Src Homology Region 2 Domain-Containing Phosphatase 1 Positively Regulates B Cell Receptor-Induced Apoptosis by Modulating Association of B Cell Linker Protein with Nck and Activation of c-Jun NH2-Terminal Kinase

Abstract

Src homology region 2 domain-containing phosphatase 1 (SHP-1) is a key mediator in lymphocyte differentiation, proliferation, and activation. We previously showed that B cell linker protein (BLNK) is a physiological substrate of SHP-1 and that B cell receptor (BCR)-induced activation of c-Jun NH2-terminal kinase (JNK) is significantly enhanced in cells expressing a form of SHP-1 lacking phosphatase activity (SHP-1-C/S). In this study, we confirmed that SHP-1 also exerts negative regulatory effects on JNK activation in splenic B cells. To further clarify the role of SHP-1 in B cells, we examined how dephosphorylation of BLNK by SHP-1 affects downstream signaling events. When a BLNK mutant (BLNKΔN) lacking the NH2-terminal region, which contains four tyrosine residues, was introduced in SHP-1-C/S-expressing WEHI-231 cells, the enhanced JNK activation was inhibited. Among candidate proteins likely to regulate JNK activation through BLNK, Nck adaptor protein was found to associate with tyrosine-phosphorylated BLNK and this association was more pronounced in SHP-1-C/S-expressing cells. Furthermore, expression of dominant-negative forms of Nck inhibited BCR-induced JNK activation. Finally, BCR-induced apoptosis was suppressed in SHP-1-C/S-expressing cells and coexpression of Nck SH2 mutants or a dominant-negative form of SEK1 reversed this phenotype. Collectively, these results suggest that SHP-1 acts on BLNK, modulating its association with Nck, which in turn negatively regulates JNK activation but exerts a positive effect on apoptosis.

SLP‐76 is recruited to CD22 and dephosphorylated by SHP‐1, thereby regulating B cell receptor‐induced c‐Jun N‐terminal kinase activation

“…These sequences of events and functional consequences are similar to what we found for BLNK. Although precise roles of JNK activation in BCR-induced apoptosis are controversial, either pro-apoptotic or antiapoptotic, as discussed previously [31], the potential role of SLP-76 in the regulation of JNK activation and apoptosis might be supported by the reduced SLP-76 expression in immature B cells where reduced SLP-76 might decrease the JNK activation, thereby facilitating apoptosis for negative selection. It is important to further elucidate as to how SLP-76 and BLNK differentially function in BCR signaling and functional significance of regulated SLP-76 expression during B cell development.…”

“…Since JNK activation is directly involved in the inhibition of BCR-induced apoptosis [31], we examined the effect of expression of these constructs on apoptosis. As shown in Fig.…”

“…The tyrosine-phosphorylated BLNK has been shown to bind Nck/NIK complex, thereby regulating JNK activa- tion [31]. We analyzed whether Nck can bind to the Nterminal region of SLP-76 upon BCR cross-linking.…”

“…Expression constructs for flag-tagged SHP-1-WT, SHP-1-C/S, Nck-WT, and NckDSH2 have been described [30,31]. To generate expression constructs for SLP-76, PCR fragments containing entire open reading frame for mouse SLP-76 were cloned in pEF-flag/stop vector (a gift of Dr. G. Koretzky).…”

“…Since SHP-1 is activated when its SH2 domains interact with tyrosine-phosphorylated peptides [29], SHP-1 bound to these coreceptors might efficiently dephosphorylate signaling molecules. Furthermore, we have previously demonstrated that dephosphorylation of BLNK by SHP-1 causes a decreased association with Nck/Nck-interacting kinase (NIK) complex, suppressed c-Jun N-terminal kinase (JNK) activation, and enhanced apoptosis [30,31]. Although we [32] and others [33,34] have previously demonstrated that SLP-76 is expressed in the mouse immature B cell line WEHI-231 and in pro-/pre-B cells, the expression and the function of SLP-76 in B cells have been largely unknown.…”