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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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

Macrophages are vital to innate immunity and express pattern recognition receptors and integrins for the rapid detection of invading pathogens. Stimulation of Dectin-1 and complement receptor 3 (CR3) activates Erk- and Akt-dependent production of reactive oxygen species (ROS). Shp2, a protein-tyrosine phosphatase encoded by Ptpn11, promotes activation of Ras-Erk and PI3K-Akt and is crucial for hematopoietic cell function; however, no studies have examined Shp2 function in particulate-stimulated ROS production. Maximal Dectin-1-stimulated ROS production corresponded kinetically to maximal Shp2 and Erk phosphorylation. Bone marrow-derived macrophages (BMMs) from mice with a conditionally deleted allele of Ptpn11 (Shp2(flox/flox);Mx1Cre+) produced significantly lower ROS levels compared with control BMMs. Although YFP-tagged phosphatase dead Shp2-C463A was strongly recruited to the early phagosome, its expression inhibited Dectin-1- and CR3-stimulated phospho-Erk and ROS levels, placing Shp2 phosphatase function and Erk activation upstream of ROS production. Further, BMMs expressing gain of function Shp2-D61Y or Shp2-E76K and peritoneal exudate macrophages from Shp2D61Y/+;Mx1Cre+ mice produced significantly elevated levels of Dectin-1- and CR3-stimulated ROS, which was reduced by pharmacologic inhibition of Erk. SIRPα (signal regulatory protein α) is a myeloid inhibitory immunoreceptor that requires tyrosine phosphorylation to exert its inhibitory effect. YFP-Shp2C463A-expressing cells have elevated phospho-SIRPα levels and an increased Shp2-SIRPα interaction compared with YFP-WT Shp2-expressing cells. Collectively, these findings indicate that Shp2 phosphatase function positively regulates Dectin-1- and CR3-stimulated ROS production in macrophages by dephosphorylating and thus mitigating the inhibitory function of SIRPα and by promoting Erk activation.

Protein tyrosine phosphorylation is thought to be important for regulation of the proliferation, differentiation, and rapid turnover of intestinal epithelial cells (IECs). The role of protein tyrosine phosphatases in such homeostatic regulation of IECs has remained largely unknown, however. Src homology 2–containing protein tyrosine phosphatase (Shp2) is a ubiquitously expressed cytoplasmic protein tyrosine phosphatase that functions as a positive regulator of the Ras–mitogen-activated protein kinase (MAPK) signaling pathway operative downstream of the receptors for various growth factors and cytokines, and it is thereby thought to contribute to the regulation of cell proliferation and differentiation. We now show that mice lacking Shp2 specifically in IECs (Shp2 CKO mice) develop severe colitis and die as early as 3 to 4 weeks after birth. The number of goblet cells in both the small intestine and colon of Shp2 CKO mice was markedly reduced compared with that for control mice. Furthermore, Shp2 CKO mice showed marked impairment of both IEC migration along the crypt-villus axis in the small intestine and the development of intestinal organoids from isolated crypts. The colitis as well as the reduction in the number of goblet cells apparent in Shp2 CKO mice were normalized by expression of an activated form of K-Ras in IECs. Our results thus suggest that Shp2 regulates IEC homeostasis through activation of Ras and thereby protects against the development of colitis. [ABSTRACT FROM AUTHOR]

Takahashi S, Tomioka M, Hiromura K, Sakairi T, Hamatani H, Watanabe M, Ikeuchi H, Kaneko Y, Maeshima A, Aoki T, Ohnishi H, Matozaki T, Nojima Y. SIRP signaling regulates podocyte structure and function. Am J Physiol Renal Physiol 305: F861-F870, 2013. First published July 10, 2013; doi:10.1152/ajprenal.00597.2012.--Signal-regulatory protein-α (SIRPα) is a transmembrane protein that contains tyrosine phosphorylation sites in its cytoplasmic region; two tyrosine phosphatases, SHP-1 and SHP-2, bind to these sites in a phosphorylationdependent manner and transduce multiple intracellular signals. Recently, SIRPα was identified as one of the major tyrosine-phosphorylated proteins in the glomeruli and found to be expressed in podocytes. In the present study, we examined the role of SIRPα expression in podocytes using knockin mice (C57BL/6 background) expressing mutant SIRPα that lacks a cytoplasmic region (SIRPα- mutant mice). Light microscopic examination revealed no apparent morphological abnormalities in the kidneys of the SIRPα-mutant mice. On the other hand, electron microscopic examination revealed abnormal podocytes with irregular major processes and wider and flattened foot processes in the SIRPα-mutant mice compared with their wild-type counterparts. Significantly impaired renal functions and slight albuminuria were demonstrated in the SIRPα-mutant mice. In addition, adriamycin injection induced massive albuminuria together with focal glomerulosclerosis in the SIRPα-mutant mice, while their wild-type counterparts were resistant to adriamycin-induced nephropathy. These data demonstrate that SIRPα is involved in the regulation of podocyte structure and function as a filtration barrier under both physiological and pathological conditions. [ABSTRACT FROM AUTHOR]

J. Neurochem. (2012) 121, 891-902. Abstract 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 ( Trec) 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

SHPS-1 is a transmembrane protein predominantly expressed in macrophages. The possible role of SHPS-1 in regulation of Toll-like receptor (TLR)-dependent production of proinflammatory cytokines by macrophages has remained unknown, however. We now show that expression either of a mutant version of mouse SHPS-1 (SHPS-1–4F) in which the four tyrosine phosphorylation sites in the cytoplasmic region are replaced by phenylalanine or of a chimeric protein comprising the extracellular and transmembrane regions of human CD8 fused to the cytoplasmic region of SHPS-1–4F (CD8–4F) markedly promoted the production of tumor necrosis factor-α (TNF-α) or interleukin-6 (IL-6) induced by lipopolysaccharide (LPS) or polyinosinic-polycytidylic acid [poly(I : C)] in RAW264.7 macrophages. In contrast, expression of a mutant form of SHPS-1 that lacks most of the cytoplasmic region did not promote such responses. Expression of SHPS-1–4F promoted the LPS- or poly(I : C)-induced activation of NF-κB. LPS and poly(I : C) each induced the tyrosine phosphorylation of SHPS-1 through a Src family kinase and the association of SHPS-1 with SHP-1 and SHP-2. These results suggest that LPS or poly(I : C) induces tyrosine phosphorylation of SHPS-1 and the association of SHPS-1 with SHP-1 and SHP-2 in a manner dependent on a Src family kinase. SHPS-1 then negatively regulates TLR4- or TLR3-dependent cytokine production through inhibition of NF-κB-dependent signaling. [ABSTRACT FROM AUTHOR]

SHPS-1 is a transmembrane protein whose cytoplasmic region undergoes tyrosine phosphorylation and then binds the protein-tyrosine phosphatase SHP-2. Formation of the SHPS-1-SHP-2 complex is implicated in regulation of cell migration. In addition, SHPS-1 and its ligand CD47 constitute an intercellular recognition system that contributes to inhibition of cell migration by cell-cell contact. The ectodomain of SHPS-1 has now been shown to be shed from cells in a reaction likely mediated by a metalloproteinase. This process was promoted by activation of protein kinase C or of Ras, and the released ectodomain exhibited minimal CD47-binding activity. Metalloproteinases catalyzed the cleavage of a recombinant SHPS-1-Fc fusion protein in vitro, and the primary cleavage site was localized to the juxtamembrane region of SHPS-1. Forced expression of an SHPS-1 mutant resistant to ectodomain shedding impaired cell migration, cell spreading, and reorganization of the actin cytoskeleton. It also increased the tyrosine phosphorylation of paxillin and FAK triggered by cell adhesion. These results suggest that shedding of the ectodomain of SHPS-1 plays an important role in regulation of cell migration and spreading by this protein. [ABSTRACT FROM AUTHOR]

Abstract: Brain-derived neurotrophic factor (BDNF) activates a variety of signaling molecules to exert various functions in the nervous system, including neuronal differentiation, survival, and regulation of synaptic plasticity. Previously, we have suggested that BIT/SHPS-1 (brain immunoglobulin-like molecule with tyrosine-based activation motifs/SHP substrate 1) is a substrate of Shp-2 and is involved in BDNF signaling in cultured cerebral cortical neurons. To elucidate the biological function of BIT/SHPS-1 in cultured cerebral cortical neurons in connection with its role in BDNF signaling, we generated recombinant adenovirus vectors expressing the wild type of rat BIT/SHPS-1 and its 4F mutant in which all tyrosine residues in the cytoplasmic domain of BIT/SHPS-1 were replaced with phenylalanine. Overexpression of wild-type BIT/SHPS-1, but not the 4F mutant, in cultured cerebral cortical neurons induced tyrosine phosphorylation of BIT/SHPS-1 itself and an association of Shp-2 with BIT/SHPS-1 even without addition of BDNF. We found that BDNF-promoted survival of cultured cerebral cortical neurons was enhanced by expression of the wild type and also 4F mutant, indicating that this enhancement by BIT/SHPS-1 does not depend on its tyrosine phosphorylation. BDNF-induced activation of mitogen-activated protein kinase was not altered by the expression of these proteins. In contrast, BDNF-induced activation of Akt was enhanced in neurons expressing wild-type or 4F mutant BIT/SHPS-1. In addition, LY294002, a specific inhibitor of phosphatidylinositol 3-kinase, blocked the enhancement of BDNF-promoted neuronal survival in both neurons expressing wild-type and 4F mutant BIT/SHPS-1. These results indicate that BIT/SHPS-1 contributes to BDNF-promoted survival of cultured cerebral cortical neurons, and that its effect depends on the phosphatidylinositol 3-kinase-Akt pathway. Our results suggest that a novel action of BIT/SHPS-1 does not occur through tyrosine phosphorylation of BIT/SHPS-1 in cultured cerebral cortical neurons. [ABSTRACT FROM AUTHOR]

BIT (brain immunoglobulin-like molecule with tyrosine-based activation motifs) is a membrane glycoprotein that has two cytoplasmic TAMs (tyrosine-based activation motifs). We previously reported that tyrosine-phosphorylated TAMs of BIT interact with the Src homology 2 domain-containing protein tyrosine phosphatase SHP-2 both in vitro and in transfected cells, and this association results in a potent stimulation of the phosphatase activity of SHP-2. Both BIT and SHP-2 are highly expressed in the mammalian brain, and they may play important roles in the regulation of synaptic function. In this study, we found that nerve growth factor (NGF) treatment of PC12 cells leads to the tyrosine phosphorylation of BIT and a subsequent complex formation between BIT and SHP-2. Furthermore, brain-derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) also induced the tyrosine phosphorylation of BIT and the association with SHP-2 in primary cultured rat neurons. Our results suggest that the BIT-SHP-2 signaling pathway is a novel signal transduction mechanism of neurons that acts in response to neurotrophic factors such as NGF, BDNF, and NT-3. [ABSTRACT FROM AUTHOR]

Abstract: 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 Ca2+/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. [Copyright &y& Elsevier]