Novel Binding Site for Src Homology 2-containing Protein-tyrosine Phosphatase-1 in CD22 Activated by B Lymphocyte Stimulation with Antigen

Chenghua Zhu,Motohiko Sato,Teruhiko Yanagisawa,Manabu Fujimoto,Takahiro Adachi,Takeshi Tsubata

doi:10.1074/jbc.m706584200

Abstract

CD22, a B lymphocyte membrane glycoprotein, contains immunoreceptor tyrosine-based inhibition motifs (ITIMs) in the cytoplasmic region and recruits Src homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) to the phosphorylated ITIMs upon ligation of B lymphocyte antigen receptor (BCR), thereby negatively regulating BCR signaling. Among the three previously identified ITIMs, both ITIMs containing tyrosine residues at position 843 (Tyr(843)) and 863 (Tyr(863)), respectively, are shown to be required for CD22 to recruit SHP-1 and regulate BCR signaling upon BCR ligation by anti-Ig antibody (Ab), indicating that CD22 has the SHP-1-binding domain at the region containing Tyr(843) and Tyr(863). Here we address the requirement of CD22 for SHP-1 recruitment and BCR regulation upon BCR ligation by antigen, which induces much stronger CD22 phosphorylation than anti-Ig Ab does. We demonstrate that the CD22 mutant in which both Tyr(843) and Tyr(863) are replaced by phenylalanine (CD22F5/6) recruits SHP-1 and regulates BCR signaling upon stimulation with antigen but not anti-Ig Ab. This result strongly suggests that CD22 contains another SHP-1 binding domain that is specifically activated upon stimulation with antigen. Both of the flanking sequences of Tyr(783) and Tyr(817) fit the consensus sequence of ITIM, and the CD22F5/6 mutant requires these tyrosine residues for SHP-1 binding and BCR regulation. Thus, these ITIMs constitute a novel conditional SHP-1-binding site of CD22 that is activated upon BCR ligation by antigen but not by anti-Ig Ab.

Highlights

B cell antigen receptor (BCR), and a crucial regulator of B cell activation [1, 2]
The flanking sequences of these tyrosine residues fit the consensus sequence of the immunoreceptor tyrosine-based inhibition motif (ITIM) [15], the motif often found at the binding sites for Src homology 2 (SH2)-containing phosphatases, including Src homology 2-containing protein-tyrosine phosphatase-1 (SHP-1) [16], and more importantly phosphotyrosyl peptides containing these tyrosine residues are capable of binding to SHP-1 in vitro [14]
In B cells stimulated with antigen but not anti-Ig Ab, the CD22 mutant in which both tyrosine residues at position 843 (Tyr843) and Tyr863 are replaced by phenylalanine is capable of recruiting SHP-1 and negatively regulating BCR signaling, suggesting that CD22 contains another SHP-1 binding domain activated by antigen stimulation other than that containing Tyr843 and Tyr863

Summary

Introduction

B cell antigen receptor (BCR), and a crucial regulator of B cell activation [1, 2]. Upon ligation of the BCR, tyrosine residues at the cytoplasmic region of CD22 are rapidly phosphorylated, thereby providing binding sites for various cytoplasmic signaling molecules, such as Src homology 2 (SH2)-containing protein-tyrosine phosphatase-1 (SHP-1), lipid phosphatase SH2containing inositol polyphosphate 5-phosphatase, the tyrosine kinase Syk, and the adaptors Grb2 and Shc [3,4,5,6]. Expression of CD22F2/5/6 failed to reduce the phosphorylation levels of various cellular substrates, including ERK and AKT, in antigen-stimulated K46␮v transfectants (Fig. 2), suggesting that Tyr783 is required for BCR regulation by CD22F5/6.

Results

Conclusion