Kazuya Mizuno

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.

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.

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.

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.

Novel Mechanism for FcϵRI-mediated Signal Transducer and Activator of Transcription 5 (STAT5) Tyrosine Phosphorylation and the Selective Influence of STAT5B over Mast Cell Cytokine Production

Background: STAT5 is a transcription factor that is vital for mast cell function. Results: Loss of Fyn kinase prevents, whereas loss of Lyn, Gab2, or SHP-1 enhances, FcϵRI-mediated STAT5 tyrosine phosphorylation. Conclusion: IgE-mediated STAT5 activation in mast cells requires Fyn kinase. Significance: Elucidating the mechanisms of mast cell activity is essential to understanding and treating allergic pathologies. Previous studies indicate that STAT5 expression is required for mast cell development, survival, and IgE-mediated function. STAT5 tyrosine phosphorylation is swiftly and transiently induced by activation of the high affinity IgE receptor, FcϵRI. However, the mechanism for this mode of activation remains unknown. In this study we observed that STAT5 co-localizes with FcϵRI in antigen-stimulated mast cells. This localization was supported by cholesterol depletion of membranes, which ablated STAT5 tyrosine phosphorylation. Through the use of various pharmacological inhibitors and murine knock-out models, we found that IgE-mediated STAT5 activation is dependent upon Fyn kinase, independent of Syk, PI3K, Akt, Brutons tyrosine kinase, and JAK2, and enhanced in the context of Lyn kinase deficiency. STAT5 immunoprecipitation revealed that unphosphorylated protein preassociates with Fyn and that this association diminishes significantly during mast cell activation. SHP-1 tyrosine phosphatase deficiency modestly enhanced STAT5 phosphorylation. This effect was more apparent in the absence of Gab2, a scaffolding protein that docks with multiple negative regulators, including SHP-1, SHP-2, and Lyn. Targeting of STAT5A or B with specific siRNA pools revealed that IgE-mediated mast cell cytokine production is selectively dependent upon the STAT5B isoform. Altogether, these data implicate Fyn as the major positive mediator of STAT5 after FcϵRI engagement and demonstrate importantly distinct roles for STAT5A and STAT5B in mast cell function.

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.

SHP-1 exhibits a pro-apoptotic function in antigen-stimulated mast cells: Positive regulation of mitochondrial death pathways and negative regulation of survival signaling pathways

Src homology region 2 domain-containing phosphatase-1 (SHP-1) is known to act as a negative signal modulator in mast cells but its roles in cell survival and cell death are poorly understood. We previously reported that SHP-1 also positively regulates mast cell activation signaling by acting as an adaptor protein. In the present study, we examined whether SHP-1 plays a role in antigen (Ag)-induced activation-induced mast cell death. Bone marrow-derived mast cells (BMMCs) from SHP-1-deficient motheaten (me) mice (me-BMMCs) were significantly less susceptible to store-operated Ca(2+) channel (SOC) activation, Ag-induced cell death and DNA fragmentation than BMMCs from their wild-type littermates (WT-BMMCs). Subsequent experiments revealed that the differences in these cellular susceptibilities to SOC activation and cell death resulted from the extent of the mitochondrial permeability transition pore (mPTP) opening. Specifically, mPTP opening was sufficiently persistent in WT-BMMCs to evoke mitochondrial integrity disruption, while mPTP opening was too transient to cause the minimal mitochondrial integrity collapse in me-BMMCs. In addition, pro-survival signaling including activation of mitogen-activated protein kinases (MAPKs) such as the extracellular signal-regulated protein kinases, c-Jun NH(2) terminal kinases and p38 and the expression of Bcl-x(L) were significantly prolonged in me-BMMCs compared with WT-BMMCs. Taken together, these data demonstrate that a lack of SHP-1 prevents the mPTP-mediated mitochondrial integrity collapse and augments anti-apoptotic signaling such as MAPKs and Bcl-x(L). These findings suggest that SHP-1 positively regulates mitochondrial death pathways and negatively regulates pro-survival signaling pathways.