Yes-associated protein (YAP) is a potent transcription coactivator acting via binding to the TEAD transcription factor, and plays a critical role in organ size regulation. YAP is phosphorylated and inhibited by the Lats kinase, a key component of the Hippo tumor suppressor pathway. Elevated YAP protein levels and gene amplification have been implicated in human cancer. In this study, we report that YAP is inactivated during embryonic stem (ES) cell differentiation, as indicated by decreased protein levels and increased phosphorylation. Consistently, YAP is elevated during induced pluripotent stem (iPS) cell reprogramming. YAP knockdown leads to a loss of ES cell pluripotency, while ectopic expression of YAP prevents ES cell differentiation in vitro and maintains stem cell phenotypes even under differentiation conditions. Moreover, YAP binds directly to promoters of a large number of genes known to be important for stem cells and stimulates their expression. Our observations establish a critical role of YAP in maintaining stem cell pluripotency.[Keywords: YAP; Hippo; stem cells; TEAD] Supplemental material is available at http://www.genesdev.org.

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Src homology 2 domain-containing protein tyrosine phosphatase (SHP) substrate-1 (SHPS-1) is a transmembrane protein that is expressed predominantly in macrophages. Its extracellular region interacts with the transmembrane ligand CD47 expressed on the surface of adjacent cells, and its cytoplasmic region binds the protein tyrosine phosphatases SHP-1 and SHP-2. Phagocytosis of IgG- or complement-opsonized RBCs by peritoneal macrophages derived from mice that express a mutant SHPS-1 protein that lacks most of the cytoplasmic region was markedly enhanced compared with that apparent with wild-type macrophages. This effect was not observed either with CD47-deficient RBCs as the phagocytic target or in the presence of blocking Abs to SHPS-1. Depletion of SHPS-1 from wild-type macrophages by RNA interference also promoted FcγR-mediated phagocytosis of wild-type RBCs. Ligation of SHPS-1 on macrophages by CD47 on RBCs promoted tyrosine phosphorylation of SHPS-1 and its association with SHP-1, whereas tyrosine phosphorylation of SHPS-1 was markedly reduced in response to cross-linking of FcγRs. Treatment with inhibitors of PI3K or of Syk, but not with those of MEK or Src family kinases, abolished the enhancement of FcγR-mediated phagocytosis apparent in macrophages from SHPS-1 mutant mice. In contrast, FcγR-mediated tyrosine phosphorylation of Syk, Cbl, or the γ subunit of FcR was similar in macrophages from wild-type and SHPS-1 mutant mice. These results suggest that ligation of SHPS-1 on macrophages by CD47 promotes the tyrosine phosphorylation of SHPS-1 and thereby prevents the FcγR-mediated disruption of the SHPS-1-SHP-1 complex, resulting in inhibition of phagocytosis. The inhibition of phagocytosis by the SHPS-1-SHP-1 complex may be mediated at the level of Syk or PI3K signaling.

SHP-2 is a cytoplasmic protein tyrosine phosphatase (PTP) that contains two

Insulin induces the translocation of vesicles containing the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane in adipocytes. SNARE proteins have been implicated in the docking and fusion of these vesicles with the cell membrane. The role of Munc18c, previously identified as an n-Sec1/ Munc18 homolog in 3T3-L1 adipocytes, in insulin-regulated GLUT4 trafficking has now been investigated in 3T3-L1 adipocytes. In these cells, Munc18c was predominantly associated with syntaxin4, although it bound both syntaxin2 and syntaxin4 to similar extents in vitro. In addition, SNAP-23, an adipocyte homolog of SNAP-25, associated with both syntaxins 2 and 4 in 3T3-L1 adipocytes. Overexpression of Munc18c in 3T3-L1 adipocytes by adenovirus-mediated gene transfer resulted in inhibition of insulin-stimulated glucose transport in a virus dose-dependent manner (maximal effect, ϳ50%) as well as in inhibition of sorbitol-induced glucose transport (by ϳ35%), which is mediated by a pathway different from that used by insulin. In contrast, Munc18b, which is also expressed in adipocytes but which did not bind to syntaxin4, had no effect on glucose transport. Furthermore, overexpression of Munc18c resulted in inhibition of insulin-induced translocation of GLUT4, but not of that of GLUT1, to the plasma membrane. These results suggest that Munc18c is involved in the insulin-dependent trafficking of GLUT4 from the intracellular storage compartment to the plasma membrane in 3T3-L1 adipocytes by modulating the formation of a SNARE complex that includes syntaxin4.Insulin stimulates glucose transport into muscle and adipose tissue by inducing the translocation of vesicles containing the glucose transporter GLUT4 from the intracellular compartment to the plasma membrane (1, 2). This process is thought to be a major contributor to the mechanism by which insulin reduces the blood concentration of glucose. The binding of insulin to its receptor on the surface of target cells results in receptor autophosphorylation and receptor-mediated tyrosine phosphorylation of several additional proteins, including insulin receptor substrates 1-4 (IRS1 to IRS4).1 The phosphorylated IRS proteins then bind other proteins, such as phosphoinositide (PI) 3-kinase, SHP-2, and GRB2, that contain SRC homology 2 (SH2) domains (3). PI 3-kinase is thought to play a role in the insulin-induced translocation of GLUT4 (4, 5); however, the mechanism by which activation of PI 3-kinase results in GLUT4 translocation remains unclear (6).The SNARE (soluble N-ethylmaleimide-sensitive fusion protein attachment protein receptor) hypothesis was initially proposed to explain the process of neurotransmitter secretion (7,8). According to this hypothesis, the docking and fusion of synaptic vesicles at the plasma membrane are initiated by the interaction of proteins, known as v-SNAREs (synaptobrevin/ VAMP), located on the vesicle surface with corresponding proteins, known as t-SNAREs (syntaxin, SNAP-25), located on the target membrane. Membrane fusion is subs...

IntroductionThe lifespan of circulating red blood cells (RBCs) is approximately 120 and 40 days in humans and mice, respectively, and is determined by their production in bone marrow (BM) and their clearance from the peripheral circulation, predominantly in the spleen and liver. [1][2][3] The production of RBCs is controlled by the primary erythropoietic regulator erythropoietin, 2,4 whereas clearance of old RBCs by the spleen is achieved mostly as a result of their specific recognition and phagocytosis by splenic macrophages. 5,6 The precise molecular mechanism by which splenic macrophages recognize senescent RBCs for phagocytosis is largely unknown, however. [1][2][3] Src homology 2 domain-containing protein tyrosine phosphatase substrate-1 (SHPS-1), 7,8 also known as signal-regulatory protein ␣, 9 brain immunoglobulin (Ig)-like molecule with tyrosinebased activation motifs, 10 and p84 neural adhesion molecule, 11 is a transmembrane protein that is especially abundant in macrophages. [12][13][14] The putative extracellular region of SHPS-1 comprises 3 Ig-like domains, and its cytoplasmic region contains 4 tyrosine phosphorylation sites that mediate the binding of Src homology 2 domain-containing protein tyrosine phosphatases designated SHP-1 and SHP-2. 7,9 Tyrosine phosphorylation of SHPS-1 is regulated by various growth factors and cytokines as well as by integrinmediated cell adhesion to extracellular matrix proteins. 7,9,[15][16][17][18] SHPS-1 thus functions as a docking protein to recruit and activate SHP-1 or SHP-2 at the cell membrane in response to extracellular stimuli. In macrophages, tyrosine-phosphorylated SHPS-1 binds SHP-1, 12,19,20 which is implicated in negative regulation of the functions of a variety of hematopoietic cells. [21][22][23] The complex of SHPS-1 and SHP-1 is thus thought to regulate macrophage functions in a negative manner.CD47 is a ligand for the extracellular region of SHPS-1. 24,25 This protein, which was originally identified in association with ␣v␤3 integrin, is also a member of the Ig superfamily, possessing an Ig-V-like extracellular domain, 5 putative membrane-spanning segments, and a short cytoplasmic tail. 26 CD47 and SHPS-1 constitute a cell-cell communication system (the CD47-SHPS-1 system) that plays important roles in a variety of cellular processes including cell migration, 27,28 adhesion of B cells, 29 and T-cell activation. 14,30 In addition, the CD47-SHPS-1 system is implicated in negative regulation of phagocytosis by macrophages. CD47 is highly expressed on the surface of RBCs, where it associates with the Rh protein complex instead of with integrins. 31 The rate of clearance of CD47-deficient RBCs from the bloodstream was found to be markedly increased compared with that of wild-type (WT) cells. 6,32 Furthermore, the phagocytosis of CD47-deficient RBCs by splenic or BM-derived macrophages was greatly enhanced Supported by a Grant-in-Aid for Scientific Research on Priority Areas Cancer; a Grant-in-Aid for Scientific Research (B); a Grant-in-Aid for Young Scie...

SHPS-1 is a receptor-type glycoprotein that binds and activates the protein-tyrosine phosphatases SHP-1 and SHP-2, and thereby negatively modulates intracellular signaling initiated by various cell surface receptors coupled to tyrosine kinases. SHPS-1 also regulates intercellular communication in the neural and immune systems through its association with CD47 (integrin-associated protein) on adjacent cells. Furthermore, recent studies with fibroblasts derived from mice expressing an SHPS-1 mutant that lacks most of the cytoplasmic region suggested that the intact protein contributes to cytoskeletal function. Mice homozygous for this SHPS-1 mutation have now been shown to manifest thrombocytopenia. These animals did not exhibit a defect in megakaryocytopoiesis or in platelet production. However, platelets were cleared from the bloodstream more rapidly in the mutant mice than in wild-type animals. Furthermore, peritoneal macrophages from the mutant mice phagocytosed red blood cells more effectively than did those from wild-type mice; in addition, they exhibited an increase both in the rate of cell spreading and in the formation of filopodia-like structures at the cell periphery. These results indicate that SHPS-1 both contributes to the survival of circulating platelets and down-regulates the macrophage phagocytic response.SHPS-1 is a transmembrane glycoprotein that is abundant in neural and myeloid tissues (1-6). This molecule is also known as SIRP␣1 (7), BIT (8), MFR (9), and p84 neural adhesion molecule (10). The cytoplasmic region of SHPS-1 contains two immunoreceptor tyrosine-based inhibitory motifs, which recruit and activate the Src homology 2 domain-containing protein-tyrosine phosphatases SHP-1 and SHP-2 in a phosphorylation-dependent manner (1, 7, 11). The putative extracellular region of this protein comprises three immunoglobulin (Ig)-like domains, of which the most amino-terminal, IgV-like domain associates with the ligand CD47, also known as integrin-associated protein (6,12,13).Tyrosine phosphorylation of SHPS-1 is induced by soluble growth factors (1,7,14,15), integrin-mediated cell adhesion (16 -18), or cross-linking of Fc␥ receptors (19). Overexpression of SHPS-1 inhibits the activation of extracellular signal-regulated kinases induced by growth factors such as insulin, epidermal growth factor, and platelet-derived growth factor (7); it also inhibits promotion of the motility and survival of glioblastoma cells by epidermal growth factor (20). Furthermore, SHPS-1 inhibits IgE-induced mediator secretion and cytokine synthesis by mast cells (21). These observations suggest that SHPS-1, presumably by recruiting SHP-1 or SHP-2, negatively modulates a wide range of cellular activation signals initiated by tyrosine kinase-coupled receptors. However, the physiological significance of these observations remains unclear.Recent studies have suggested that SHPS-1, through its association with CD47, contributes to cellular functions that depend on intercellular communication, including T cell activation (13),...

Both syntaxin4 and VAMP2 are implicated in insulin regulation of glucose transporter-4 (GLUT4) trafficking in adipocytes as target (t) soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNARE) and vesicle (v)-SNARE proteins, respectively, which mediate fusion of GLUT4-containing vesicles with the plasma membrane. Synaptosome-associated 23-kDa protein (SNAP23) is a widely expressed isoform of SNAP25, the principal t-SNARE of neuronal cells, and colocalizes with syntaxin4 in the plasma membrane of 3T3-L1 adipocytes. In the present study, two SNAP23 mutants, SNAP23-⌬C8 (amino acids 1 to 202) and SNAP23-⌬C49 (amino acids 1 to 161), were generated to determine whether SNAP23 is required for insulin-induced translocation of GLUT4 to the plasma membrane in 3T3-L1 adipocytes. Wild-type SNAP23 (SNAP23-WT) promoted the interaction between syntaxin4 and VAMP2 both in vitro and in vivo. Although SNAP23-⌬C49 bound to neither syntaxin4 nor VAMP2, the SNAP23-⌬C8 mutant bound to syntaxin4 but not to VAMP2. In addition, although SNAP23-⌬C8 bound to syntaxin4, it did not mediate the interaction between syntaxin4 and VAMP2. Moreover, overexpression of SNAP23-⌬C8 in 3T3-L1 adipocytes by adenovirus-mediated gene transfer inhibited insulin-induced translocation of GLUT4 but not that of GLUT1. In contrast, overexpression of neither SNAP23-WT nor SNAP23-⌬C49 in 3T3-L1 adipocytes affected the translocation of GLUT4 or GLUT1. Together, these results demonstrate that SNAP23 contributes to insulin-dependent trafficking of GLUT4 to the plasma membrane in 3T3-L1 adipocytes by mediating the interaction between t-SNARE (syntaxin4) and v-SNARE (VAMP2).A primary function of insulin is to stimulate the transport of glucose into target tissues, prominent among which are skeletal muscle, cardiac muscle, and adipose tissue. Insulin achieves this effect by inducing the translocation of GLUT4 glucose transporters from an intracellular vesicular compartment to the plasma membrane. Under basal conditions, GLUT4 cycles slowly between this intracellular compartment and the plasma membrane (1, 2). However, activation of insulin receptors triggers a large increase in the rate of exocytosis of GLUT4-containing vesicles and a smaller decrease in the rate of GLUT4 internalization by endocytosis (3-5), with the former action likely contributing most to the insulin-induced increase in the amount of GLUT4 in the plasma membrane (6).Intracellular membrane fusion is mediated by evolutionarily conserved membrane proteins known as soluble N-ethylmaleimide-sensitive factor (NSF) 1 attachment protein receptors (SNAREs) (7,8). SNARE proteins that contribute to neuronal exocytosis include the synaptic vesicle protein synaptobrevin (also referred to as VAMP) and the plasma membrane proteins synaptosome-associated 25-kDa protein and syntaxin1A. These proteins readily assemble into a stable ternary complex; however, disassembly of this complex can be reversibly induced by the ATPase NSF in conjunction with soluble cofactors termed SNAPs (soluble NSF-att...