Shinya Kusakari

CD47 Promotes Neuronal Development through Src- and FRG/Vav2-Mediated Activation of Rac and Cdc42

The development of axons and dendrites is controlled by small GTP-binding proteins of the Rho family, but the upstream signaling mechanisms responsible for such regulation remain unclear. We have now investigated the role of the transmembrane protein cluster of differentiation 47 (CD47) in this process with hippocampal neurons. CD47-deficient neurons manifested markedly impaired development of dendrites and axons, whereas overexpression of CD47 promoted such development. Interaction of SH2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1) with CD47 also induced the formation of dendritic filopodia and spines. These effects of CD47 were prevented by inhibition of either cell division cycle 42 (Cdc42) or Rac. In CD47-deficient neurons, autophosphorylation of Src was markedly reduced. In addition, overexpression of CD47 promoted the autophosphorylation of Src. Inhibition of Src family kinases indeed prevented CD47-promoted dendritic development. Inhibition of either FGD1-related Cdc42-guanine nucleotide exchange factor (GEF) (FRG) or Vav2, which is a GEF for Cdc42 and Rac and is activated by Src, also prevented the effects of CD47 on dendritic development. These results indicate that CD47 promotes development of dendrites and axons in hippocampal neurons in a manner dependent, at least in part, on activation of Cdc42 and Rac mediated by Src as well as by FRG and Vav2.

Dendritic Cell-Specific Ablation of the Protein Tyrosine Phosphatase Shp1 Promotes Th1 Cell Differentiation and Induces Autoimmunity

Dendritic cells (DCs) promote immune responses to foreign Ags and immune tolerance to self-Ags. Deregulation of DCs is implicated in autoimmunity, but the molecules that regulate DCs to protect against autoimmunity have remained unknown. In this study, we show that mice lacking the protein tyrosine phosphatase Shp1 specifically in DCs develop splenomegaly associated with more CD11c+ DCs. Splenic DCs from the mutant mice showed upregulation of CD86 and CCR7 expression and of LPS-induced production of proinflammatory cytokines. The mice manifested more splenic Th1 cells, consistent with the increased ability of their DCs to induce production of IFN-γ by Ag-specific T cells in vitro. The number of splenic CD5+CD19+ B-1a cells and the serum concentrations of Igs M and G2a were also increased in the mutant mice. Moreover, aged mutant mice developed glomerulonephritis and interstitial pneumonitis together with increased serum concentrations of autoantibodies. Shp1 is thus a key regulator of DC functions that protects against autoimmunity.

SAP-1 is a microvillus-specific protein tyrosine phosphatase that modulates intestinal tumorigenesis.

SAP‐1 (PTPRH) is a receptor‐type protein tyrosine phosphatase (RPTP) with a single catalytic domain in its cytoplasmic region and fibronectin type III‐like domains in its extracellular region. The cellular localization and biological functions of this RPTP have remained unknown, however. We now show that mouse SAP‐1 mRNA is largely restricted to the gastrointestinal tract and that SAP‐1 protein localizes to the microvilli of the brush border in gastrointestinal epithelial cells. The expression of SAP‐1 in mouse intestine is minimal during embryonic development but increases markedly after birth. SAP‐1‐deficient mice manifested no marked changes in morphology of the intestinal epithelium. In contrast, SAP‐1 ablation inhibited tumorigenesis in mice with a heterozygous mutation of the adenomatous polyposis coli gene. These results thus suggest that SAP‐1 is a microvillus‐specific RPTP that regulates intestinal tumorigenesis.

Stress-evoked tyrosine phosphorylation of signal regulatory protein α regulates behavioral immobility in the forced swim test.

Severe stress induces changes in neuronal function that are implicated in stress-related disorders such as depression. The molecular mechanisms underlying the response of the brain to stress remain primarily unknown, however. Signal regulatory protein α (SIRPα) is an Ig-superfamily protein that undergoes tyrosine phosphorylation and binds the protein tyrosine phosphatase Shp2. Here we show that mice expressing a form of SIRPα that lacks most of the cytoplasmic region manifest prolonged immobility (depression-like behavior) in the forced swim (FS) test. FS stress induced marked tyrosine phosphorylation of SIRPα in the brain of wild-type mice through activation of Src family kinases. The SIRPα ligand CD47 was important for such SIRPα phosphorylation, and CD47-deficient mice also manifested prolonged immobility in the FS test. Moreover, FS stress-induced tyrosine phosphorylation of both the NR2B subunit of the NMDA subtype of glutamate receptor and the K+-channel subunit Kvβ2 was regulated by SIRPα. Thus, tyrosine phosphorylation of SIRPα is important for regulation of depression-like behavior in the response of the brain to stress.

Signal Regulatory Protein α Regulates the Homeostasis of T Lymphocytes in the Spleen

The molecular basis for formation of lymphoid follicle and its homeostasis in the secondary lymphoid organs remains unclear. Signal regulatory protein α (SIRPα), an Ig superfamily protein that is predominantly expressed in dendritic cells or macrophages, mediates cell–cell signaling by interacting with CD47, another Ig superfamily protein. In this study, we show that the size of the T cell zone as well as the number of CD4+ T cells were markedly reduced in the spleen of mice bearing a mutant (MT) SIRPα that lacks the cytoplasmic region compared with those of wild-type mice. In addition, the expression of CCL19 and CCL21, as well as of IL-7, which are thought to be important for development or homeostasis of the T cell zone, was markedly decreased in the spleen of SIRPα MT mice. By the use of bone marrow chimera, we found that hematopoietic SIRPα is important for development of the T cell zone as well as the expression of CCL19 and CCL21 in the spleen. The expression of lymphotoxin and its receptor, lymphotoxin β receptor, as well as the in vivo response to lymphotoxin β receptor stimulation were also decreased in the spleen of SIRPα MT mice. CD47-deficient mice also manifested phenotypes similar to SIRPα MT mice. These data suggest that SIRPα as well as its ligand CD47 are thus essential for steady-state homeostasis of T cells in the spleen.

Trans-endocytosis of CD47 and SHPS-1 and its role in regulation of the CD47–SHPS-1 system

CD47 and SHPS-1 are transmembrane proteins that interact with each other through their extracellular regions and constitute a bidirectional cell-cell communication system (the CD47–SHPS-1 system). We have now shown that the trans-interaction of CD47 and SHPS-1 that occurred on contact of CD47-expressing CHO cells and SHPS-1-expressing CHO cells resulted in endocytosis of the ligand-receptor complex into either cell type. Such trans-endocytosis of CD47 by SHPS-1-expressing cells was found to be mediated by clathrin and dynamin. A juxtamembrane region of SHPS-1 was indispensable for efficient trans-endocytosis of CD47, which was also regulated by Rac and Cdc42, probably through reorganization of the actin cytoskeleton. Inhibition of trans-endocytosis of CD47 promoted the aggregation of CD47-expressing cells with the cells expressing SHPS-1. Moreover, CD47 expressed on the surface of cultured mouse hippocampal neurons was shown to undergo trans-endocytosis by neighboring astrocytes expressing endogenous SHPS-1. These results suggest that trans-endocytosis of CD47 is responsible for removal of the CD47–SHPS-1 complex from the cell surface and hence regulates the function of the CD47–SHPS-1 system, at least in neurons and glial cells.

Promotion of cell spreading and migration by vascular endothelial-protein tyrosine phosphatase (VE-PTP) in cooperation with integrins

Vascular endothelial‐protein tyrosine phosphatase (VE‐PTP) is a receptor‐type protein tyrosine phosphatase with a single catalytic domain in its cytoplasmic region and multiple fibronectin type III‐like domains in its extracellular region. VE‐PTP is expressed specifically in endothelial cells and is implicated in regulation of angiogenesis. The molecular basis for such regulation by VE‐PTP has remained largely unknown, however. We now show that forced expression of VE‐PTP promoted cell spreading as well as formation of lamellipodia and filopodia in cultured fibroblasts plated on fibronectin. These effects of VE‐PTP on cell morphology required its catalytic activity as well as activation of integrins and Ras. In addition, VE‐PTP‐induced cell spreading and lamellipodium formation were prevented by inhibition of Src family kinases or of Rac or Cdc42. Indeed, forced expression of VE‐PTP increased the level of c‐Src phosphorylation at tyrosine‐416. Moreover, the VE‐PTP‐induced changes in cell morphology were suppressed by expression of dominant negative forms of FRG or Vav2, both of which are guanine nucleotide exchange factors for Rho family proteins and are activated by tyrosine phosphorylation. Forced expression of VE‐PTP also enhanced fibronectin‐dependent migration of cultured fibroblasts. Conversely, depletion of VE‐PTP by RNA interference in human umbilical vein endothelial cells or mouse endothelioma cells inhibited cell spreading on fibronectin. These results suggest that VE‐PTP, in cooperation with integrins, regulates the spreading and migration of endothelial cells during angiogenesis. J. Cell. Physiol. 224:195–204, 2010

Tyrosine phosphorylation of R3 subtype receptor‐type protein tyrosine phosphatases and their complex formations with Grb2 or Fyn

Post‐translational modification of protein tyrosine phosphatases (PTPs) is implicated in functional modulation of these enzymes. Stomach cancer–associated protein tyrosine phosphatase‐1 (SAP‐1), as well as protein tyrosine phosphatase receptor type O (PTPRO) and vascular endothelial‐protein tyrosine phosphatase (VE‐PTP) are receptor‐type PTPs (RPTPs), which belong to the R3 subtype RPTP family. Here, we have shown that the carboxyl (COOH)‐terminal region of SAP‐1 undergoes tyrosine phosphorylation by the treatment with a PTP inhibitor. Src family kinases are important for the tyrosine phosphorylation of SAP‐1. Either Grb2 or Fyn, through their Src homology‐2 domains, bound to the tyrosine‐phosphorylated SAP‐1. Moreover, both PTPRO and VE‐PTP underwent tyrosine phosphorylation in their COOH‐terminal regions. Tyrosine phosphorylation of VE‐PTP or PTPRO also promoted their complex formations with Grb2 or Fyn. Forced expression of SAP‐1, PTPRO or VE‐PTP promoted cell spreading and lamellipodium formation of fibroblasts that expressed an activated form of Ras. In contrast, such effects of non‐tyrosine‐phosphorylated forms of these RPTPs were markedly smaller than those of wild‐type RPTPs. Our results thus suggest that tyrosine phosphorylation of R3 subtype RPTPs promotes their complex formations with Grb2 or Fyn and thus participates in the regulation of cell morphology.

Shp2 in forebrain neurons regulates synaptic plasticity, locomotion, and memory formation in mice.

ABSTRACT Shp2 (Src homology 2 domain-containing protein tyrosine phosphatase 2) regulates neural cell differentiation. It is also expressed in postmitotic neurons, however, and mutations of Shp2 are associated with clinical syndromes characterized by mental retardation. Here we show that conditional-knockout (cKO) mice lacking Shp2 specifically in postmitotic forebrain neurons manifest abnormal behavior, including hyperactivity. Novelty-induced expression of immediate-early genes and activation of extracellular-signal-regulated kinase (Erk) were attenuated in the cerebral cortex and hippocampus of Shp2 cKO mice, suggestive of reduced neuronal activity. In contrast, ablation of Shp2 enhanced high-K+-induced Erk activation in both cultured cortical neurons and synaptosomes, whereas it inhibited that induced by brain-derived growth factor in cultured neurons. Posttetanic potentiation and paired-pulse facilitation were attenuated and enhanced, respectively, in hippocampal slices from Shp2 cKO mice. The mutant mice also manifested transient impairment of memory formation in the Morris water maze. Our data suggest that Shp2 contributes to regulation of Erk activation and synaptic plasticity in postmitotic forebrain neurons and thereby controls locomotor activity and memory formation.

Protein tyrosine phosphatase SAP-1 protects against colitis through regulation of CEACAM20 in the intestinal epithelium

Significance Much attention has been recently paid to the role of intestinal epithelial cells in the homeostatic regulation of intestinal immunity. Here we show that ablation of stomach-cancer–associated protein tyrosine phosphatase 1 (SAP-1) markedly increased the severity of colitis in interleukin (IL)-10–deficient mice, suggesting that SAP-1 protects against colitis in a cooperative manner with IL-10. We also identify carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvilli-specific membrane protein, as a dephosphorylation target for SAP-1. Indeed, tyrosine phosphorylation of CEACAM20 promotes the binding of spleen tyrosine kinase (Syk) and activation of nuclear factor-κB (NF-κB), thereby inducing production of chemokines such as IL-8. Thus, we propose a mechanism by SAP-1 and CEACAM20 in the intestinal epithelium for regulation of the intestinal immunity. Intestinal epithelial cells contribute to regulation of intestinal immunity in mammals, but the detailed molecular mechanisms of such regulation have remained largely unknown. Stomach-cancer–associated protein tyrosine phosphatase 1 (SAP-1, also known as PTPRH) is a receptor-type protein tyrosine phosphatase that is localized specifically at microvilli of the brush border in gastrointestinal epithelial cells. Here we show that SAP-1 ablation in interleukin (IL)-10–deficient mice, a model of inflammatory bowel disease, resulted in a marked increase in the severity of colitis in association with up-regulation of mRNAs for various cytokines and chemokines in the colon. Tyrosine phosphorylation of carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20, an intestinal microvillus-specific transmembrane protein of the Ig superfamily, was greatly increased in the intestinal epithelium of the SAP-1–deficient animals, suggesting that this protein is a substrate for SAP-1. Tyrosine phosphorylation of CEACAM20 by the protein tyrosine kinase c-Src and the consequent association of CEACAM20 with spleen tyrosine kinase (Syk) promoted the production of IL-8 in cultured cells through the activation of nuclear factor-κB (NF-κB). In addition, SAP-1 and CEACAM20 were found to form a complex through interaction of their ectodomains. SAP-1 and CEACAM20 thus constitute a regulatory system through which the intestinal epithelium contributes to intestinal immunity.