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

The substrate specificity of the catalytic domain of SHP-1, an important regulator in the proliferation and development of hematopoietic cells, is critical for understanding the physiological functions of SHP-1. Here we report the crystal structures of the catalytic domain of SHP-1 complexed with two peptide substrates derived from SIRP␣, a member of the signal-regulatory proteins. We show that the variable ␤5-loop-␤6 motif confers SHP-1 substrate specificity at the P-4 and further Nterminal subpockets. We also observe a novel residue shift at P-2, the highly conserved subpocket in proteintyrosine phosphatases. Our observations provide new insight into the substrate specificity of SHP-1. Protein-tyrosine phosphatases (PTPs)1 consist of a diverse family of enzymes that play crucial roles in cell growth, differentiation, and transformation (1-3). They can be broadly divided into membrane-bound, receptor-like PTPs, and cytosolic PTPs. The cytosolic PTPs contain only one catalytic domain, whereas the membrane-bound receptor-like PTPs usually contain two tandem catalytic domains. The catalytic domains of PTPs are highly conserved in their three-dimensional structures (4 -7). However, they have remarkably different substrate specificity (3, 8 -10), which is still not well understood. Previous studies using various synthetic phosphotyrosyl peptides failed to identify a shared by PTP substrate because the peptides studied were not derived from physiological substrates of PTPs. In the present study, we have addressed the structural basis for the substrate specificity of PTPs using SHP-1 and its physiological substrate SIRP␣/SHPS-1 as a model. SIRP␣ is a transmembrane protein of the signal-regulatory protein family. Its extracellular domain contains three immunoglobulin domains, and its cytoplasmic domain contains four phosphotyrosine sites (Tyr(P) 427 , Tyr(P) 452 , Tyr(P) 469 , and Tyr(P) 495 ). SHP-1 is expressed primarily in hematopoietic cells, and contains two Src homology 2 (SH2) domains, a neighboring catalytic domain, and a C-terminal tail. Its phosphatase activity is inhibited by both the SH2 domains and the C-terminal tail (11,12). SHP-1 is activated upon the binding of its tandem SH2 domains to immunoreceptor tyrosine-based inhibitory motifs. Domain-swapping studies on SHP-1 and its analogue, SHP-2, have shown that the catalytic domains of SHP-1 and SHP-2 have distinct substrate specificity (9, 10), and therefore illustrate that the dissection of the structural basis for the substrate specificity of SHP-1 is fundamental to the understanding of its physiological functions. The identification of the substrates of SHP-1 (i.e. SIRP␣, CD22, and CD72; Refs. 13-15) has made it possible for us to probe this structural basis. The results of this probe are presented below. EXPERIMENTAL PROCEDURESCrystallization and Data Collection-The C455S mutant of the SHP-1 catalytic domain (245-532) was cloned, expressed, and purified as described elsewhere (16). The phosphotyrosyl decapeptides were synthesized and purified to ...

Section: Identification Of In Vitromentioning

confidence: 99%

“…SIRP␣, CD22, and CD72; Refs. [13][14][15] has made it possible for us to probe this structural basis. The results of this probe are presented below.…”

mentioning

confidence: 99%

Structural Basis for Substrate Specificity of Protein-tyrosine Phosphatase SHP-1

Yang¹,

Cheng²,

Niu³

et al. 2000

“…Antibody to the extracellular domain of SIRPα1 (αSIRP-Ex1) was raised against a glutathione S-transferase fusion protein encoding the first immunoglobulin-like domain (Ex1) and used at dilutions of 1:500 for immunoprecipitation and 1:1000 for Western blotting (1). Antibody to SHPS-1 (αSHPS-1), against a glutathione S-transferase fusion protein containing amino acids 403-511 of SHPS-1 (16), was kindly provided by B. Neel (Beth Israel Deaconess Hospital, Boston, MA) and was used at dilutions of 1:500 for immunoprecipitation and 1:1000 for Western blotting. Antibody to the doubly phosphorylated active form of ERK (αpERK) was from Promega (# V8031) and used at a dilution of 1:5000 for blotting.…”

Section: Introductionmentioning

confidence: 99%

Negative Regulation of Growth Hormone Receptor/JAK2 Signaling by Signal Regulatory Protein α

Stofega

Argetsinger²,

Wang³

et al. 2000

SUMMARYSIRPs (signal regulatory proteins) are receptor-like transmembrane proteins, the majority of which contain a cytoplasmic proline-rich region and four cytoplasmic tyrosines that, when phosphorylated, bind SH2 domain-containing protein tyrosine phosphatases (SHP). We Consistent with reduced JAK2 activity, overexpression of wild-type SIRPα1 but not SIRP 4YF reduces GH-induced phosphorylation of ERKs 1 and 2, STAT3 and STAT5B. These results suggest that SIRPα1 is a negative regulator of GH signaling and that the ability of SIRPα1

“…For example, treatment of tissue-cultured cells with growth factors (including plateletderived growth factor and epidermal growth factor), growth hormone, insulin, colony-stimulating factor, lysophosphatidic acid, etc., has been shown to induce phosphorylation of tyrosines on the intracellular immunoreceptor tyrosine-based inhibitory motif domain of SIRP␣, resulting in binding to Src homology 2 domain-containing tyrosine phosphatase-1 or 2 (SHP-1 and 2) (24,27,31). SIRP␣ binding to SHP-1 or SHP-2 mediates positive or negative signals that regulate a variety of cellular functions, respectively (27,32,33).…”

mentioning

confidence: 99%

Signal Regulatory Protein (SIRPα), a Cellular Ligand for CD47, Regulates Neutrophil Transmigration

Liu¹,

Bühring²,

Zen³

et al. 2002

189

232

Recent studies have demonstrated that CD47 plays an important role in regulating human neutrophil (PMN) chemotaxis. Two ligands for CD47, thrombospondin and SIRP␣, have been described. However, it is not known if SIRP-CD47 interactions play a role in regulating PMN migration. In this study, we show that SIRP␣1 directly binds to the immunoglobulin variable domain loop of purified human CD47 and that such SIRP-CD47 interactions regulate PMN transmigration. Specifically, PMN migration across both human epithelial monolayers and collagen-coated filters was partially inhibited by anti-SIRP monoclonal antibodies. Similar kinetics of inhibition were observed for PMN transmigration in the presence of soluble, recombinant CD47 consisting of the SIRP-binding loop. In contrast, anti-CD47 monoclonal antibodies inhibited PMN transmigration by markedly different kinetics. Results of signal transduction experiments suggested differential regulation of PMN migration by SIRP versus CD47 by phosphatidylinositol 3-kinase and tyrosine kinases, respectively. Immunoprecipitation followed by Western blotting after SDS-PAGE under nonreducing conditions suggested that several SIRP protein species may be present in PMN. Stimulation of PMN with fMLP resulted in increased surface expression of these SIRP proteins, consistent with the existence of intracellular pools. Taken together, these results demonstrate that PMN migration is regulated by CD47 through SIRP␣-dependent and SIRP␣-independent mechanisms.