Mami Ogimoto

Src Homology Region 2 (SH2) Domain-Containing Phosphatase-1 Dephosphorylates B Cell Linker Protein/SH2 Domain Leukocyte Protein of 65 kDa and Selectively Regulates c-Jun NH2-Terminal Kinase Activation in B Cells

Src homology region 2 (SH2) domain-containing phosphatase-1 (SHP-1) is a cytosolic protein tyrosine phosphatase containing two SH2 domains in its NH2 terminus. That immunological abnormalities of the motheaten and viable motheaten mice are caused by mutations in the gene encoding SHP-1 indicates that SHP-1 plays important roles in lymphocyte differentiation, proliferation, and activation. To elucidate molecular mechanisms by which SHP-1 regulates BCR-mediated signal transduction, we determined SHP-1 substrates in B cells using the substrate-trapping approach. When the phosphatase activity-deficient form of SHP-1, in which the catalytic center cysteine (C453) was replaced with serine (SHP-1-C/S), was introduced in WEHI-231 cells, tyrosine phosphorylation of a protein of about 70 kDa was strongly enhanced. Immunoprecipitation and Western blot analyses revealed that this protein is the B cell linker protein (BLNK), also named SH2 domain leukocyte protein of 65 kDa, and that upon tyrosine phosphorylation BLNK binds to SHP-1-C/S in vitro. In vitro kinase assays demonstrated that hyperphosphorylation of BLNK in SHP-1-C/S-expressing cells was not due to enhanced activity of Lyn or Syk. Furthermore, BCR-induced activation of c-Jun NH2-terminal kinase was shown to be significantly enhanced in SHP-1-C/S transfectants. Taken collectively, our results suggest that BLNK is a physiological substrate of SHP-1 in B cells and that SHP-1 selectively regulates c-Jun NH2-terminal kinase activation.

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

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.

Developmental regulation of gene expression for the MPTPδ isoforms in the central nervous system and the immune system

MPTPδ is a murine transmembrane protein tyrosine phosphatase which has three isoforms, types A–C, differing in the structure of the extracellular regions. In this study, we examined MPTPδ isoform expression in the brain and the immune system at discrete developmental or differentiation stages. RT‐PCR analysis demonstrated that another isoform, type D, is transcribed from the MPTPδ gene. In the brain, only type D was expressed until postnatal day 7 (P7), but after P14, all four isoforms were detected. In contrast, the spleen, thymus and all the hematopoietic cell lines examined express only types B and C isoforms. An in situ hybridization study showed that MPTPδ mRNA is diffusely expressed throughout the spleen, but its expression in the thymus is restricted to the medullary region.

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

Despite the important role in the development and activation of T cells, NK cells, mast cells, and macrophages, the expression and function of SLP‐76 in B cells have been largely unknown. Here we demonstrate that SLP‐76 is expressed in all mouse B cell lines tested and in normal splenic B cells, and serves as an SHP‐1 substrate. Dephosphorylation of SLP‐76 by SHP‐1 inhibits its association with Nck, down‐regulating c‐Jun N‐terminal kinase (JNK) activation and exerting a positive effect on apoptosis. Knockdown of SLP‐76 in WEHI‐231 cells by small interfering RNA attenuated JNK activation, but showed little effects on extracellular signal‐regulated kinase (ERK) or p38 activation. Although WEHI‐231 does not express linker for activation of T cells (LAT), SLP‐76 localizes in membrane fraction, which increases following B cell receptor (BCR) cross‐linking. Further analyses revealed that SLP‐76 complexed with Gads is associated with tyrosine‐phosphorylated CD22 through the SH2 domains of SLP‐76 and Gads. Given that SHP‐1 binds to CD22 upon BCR ligation, our findings suggest that dephosphorylation of SLP‐76 recruited to CD22 by SHP‐1 inhibits BCR‐induced JNK activation, dictating apoptosis.

Requirement of PEST domain tyrosine phosphatase PEP in B cell antigen receptor‐induced growth arrest and apoptosis

Signaling events leading to B cell growth or apoptosis are beginning to be unravelled, but detailed information is still lacking. To identify signaling molecules involved in B cell antigen receptor (BCR)‐initiated pathways, we used the immature B cell line, WEHI‐231, to investigate protein tyrosine phosphatases (PTP) whose expression was modulated by BCR ligation. Among the PTP cloned by reverse transcription‐PCR, mRNA expression of the proline‐, glutamic acid‐, serine‐ and threonine‐rich (PEST) domain phosphatase (PEP) was selectively elevated 3.1‐fold within 3 h after anti‐IgM antibody stimulation. In contrast, expression of another PEST domain phosphatase, PTP‐PEST, was unaffected. Western blot analysis revealed that 71 % of PEP was located in the cytosolic fraction, while 29 % was in the membrane fraction. To examine the direct contribution made by PEP to BCR‐initiated signal transduction, we transfected an antisense PEP cDNA into WEHI‐231 cells. Two stable clones were established in which PEP expression was reduced by 34 % and 47 %, respectively. Strikingly, BCR‐mediated inhibition of DNA synthesis was significantly rescued in the clones, and G1 phase cell cycle arrest and apoptosis were almost completely ablated. Considered collectively, these results indicate that PEP is a positive, crucial regulator in determining B cell fate triggered by BCR engagement.

SHP‐1 is involved in neuronal differentiation of P19 embryonic carcinoma cells

Accumulating evidence suggests that tyrosine phosphorylation plays an important role in the development of the central nervous system and in the differentiation of neuronal cells. To identify protein tyrosine phosphatases (PTPs) that might regulate signaling events leading to neuronal cell differentiation, we cloned PTP genes from the murine P19 embryonic carcinoma cell line and examined the change of their expression during differentiation. P19 cells are known to be pluripotent and the aggregate formation and subsequent replating in the presence of retinoic acid (RA) induce growth arrest and neuronal differentiation. The results demonstrated that among several PTP genes expressed in P19 cells, a cytosolic Src homology region 2 domain‐containing PTP, SHP‐1, is expressed highly in undifferentiated P19 cells, but is reduced to an undetectable level at day 3 after replating in the presence of RA. Further, SHP‐1 was tyrosine‐phosphorylated and activated at day 1 after replating. When ectopic SHP‐1 was constitutively expressed, P19 cells continued to proliferate and failed to differentiate upon stimulation with RA. Collectively, these results suggest that the regulated expression and activity of SHP‐1 may be involved in the neuronal differentiation of P19 cells.

CD45 Is Required for CD40-induced Inhibition of DNA Synthesis and Regulation of c-Jun NH2-terminal Kinase and p38 in BAL-17 B Cells

Stimulation of B cell antigen receptor (BCR) may induce proliferation, differentiation, or apoptosis, depending upon the maturational stage of the cell and the presence or absence of signals transmitted via coreceptors. One such signal is delivered via CD40; for instance, ligation of CD40 rescues B cells from BCR-induced apoptosis. Here we show that, in contrast to WEHI-231 cells, CD40 ligation did not reverse BCR-induced growth inhibition in the BAL-17 mature B cell line and CD40 ligation itself inhibited proliferation. This inhibitory signaling was not observed in CD45-deficient cells. Further analyses demonstrate that transfection of dominant-negative form of SEK1 or treatment with SB203580 strongly reduced CD40-induced inhibition of BAL-17 proliferation, suggesting a requirement for c-Jun NH2-terminal kinase and p38 in CD40-induced inhibition of proliferation. Interestingly, CD40-initiated activation of c-Jun NH2-terminal kinase and p38 was enhanced and sustained in CD45-deficient cells, and these phenotypes were reversed by transfecting CD45 gene. However, CD40-mediated induction of cell surface molecules was not affected in CD45-deficient cells. Taken collectively, these results suggest that CD45 exerts a decisive effect on selective sets of CD40-mediated signaling pathways, dictating B cell fate.

Opposing regulation of B cell receptor-induced activation of mitogen-activated protein kinases by CD45

In this study, we examined the contribution made by CD45 to B cell antigen receptor (BCR)‐induced activation of mitogen‐activated protein kinase (MAPK) family members. We found that CD45 negatively regulated BCR‐induced c‐Jun NH2‐terminal kinase (JNK) and p38 activation in immature WEHI‐231 cells, whereas in mature BAL‐17 cells, CD45 positively regulated JNK and p38 activation and negatively regulated extracellular signal‐regulated kinase activity. Furthermore, cooperative action of JNK and p38 dictated BCR‐induced inhibition of growth. Thus, CD45 appears to differentially regulate BCR‐induced activation of MAPK members, and can exert opposing effects on JNK and p38 in different cellular milieu, controlling the B cell fate.