Protein tyrosine phosphatase Shp2 positively regulates cold stress-induced tyrosine phosphorylation of SIRPα in neurons.

The membrane protein SIRPα is a cold stress-responsive signaling molecule in neurons. Cold stress directly induces tyrosine phosphorylation of SIRPα in its cytoplasmic region, and phosphorylated SIRPα is involved in regulating experience-dependent behavioral changes in mice. Here, we examined the mechanism of cold stress-induced SIRPα phosphorylation in vitro and in vivo. The levels of activated Src family protein tyrosine kinases (SFKs), which phosphorylate SIRPα, were not increased by lowering the temperature in cultured neurons. Although the SFK inhibitor dasatinib markedly reduced SIRPα phosphorylation, low temperature induced an increase in SIRPα phosphorylation even in the presence of dasatinib, suggesting that SFK activation is not required for low temperature-induced SIRPα phosphorylation. However, in the presence of pervanadate, a potent inhibitor of protein tyrosine phosphatases (PTPases), SIRPα phosphorylation was significantly reduced by lowering the temperature, suggesting that either the inactivation of PTPase(s) that dephosphorylate SIRPα or increased protection of phosphorylated SIRPα from the PTPase activity is important for low temperature-induced SIRPα phosphorylation. Inactivation of PTPase Shp2 by the allosteric Shp2 inhibitor SHP099, but not by the competitive inhibitor NSC-87877, reduced SIRPα phosphorylation in cultured neurons. Shp2 knockout also reduced SIRPα phosphorylation in the mouse brain. Our data suggest that Shp2, but not SFKs, positively regulates cold stress-induced SIRPα phosphorylation in a PTPase activity-independent manner.

Calmodulin-like skin protein protects against spatial learning impairment in a mouse model of Alzheimer disease.

Humanin and calmodulin-like skin protein (CLSP) inhibits Alzheimer disease (AD)-related neuronal cell death via the heterotrimeric humanin receptor in vitro. It has been suggested that CLSP is a central agonist of the heterotrimeric humanin receptor in vivo. To investigate the role of CLSP in the AD pathogenesis in vivo, we generated mouse CLSP-1 transgenic mice, crossed them with the APPswe/PSEN1dE9 mice, a model mouse of AD, and examined the effect of CLSP over-expression on the pathological phenotype of the AD mouse model. We found that over-expression of the mouse CLSP-1 gene attenuated spatial learning impairment, the loss of a presynaptic marker synaptophysin, and the inactivation of STAT3 in the APPswe/PSEN1dE9 mice. On the other hand, CLSP over-expression did not affect levels of Aß, soluble Aß oligomers, or gliosis. These results suggest that the CLSP-mediated attenuation of memory impairment and synaptic loss occurs in an Aß-independent manner. The results of this study may serve as a hint to the better understanding of the AD pathogenesis and the development of AD therapy.

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

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.

An Alzheimer Disease-linked Rare Mutation Potentiates Netrin Receptor Uncoordinated-5C-induced Signaling That Merges with Amyloid ß Precursor Protein Signaling.

A missense mutation (T835M) in the uncoordinated-5C (UNC5C) netrin receptor gene increases the risk of late-onset Alzheimer disease (AD) and also the vulnerability of neurons harboring the mutation to various insults. The molecular mechanisms underlying T835M-UNC5C-induced death remain to be elucidated. In this study, we show that overexpression of wild-type UNC5C causes low-grade death, which is intensified by an AD-linked mutation T835M. An AD-linked survival factor, calmodulin-like skin protein (CLSP), and a natural ligand of UNC5C, netrin1, inhibit this death. T835M-UNC5C-induced neuronal cell death is mediated by an intracellular death-signaling cascade, consisting of death-associated protein kinase 1/protein kinase D/apoptosis signal-regulating kinase 1 (ASK1)/JNK/NADPH oxidase/caspases, which merges at ASK1 with a death-signaling cascade, mediated by amyloid ß precursor protein (APP). Notably, netrin1 also binds to APP and partially inhibits the death-signaling cascade, induced by APP. These results may provide new insight into the amyloid ß-independent pathomechanism of AD.

Shear stress-induced redistribution of vascular endothelial-protein-tyrosine phosphatase (VE-PTP) in endothelial cells and its role in cell elongation.

Vascular endothelial cells (ECs) are continuously exposed to shear stress (SS) generated by blood flow. Such stress plays a key role in regulation of various aspects of EC function including cell proliferation and motility as well as changes in cell morphology. Vascular endothelial-protein-tyrosine phosphatase (VE-PTP) is an R3-subtype PTP that possesses multiple fibronectin type III-like domains in its extracellular region and is expressed specifically in ECs. The role of VE-PTP in EC responses to SS has remained unknown, however. Here we show that VE-PTP is diffusely localized in ECs maintained under static culture conditions, whereas it undergoes rapid accumulation at the downstream edge of the cells relative to the direction of flow in response to SS. This redistribution of VE-PTP triggered by SS was found to require its extracellular and transmembrane regions and was promoted by integrin engagement of extracellular matrix ligands. Inhibition of actin polymerization or of Cdc42, Rab5, or Arf6 activities attenuated the SS-induced redistribution of VE-PTP. VE-PTP also underwent endocytosis in the static and SS conditions. SS induced the polarized distribution of internalized VE-PTP. Such an effect was promoted by integrin engagement of fibronectin but prevented by inhibition of Cdc42 activity or of actin polymerization. In addition, depletion of VE-PTP by RNA interference in human umbilical vein ECs blocked cell elongation in the direction of flow induced by SS. Our results suggest that the polarized redistribution of VE-PTP in response to SS plays an important role in the regulation of EC function by blood flow.

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.

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 ß receptor, as well as the in vivo response to lymphotoxin ß 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.

Hypothermia-induced tyrosine phosphorylation of SIRPα in the brain.

Signal regulatory protein α (SIRPα) is a neuronal membrane protein that undergoes tyrosine phosphorylation in the brain of mice in response to forced swim (FS) stress in cold water, and this response is implicated in regulation of depression-like behavior in the FS test. We now show that subjection of mice to the FS in warm (37 °C) water does not induce the tyrosine phosphorylation of SIRPα in the brain. The rectal temperature (T(rec) ) of mice was reduced to 27° to 30 °C by performance of the FS for 10 min in cold water, whereas it was not affected by the same treatment in warm water. The level of tyrosine phosphorylation of SIRPα in the brain was increased by administration of ethanol or picrotoxin, starvation, or cooling after anesthesia, all of which also induced hypothermia. Furthermore, the tyrosine phosphorylation of SIRPα in cultured hippocampal neurons was induced by lowering the temperature of the culture medium. CD47, a ligand of SIRPα, as well as Src family kinases or SH2 domain-containing protein phosphatase 2 (Shp2), might be important for the basal and the hypothermia-induced tyrosine phosphorylation of SIRPα. Hypothermia is therefore likely an important determinant of both the behavioral immobility and tyrosine phosphorylation of SIRPα observed in the FS test.

Hypothermia-dependent and -independent effects of forced swim on the phosphorylation states of signaling molecules in mouse hippocampus.

Forced swim (FS) stress induces diverse biochemical responses in the brain of rodents. Here, we examined the effect of hypothermia induced by FS in cold water on the phosphorylation of FS-sensitive signaling molecules in the mouse brain. As we have shown previously, FS in cold water induced a significant increase in the level of tyrosine phosphorylation of SIRPα, a neuronal membrane protein, in mouse hippocampus, while such effect of FS was markedly reduced in mice subjected to FS in warm water. FS in cold water also induced phosphorylation of mitogen-activated protein kinase kinase (MEK) as well as of cAMP response element-binding protein (CREB), or dephosphorylation of α isoform of Ca(2+)/calmodulin-dependent protein kinase II (αCaMKII) in the hippocampus. These effects of FS on the phosphorylation of these molecules were also lost in mice subjected to FS in warm water. Genetic ablation of SIRPα did not change the phosphorylation states of these molecules in the brain. Forced cooling of anesthetized mice, which induced a marked increase in the phosphorylation of SIRPα, induced dephosphorylation of αCaMKII in the brain, while the same treatment did not affect the phosphorylation level of MEK and CREB. Hibernation also induced an increase and a decrease of the phosphorylation of SIRPα and αCaMKII, respectively, in the brain of chipmunk. These results suggest that hypothermia is a major element that determines the levels of phosphorylation of αCaMKII and SIRPα during the FS in cold water, while it is not for the phosphorylation levels of MEK and CREB.

CLSPCOL rescues Alzheimer's disease mouse models.

Calmodulin-like skin protein (CLSP) inhibits Alzheimer's disease (AD)-related neurotoxicity. The activity of CLSP is reduced in AD. To restore the CLSP activity, we developed a hybrid peptide named CLSPCOL, consisting of CLSP(1-61) and the collagen-homologous region (COL) of adiponectin. It was previously shown that the CLSPCOL-mediated restoration of the reduced CLSP activity alleviated memory impairment and neuronal synaptic loss in APPswe/PS1dE9 double transgenic mice (APP/PS1 mice) at an advanced phase. Here, we examined whether CLSPCOL is effective against the memory impairment of the APP/PS1 mice at an early phase, and the memory impairment, caused by the temporal disturbance of the cholinergic neurotransmission, that mimics a part of AD-linked neuronal abnormality. The CLSPCOL-mediated restoration of the CLSP activity corrected the impairment in acquisition of fear-conditioned memory at an early-phase AD model. A single subcutaneous injection of CLSPCOL rescued the short-term working memory impairment, caused by subcutaneous injection of scopolamine. We have concluded that CLSPCOL is a promising disease-modifying therapeutic agent for not only the advanced phase but also the early-phase AD. It also serves as a symptomatic modifier of AD by potentiating the cholinergic neurotransmission.

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 alpha (SIRPalpha) 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 SIRPalpha 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 SIRPalpha in the brain of wild-type mice through activation of Src family kinases. The SIRPalpha ligand CD47 was important for such SIRPalpha 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 Kvbeta2 was regulated by SIRPalpha. Thus, tyrosine phosphorylation of SIRPalpha is important for regulation of depression-like behavior in the response of the brain to stress.

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.

Restoration of the reduced CLSP activity alleviates memory impairment in Alzheimer disease.

Calmodulin-like skin protein (CLSP), a secreted peptide, inhibits neuronal death in cell-based Alzheimer's disease (AD) models and transgenic overexpression of the CLSP gene suppresses synaptic loss and memory impairment in AD model mice, APPswe/PS1dE9 double transgenic mice (APP/PS1 mice). Despite the anticipated role of CLSP as an AD-suppressing factor, it remains unanswered whether the insufficiency of the CLSP activity is linked to the AD pathogenesis. In this study, we first show that adiponectin, a CLSP potentiator/protector, dominantly determines the CLSP activity in the central nervous system where there are sufficient concentrations of CLSP, higher concentrations of CLSP inhibitors such as apolipoprotein E, and smaller concentrations of adiponectin. We next show that both the levels of brain adiponectin and the intraneuronal levels of SH3BP5, an important effector of the CLSP signal, are reduced in both AD patients and APP/PS1 mice. Finally, the restoration of the CLSP activity by subcutaneous injection of a hybrid peptide named CLSPCOL consisting of CLSP(1-61) and the collagen-homologous region of adiponectin, which has more potent neuroprotective activity than CLSP, is insensitive to the suppression by the CLSP inhibitors, and is efficiently recruited into brains, alleviates dementia and synaptic loss in the aged APP/PS1 mice. Collectively, these results suggest that the reduction in the CLSP activity, likely caused by the reduction in the levels of adiponectin, leads to the insufficient protection of neurons from neurotoxicity in the AD brains and the restoration of the CLSP activity is a promising strategy for the treatment of AD.

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.

Requirement of SIRPα for protective immunity against Leishmania major.

Signal regulatory protein α (SIRPα) is a transmembrane protein that binds the protein tyrosine phosphatases SHP-1 and SHP-2 through its cytoplasmic region and is abundantly expressed on dendritic cells and macrophages. Wild-type (WT) C57BL/6 mice are known to be resistant to Leishmania major infection. We here found that C57BL/6 mice that express a mutant version of SIRPα lacking most of the cytoplasmic region manifested increased susceptibility to L. major infection, characterized by the marked infiltration of inflammatory cells in the infected lesions. The numbers of the parasites in footpads, draining lymph nodes and spleens were also markedly increased in the infected SIRPα mutant mice, compared with those for the infected WT mice. In addition, soluble leishmanial antigen-induced production of IFN-γ by splenocytes of the infected SIRPα mutant mice was markedly reduced. By contrast, the ability of macrophages of SIRPα mutant mice to produce nitric oxide in response to IFN-γ was almost equivalent to that of macrophages from WT mice. These results suggest that SIRPα is indispensable for protective immunity against L. major by the induction of Th1 response.

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.

[Practical training of pharmacokinetics with an web-based simulation application].

A computer simulation application on pharmacokinetics, which we developed using a software, named "Stella®", has been successfully used for the virtual training of pharmacokinetics at multiple medical schools. The training course using Stella® has encouraged the medical students to optimize drug administration for individual patients on the computers. Importantly, the virtual training is free of any concern on human and animal ethics. The simulation application has been freely provided for medical schools without any restrictions and charge. For many years, it has been under constant version-upgrade in response to updates of the operating systems (OS) of personal computers or the software. Very recently, major updates of the OS and the software, and the emergence of tablet- and smartphones-type computers have been prompting us to perform a major revision of the simulation application. Here, we introduce the new version of the "web-based" simulation application that is available through any device including personal computers, tablets, and smartphones irrespective of the OSs (Microsoft Windows and Macintosh, Android, and iOS), without any extra charge unless the modification is required. We believe that the new-version of web-based simulation application will be useful not only for medical, nursing and pharmacy students, but also for medical workers who need to simulate drug pharmacokinetics on the computers before they administer drugs to the patients.

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.

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

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.

Tyrosine Phosphorylation of Carcinoembryonic Antigen-related Cell Adhesion Molecule 20 and Its Functional Role.

Carcinoembryonic antigen-related cell adhesion molecule (CEACAM) 20 is an immunoglobulin-superfamily transmembrane protein that contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic region. However, the mechanism for tyrosine phosphorylation of, or the physiological function of, this protein remains largely unknown. Here we have shown that CEACAM20 is indeed tyrosine-phosphorylated by either treatment with pervanadate or forced expression of c-Src. In addition, Tyr522, Tyr559 or Tyr570, the latter two of which are within the ITAM, is likely important for such tyrosine phosphorylation. Forced expression of Myc-tagged wild-type CEACAM20 promoted the phagocytic activity of cultured cells for microbeads coupled with anti-Myc antibodies. By contrast, such phagocytic activity was markedly reduced when a mutant form of CEACAM20, in which Tyr559 and Tyr570 were substituted with phenylalanine, was expressed. Furthermore, the CEACAM20-mediated phagocytic activity was markedly prevented by the treatment with an inhibitor for either Src family kinases (SFKs), Syk, phosphoinositide 3-kinase (PI3K) or phospholipase C-γ (PLCγ). Inhibition of actin polymerization by Cytochalasin D significantly inhibited the CEACAM20-mediated phagocytosis. These results thus suggest that tyrosine phosphorylation of CEACAM20 likely promotes phagocytic activity of the cells. The CEACAM20-mediated phagocytic activity requires the activation of SFKs, Syk, PI3K or PLCγ.