RhoE belongs to the Rho GTPase family, the members of which control actin cytoskeletal dynamics. RhoE induces stress fiber disassembly in a variety of cell types, whereas RhoA stimulates stress fiber assembly. The similarity of RhoE and RhoA sequences suggested that RhoE might compete with RhoA for interaction with its targets. Here, we show that RhoE binds ROCK I but none of the other RhoA targets tested. The interaction of RhoE with ROCK I was confirmed by coimmunoprecipitation of the endogenous proteins, and the two proteins colocalized on the trans-Golgi network in COS-7 cells. Although RhoE and RhoA were not able to bind ROCK I simultaneously, RhoE bound to the amino-terminal region of ROCK I encompassing the kinase domain, at a site distant from the carboxy-terminal RhoA-binding site. Overexpression of RhoE inhibited ROCK I-induced stress fiber formation and phosphorylation of the ROCK I target myosin light chain phosphatase. These data suggest that RhoE induces stress fiber disassembly by directly binding ROCK I and inhibiting it from phosphorylating downstream targets.

The Notch receptor and its ligands are key components in a core metazoan signaling pathway that regulates the spatial patterning, timing and outcome of many cell-fate decisions. Ligands contain a disulfide-rich Delta/Serrate/LAG-2 (DSL) domain required for Notch trans-activation or cis-inhibition. Here we report the X-ray structure of a receptor binding region of a Notch ligand, the DSL-EGF3 domains of human Jagged-1 (J-1(DSL-EGF3)). The structure reveals a highly conserved face of the DSL domain, and we show, by functional analysis of Drosophila melanogster ligand mutants, that this surface is required for both cis- and trans-regulatory interactions with Notch. We also identify, using NMR, a surface of Notch-1 involved in J-1(DSL-EGF3) binding. Our data imply that cis- and trans-regulation may occur through the formation of structurally distinct complexes that, unexpectedly, involve the same surfaces on both ligand and receptor.

Activating and inhibitory receptors control natural killer (NK) cell activity. T-cell immunoglobulin and ITIM (immunoreceptor tyrosine-based inhibition motif) domain (TIGIT) was recently identified as a new inhibitory receptor on T and NK cells that suppressed their effector functions. TIGIT harbors the immunoreceptor tail tyrosine (ITT)-like and ITIM motifs in its cytoplasmic tail. However, how its ITT-like motif functions in TIGIT-mediated negative signaling is still unclear. Here, we show that TIGIT/PVR (poliovirus receptor) engagement disrupts granule polarization leading to loss of killing activity of NK cells. The ITT-like motif of TIGIT has a major role in its negative signaling. After TIGIT/PVR ligation, the ITT-like motif is phosphorylated at Tyr225 and binds to cytosolic adapter Grb2, which can recruit SHIP1 to prematurely terminate phosphatidylinositol 3-kinase (PI3K) and MAPK signaling, leading to downregulation of NK cell function. In support of this, Tyr225 or Asn227 mutation leads to restoration of TIGIT/PVR-mediated cytotoxicity, and SHIP1 silencing can dramatically abolish TIGIT/PVR-mediated killing inhibition. Meanwhile, normal cells are kept away from their cytotoxicity. Therefore, the discrimination between 'self' normal cells and 'nonself' abnormal cells has to be precisely recognized by NK cells. [5][6][7] A very large repertoire of receptors, both activating and inhibitory, is proved to be critical in NK cell function. The best-known inhibitory receptors of NK cells are the killer-cell immunoglobulin-like receptor family, whose physical ligands are MHC-I molecules that are expressed on self normal cells to protect them from NK cell lysis. 8 Other non-MHC-I inhibitory receptors, which do not associate with MHC-I molecules, are also expressed on NK cells. 9 However, their physiological and pathological significances have not been defined yet.T-cell immunoglobulin and ITIM domain (TIGIT) was recently identified as an inhibitory receptor that is expressed mainly on NK cells, activated CD4 and CD8 T cells. 10-12 TIGIT harbors one extracellular immunoglobulin domain, a type 1 transmembrane region, and an immunoglobulin tail tyrosine (ITT)-like phosphorylation motif followed by an ITIM (immunoreceptor tyrosine-based inhibition motif) of the cytoplasmic tail. 13 The physical ligands of TIGIT were identified as the poliovirus receptor (PVR, or CD155) and the PVRL2 (Nectin2, or CD112). 10,12 TIGIT can bind to PVR of human dendritic cells to enhance interleukin 10 (IL-10) production, which inhibits T-cell activation. 10 Kuchroo et al. 14 showed that TIGIT harbors a T-cell-intrinsic inhibitory function to suppress T-cell activation.Moreover, TIGIT can inhibit NK cell cytolysis through engagement with PVR or PVRL2. 11 TIGIT-deficient mice are more susceptible to autoimmune diseases. 14,15 However, the inhibitory mechanism mediated by TIGIT has not been elucidated.TIGIT contains a classical ITIM motif, which recruits either Src homology (SH) 2 domain-containing protein tyrosine phosphatases SHP1 and SHP...

We recently showed that ASPP1 and ASPP2 stimulate the apoptotic function of p53. We show here that ASPP1 and ASPP2 also induce apoptosis independently of p53. By binding to p63 and p73 in vitro and in vivo, ASPP1 and ASPP2 stimulate the transactivation function of p63 and p73 on the promoters of Bax, PIG3, and PUMA but not mdm2 or p21 WAF-1/CIP1 . The expression of ASPP1 and ASPP2 also enhances the apoptotic function of p63 and p73 by selectively inducing the expression of endogenous p53 target genes, such as PIG3 and PUMA, but not mdm2 or p21 WAF-1/CIP1 . Removal of endogenous p63 or p73 with RNA interference demonstrated that (16) the p53-independent apoptotic function of ASPP1 and ASPP2 is mediated mainly by p63 and p73. Hence, ASPP1 and ASPP2 are the first two identified common activators of all p53 family members. All these results suggest that ASPP1 and ASPP2 could suppress tumor growth even in tumors expressing mutant p53.The p53 gene is mutated in around 35 to 40% of human tumors. Pathways that activate p53 are also disrupted in many other tumors. The p53 protein modulates cellular functions, such as gene transcription, DNA synthesis, DNA repair, cell cycle arrest, senescence, and apoptosis. Mutations of the gene may result in inhibited protein function, and it is this dysfunction that is linked to tumor progression and genetic instability. In response to a variety of cellular stresses, p53 is posttranslationally modified, and protein levels increase dramatically. Activation of the protein results in either arrest of the cell at G 1 or commitment to death through apoptosis. Research has demonstrated the role of p53 transcriptional transactivation in cell cycle arrest through the up-regulation of the p21 WAF-1/CIP1 cyclin-dependent kinase inhibitor (cdki). However, many reports have shown that p53 can induce apoptosis by both transcription-dependent and -independent mechanisms (19,20).p53 is a member of a family of three proteins: p53, p63, and p73. p63 and p73 have more than 60% amino acid identity within the DNA binding region of p53 (12,13,22). DNA binding specificity among p53 family members is very similar but not identical. As a result, a large number of p53 target genes are also transactivated by p63 and p73. Hence, p63 and p73 share some p53 functions, such as cell cycle arrest and apoptosis. However, there are many other structural and functional differences between p53, p63, and p73. For example, mutations in p63 and p73 are rare in human cancer. Studies of p53-, p63-, and p73-deficient mice established that the expression of p63 and p73 is more important for mouse development than the expression of p53 and that the loss of p73 or p63 does not predispose mice to cancer (21). Cellular regulators of p53, such as mdm2, do not have the same effects on p63 and p73.While the binding of mdm2 to p53 inhibits the transactivation function of p53 and targets it for degradation (11,14), it fails to target p63 and p73 for degradation (4,8). In contrast, the binding of mdm2 to p63 stimulates the transactivati...

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