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

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.Receptor-mediated activation of cellular responses is often initiated by the activation of tyrosine kinases. The signal is propagated by the sequential recruitment of proteins to the phosphorylated targets of the kinases. SH2 (Src homology 2) domains are protein modules that specifically bind to phosphotyrosine residues and are found in a variety of proteins such as protein kinases and adapter molecules. A general feature of all SH2 domains is a conserved pocket that binds the phosphotyrosine moiety. The specificity of the interaction between individual SH2 domains and particular phosphoproteins is generally determined by three to six residues following the phosphotyrosine and often incorporates a hydrophobic residue at the third position (reviewed in Ref. 1). This specificity is provided by specific pockets that bind the phosphotyrosine and ϩ3 residues as revealed by structural studies of several SH2 domains complexed with phosphopeptides (1).A small family of protein-tyrosine phosphatases contain SH2 domains. This family is composed of mammalian SHP-1 and SHP-2 and the Drosophila homologue of SHP-2, corkscrew. These protein-tyrosine phosphatases are important regulators of many cellular signaling processes (reviewed in Ref. 2). SHP-2 is broadly expressed and is important for activation signals through several different growth factor receptors. In contrast, SHP-1 is expressed predominantly in hematopoietic cells and has been implicated in inhibition of signaling through growth factor, cytokine, and antigen rec...

Functional Consequences of Interactions between Human NKR-P1A and Its Ligand LLT1 Expressed on Activated Dendritic Cells and B Cells

“…Previous data have suggested that this -2 position is important for inhibitory function and mutating the -2 position to an alanine in an ITIM resulted in decreased inhibitory potential (35). This noncanonical ITIM likely explains the weak inhibitory potential of NKR-P1A.…”

The Carboxyl-terminal Region of Biliary Glycoprotein Controls Its Tyrosine Phosphorylation and Association with Protein-tyrosine Phosphatases SHP-1 and SHP-2 in Epithelial Cells

“…Although they are positioned outside the classical ITIM consensus sequence, they appear to play a determining role in the association of either of the Tyr phosphatases. Several reports have suggested that residues ϩ3, ϩ4, and ϩ5 relative to the ITIM sequences can dramatically affect either maximal SHP-1 or SHP-2 binding or levels of activity (31,46,47). Substitutions at positions ϩ1, ϩ2, ϩ4, and ϩ5 relative to the first ITIM of the natural killer cell receptor decreased phosphatase activation and presumably binding by approximately 50% (31).…”

“…This includes a phosphorylated Tyr residue preceded by a Val, Ile, or Leu residue at position Ϫ2 and followed by a critical Leu or Val residue at position ϩ3 (30,31). In addition, as defined using mutated phosphopeptides, other residues such as those positioned at Ϫ4 as well as ϩ1, ϩ2, ϩ4, and ϩ5 relative to the Tyr residue within the ITIM might also contribute to the effective binding of SHP-1 with the natural killer cell inhibitory receptors (31). The ITIM consensus sequence is found in a number of hemopoietic cell-surface receptors such as Fc␥ receptor IIB, the B cell accessory receptor CD22, and the natural killer cell inhibitory receptors.…”

A novel family of Ig-like receptors for HLA class I molecules that modulate function of lymphoid and myeloid cells

“…The cytoplasmic tails of ILT2, -3, -4, and -5 contain three to four tyrosine-based motifs similar to those found in KIRs, although only some of them fit the V/I-x-Y-x-x-L motif proposed to be required for SHP-1 binding [53]. To determine whether ILTs also recruit SHP phosphatases, ILT2, ILT3, and ILT4 were immunoprecipitated from NK cells, B cells, and monocytes, either resting or stimulated with pervanadate, which induces substantial tyrosine phosphorylation of cellular substrates.…”