Hidetaka Yakura

Identification of CD72 as a lymphocyte receptor for the class IV semaphorin CD100: a novel mechanism for regulating B cell signaling.

We have identified the lymphocyte semaphorin CD100/Sema4D as a CD40-inducible molecule by subtractive cDNA cloning. CD100 stimulation significantly enhanced the effects of CD40 on B cell responses. Administration of soluble CD100 markedly accelerated in vivo antigen-specific antibody responses. CD100 receptors with different binding affinities were detected on renal tubular cells (K(d) = approximately 1 x 10(-9)M) and lymphocytes (K(d) = approximately 3 x 10(-7)M). Expression cloning revealed that the CD100 receptor on lymphocytes is CD72, a negative regulator of B cell responsiveness. CD72 thus represents a novel class of semaphorin receptors. CD100 stimulation induced tyrosine dephosphorylation of CD72 and dissociation of SHP-1 from CD72. Our findings indicate that CD100 plays a critical role in immune responses by the novel mechanism of turning off negative signaling by CD72.

Impaired learning with enhanced hippocampal long‐term potentiation in PTPδ‐deficient mice

Protein tyrosine phosphatase δ (PTPδ) is a receptor‐type PTP expressed in the specialized regions of the brain including the hippocampal CA2 and CA3, B lymphocytes and thymic medulla. To elucidate the physiological roles of PTPδ, PTPδ‐deficient mice were produced by gene targeting. It was found that PTPδ‐deficient mice were semi‐lethal due to insufficient food intake. They also exhibited learning impairment in the Morris water maze, reinforced T‐maze and radial arm maze tasks. Interestingly, although the histology of the hippocampus appeared normal, the magnitudes of long‐term potentiation (LTP) induced at hippocampal CA1 and CA3 synapses were significantly enhanced in PTPδ‐deficient mice, with augmented paired‐pulse facilitation in the CA1 region. Thus, it was shown that PTPδ plays important roles in regulating hippocampal LTP and learning processes, and that hippocampal LTP does not necessarily positively correlate with spatial learning ability. To our knowledge, this is the first report of a specific PTP involved in the regulation of synaptic plasticity or in the processes regulating learning and memory.

CD72 Negatively Regulates Signaling Through the Antigen Receptor of B Cells

The immunoreceptor tyrosine-based inhibition motif (ITIM) is found in various membrane molecules such as CD22 and the low-affinity Fc receptor for IgG in B cells and the killer cell-inhibitory receptor and Ly-49 in NK cells. Upon tyrosine phosphorylation at the ITIMs, these molecules recruit SH2 domain-containing phosphatases such as SH2-containing tyrosine phosphatase-1 and negatively regulate cell activity. The B cell surface molecule CD72 carries an ITIM and an ITIM-like sequence. We have previously shown that CD72 is phosphorylated and recruits SH2-containing tyrosine phosphatase-1 upon cross-linking of the Ag receptor of B cells (BCR). However, whether CD72 modulates BCR signaling has not yet been elucidated. In this paper we demonstrate that expression of CD72 down-modulates both extracellular signal-related kinase (ERK) activation and Ca2+ mobilization induced by BCR ligation in the mouse B lymphoma line K46μmλ, whereas BCR-mediated ERK activation was not reduced by the ITIM-mutated form of CD72. Moreover, coligation with CD72 with BCR reduces BCR-mediated ERK activation in spleen B cells of normal mice. These results indicate that CD72 negatively regulates BCR signaling. CD72 may play a regulatory role in B cell activation, probably by setting a threshold for BCR signaling.

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.

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.

Deficiency of SHP1 leads to sustained and increased ERK activation in mast cells, thereby inhibiting IL-3-dependent proliferation and cell death.

SHP-1 plays an important role for the regulation of signaling from various hematopoietic cell receptors. In this study, we examined IL-3-induced cell proliferation and IL-3 depletion-induced apoptosis in bone marrow-derived mast cells (BMMC) established from motheaten (me) that lack SHP-1 expression, viable motheaten (me(v)) expressing phosphatase-deficient SHP-1, and wild-type (WT) mice. When BMMC were stimulated with IL-3, increased ERK activation was evident in resting state and sustained in me-BMMC relative to WT-BMMC. ERK is known to be involved in the regulation of cell proliferation and apoptosis in some cells. In accordance with sustained ERK activation, apoptosis was decreased in me- and me(v)-BMMC compared with WT-BMMC. In contrast to the predicted role of ERK as a pro-survival molecule, IL-3-induced cell proliferation was much lower in me- and me(v)-BMMC than WT-BMMC. Stimulation with lower concentration of IL-3 or addition of PD98059, a MEK inhibitor, to the culture resulted in the suppression of decreased apoptosis and cell proliferation in me- and me(v)-BMMC. Collectively, these results suggest that SHP-1 positively regulates IL-3-dependent mast cell proliferation and apoptosis by inhibiting ERK activity through its phosphatase activity. Furthermore, our results indicate that ERK would act as a negative regulator for cell proliferation and induce apoptosis when its activity is highly increased.

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.