A Novel Phosphotyrosine Motif with a Critical Amino Acid at Position −2 for the SH2 Domain-mediated Activation of the Tyrosine Phosphatase SHP-1

Taolin Yi,Wentian Yang,Deborah N. Burshtyn,Eric O. Long

doi:10.1074/jbc.272.20.13066

Abstract

SHP-1 is a protein-tyrosine phosphatase associated with inhibition of activation pathways in hematopoietic cells. The catalytic activity of SHP-1 is regulated by its two SH2 (Src homology 2) domains; phosphotyrosine peptides that bind to the SH2 domains activate SHP-1. The consensus sequence (I/V)XYXX(L/V) is present in the cytoplasmic tails of several lymphocyte receptors that interact with the second SH2 domain of SHP-1. In several of these receptors, there are two or three occurrences of the motif. Here we show that the conserved hydrophobic amino acid preceding the phosphotyrosine is critical for binding to and activation of SHP-1 by peptides corresponding to sequences from killer cell inhibitory receptors. The interaction of most SH2 domains with phosphopeptides requires only the phosphotyrosine and the three residues downstream of the tyrosine. In contrast, the shortest peptide able to bind or activate SHP-1 also included the two residues upstream of the phosphotyrosine. A biphosphopeptide corresponding to the cytoplasmic tail of a killer cell inhibitory receptor with the potential to interact simultaneously with both SH2 domains of SHP-1 was the most potent activator of SHP-1. The hydrophobic residue upstream of the tyrosine was also critical in the context of the biphosphopeptide. The contribution of a hydrophobic amino acid two residues upstream of the tyrosine in the SHP-1-binding motif may be an important feature that distinguishes inhibitory receptors from those that provide activation signals.

Highlights

A Novel Phosphotyrosine Motif with a Critical Amino Acid at Position ؊2 for the SH2 Domain-mediated Activation of the Tyrosine Phosphatase SHP-1*
Binding of SHP-1 to Phosphotyrosine Peptides Derived from killer cell inhibitory receptors (KIR)—To assess the relative specificity of the cytoplasmic tail sequences of KIR and Fc␥RIIB for SHP-1, proteins that associate with phosphopeptides coupled to beads were analyzed by silver staining after separation by SDS-polyacrylamide gel electrophoresis
Phosphopeptides derived from the first tyrosine in the KIR cytoplasmic tail or corresponding to the biphosphorylated KIR sequence each bound to a single major protein that corresponded in size to SHP-1

Summary

EXPERIMENTAL PROCEDURES

Peptides—All peptides were purchased from Quality Controlled Biochemicals, Inc. (Hopkinton, MA) and supplied at a purity of Ͼ98%. Purified GST-SHP-1 was dialyzed into phosphate-buffered saline, pH 7.4; GST-catSHP-1 was dialyzed into 0.15 M NaCl and 10 mM Tris-HCl, pH 8. SHP-1 Binding Assay—Cell lysate was prepared at a concentration of 107 Jurkat cells in 1 ml of lysis buffer (1% Triton X-100, 0.15 M NaCl, 2 mM NaVO3, 5 mM iodoacetamide, 1 mM phenylmethylsulfonyl fluoride, 10 ␮g/ml aprotinin, 10 ␮g/ml leupeptin, and 20 mM Tris-HCl, pH 8). A 100-␮l volume of peptide was added to 100 ␮l of lysate or GST-SH2C(SHP-1) diluted in lysis buffer. Samples were washed three times in lysis buffer and analyzed by SDS-polyacrylamide gel electrophoresis and Western blotting with anti-SHP-1 antibodies or with antiGST antibodies (Upstate Biotechnology, Inc., Lake Placid, NY) using the chemiluminescence detection system (Pierce)

RESULTS

A Phosphopeptide Motif for Activation of SHP-1

DISCUSSION