PDGFRB (platelet derived growth factor receptors alpha and beta) are frequently expressed on malignant hematopoietic cells and regulate various cellular responses such as development, proliferation, differentiation, cell survival and cellular transformation. Stimulation by either autocrine loops or constitutional activation by chromosomal translocation (i.e. chronic myelomonocytic leukemia [CMML, TEL-PDGFRB] or chronic eosinophilic leukemia [CEL, FIP1L1-PDGFRA]) makes them important factors in development of hematopoietic disorders. Normally, interaction with the ligand PDGF, induces dimerization of two distinct receptor subunits, resulting in activation of the intracellular tyrosine kinase domain and phosphorylation of tyrosine residues, thereby creating binding sites for several molecules containing Src homology 2 (SH2) domains. We hypothesized that one such protein may be the adaptor Lnk, a negative regulator of several hematopoietic cytokine receptors including MPL, EpoR and c-Kit. Lnk belongs to a family of proteins sharing several structural motifs including a SH2 domain, a pleckstrin homology domain (PH) and a dimerization domain (DD). The SH2 domain is known to be essential for its inhibitory effect which can be abolished by the point mutation R392E. We investigated the ability of Lnk to bind to PDGFRA, PDGFRB, FIP1L1-PDGFRA and TEL-PDGFRB. To determine the domain of Lnk that is responsible for the binding, we constructed a series of V5-tagged Lnk mutants including: a mutation in the SH2 domain (R392E); deletion of the SH2 domain; deletion of the PH and SH2 domains and a construct only containing the DD domain. 293T cells were co-transfected with cDNAs encoding either PDGFRA, PDGFRB or one of the translocation products and either wild-type or mutant Lnk. Whole cell lysates were used to perform immunoprecipitation with either V5-tag or PDGFR antibodies. Binding of Lnk and PDGFR was detected by Western blot probed with PDGFR or V5-tag antibodies. NIH3T3 cells were transfected either with empty vector or Lnk cDNA, transfectants were selected for 5 days with G418, serum starved for 16 hours and induced with PDGF for 10 minutes. Phosphorylation of downstream targets of PDGFRA and PDGFRB was detected by Western blot. Our data showed that Lnk bound to PDGFRA and PDGFRB only after exposure of the cells to PDGF and to the FIP1L1-PDGFRA fusion protein independent of PDGF exposure. Mutation or deletion of the Lnk SH2 domain abolished binding completely in PDGFRA and FIP1L1-PDGFRA, but just partly in PDGFRB. Expression of Lnk in NIH3T3 cells inhibited phosphorylation of ERK after treatment with PDGF. In other experiments, we determined that Lnk bound the juxtamembrane region of this class of receptors. Interestingly, the TEL-PDGFRB fusion protein was unable to bind Lnk, although its breakpoint in PDGFRB is distal to the juxtamembrane domain and the whole intracellular region of PDGFRB is included in the fusion protein. Further exploration of the mechanisms by which Lnk affects wild-type or PDGFR fusion product will provide insight into the molecular pathophysiology of myeloid disorders and could help develop new treatments.

scholarly journals Identification of Major Binding Proteins and Substrates for the SH2-Containing Protein Tyrosine Phosphatase SHP-1 in Macrophages

1998 ◽  
Vol 18 (7) ◽  
pp. 3838-3850 ◽  
Author(s):  
John F. Timms ◽  
Kristen Carlberg ◽  
Haihua Gu ◽  
Haiyan Chen ◽  
Shubhangi Kamatkar ◽  
...  
Keyword(s):  
Protein Tyrosine Phosphatase ◽  
Kinase Activity ◽  
Tyrosine Phosphatase ◽  
Regulatory Protein ◽  
Protein Tyrosine ◽  
Sh2 Domains ◽  
Receptor Complexes ◽  
Multiple Signaling ◽  
Tyrosyl Phosphorylation ◽  
Stimulating Factor

ABSTRACT The protein tyrosine phosphatase SHP-1 is a critical regulator of macrophage biology, but its detailed mechanism of action remains largely undefined. SHP-1 associates with a 130-kDa tyrosyl-phosphorylated species (P130) in macrophages, suggesting that P130 might be an SHP-1 regulator and/or substrate. Here we show that P130 consists of two transmembrane glycoproteins, which we identify as PIR-B/p91A and the signal-regulatory protein (SIRP) family member BIT. These proteins also form separate complexes with SHP-2. BIT, but not PIR-B, is in a complex with the colony-stimulating factor 1 receptor (CSF-1R), suggesting that BIT may direct SHP-1 to the CSF-1R. BIT and PIR-B bind preferentially to substrate-trapping mutants of SHP-1 and are hyperphosphorylated in macrophages from motheaten viable mice, which express catalytically impaired forms of SHP-1, indicating that these proteins are SHP-1 substrates. However, BIT and PIR-B are hypophosphorylated in motheaten macrophages, which completely lack SHP-1 expression. These data suggest a model in which SHP-1 dephosphorylates specific sites on BIT and PIR-B while protecting other sites from dephosphorylation via its SH2 domains. Finally, BIT and PIR-B associate with two tyrosyl phosphoproteins and a tyrosine kinase activity. Tyrosyl phosphorylation of these proteins and the level of the associated kinase activity are increased in the absence of SHP-1. Our data suggest that BIT and PIR-B recruit multiple signaling molecules to receptor complexes, where they are regulated by SHP-1 and/or SHP-2.

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