Glutamate Receptor-interacting Protein 1 Protein Binds to the Microtubule-associated Protein

To identify the interaction proteins for the α-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA) receptor subunit glutamate receptor-interacting protein 1 (GRIP1), GRIP1 interactions with microtubule-associated protein (MAP)-1B light chain (LC) were investigated. GRIP1 interacts with MAP-1A and MAP-1B in the yeast two-hybrid assay, as is indicated also by glutathione S-transferase (GST) pull-down and coimmunoprecipitation with MAP-1B LC antibody in brain fractions. These results suggest a novel mechanism for localizing AMPA receptors to synaptic sites.     

多功能蛋白质COMMD1

COMMD蛋白家族是近年来新发现的一组新颖的因子,它们结构独特,在生物进化过程中高度保守且有着某些共同的功能性质.它们广泛存在于多细胞生物中,最典型的特征是它们的羧基端存在一个高度保守而独特的结构-COMMD结构域,为蛋白质间的相互作用提供了关键界面.目前研究已证实,COMMD蛋白家族有10个成员,即COMMD1~10.COMMD1是COMMD蛋白家族中最先被证实且研究最为深入的蛋白质,在不同物种中广泛表达,且在人类的不同组织表达存在差异.COMMD1是1个多效性因子,参与许多生理活动,包括对铜代谢、转录因子NF-κB及低氧诱导因子1（HIF-1）的调节等.COMMD1参与铜的内稳态调节,是转录调节因子NF-κB及HIF-1的抑制物,通过对它们的抑制而介导多种基因的表达.本文就COMMD1的结构、表达、调节及生物效应等做一综述.     

Coronin-1C Protein and Caveolin Protein Provide Constitutive and Inducible Mechanisms of Rac1 Protein Trafficking

Sustained directional fibroblast migration requires both polarized activation of the protrusive signal, Rac1, and redistribution of inactive Rac1 from the rear of the cell so that it can be redistributed or degraded. In this work, we determine how alternative endocytic mechanisms dictate the fate of Rac1 in response to the extracellular matrix environment. We discover that both coronin-1C and caveolin retrieve Rac1 from similar locations at the rear and sides of the cell. We find that coronin-1C-mediated extraction, which is responsible for Rac1 recycling, is a constitutive process that maintains Rac1 protein levels within the cell. In the absence of coronin-1C, the effect of caveolin-mediated endocytosis, which targets Rac1 for proteasomal degradation, becomes apparent. Unlike constitutive coronin-1C-mediated trafficking, caveolin-mediated Rac1 endocytosis is induced by engagement of the fibronectin receptor syndecan-4. Such an inducible endocytic/degradation mechanism would predict that, in the presence of fibronectin, caveolin defines regions of the cell that are resistant to Rac1 activation but, in the absence of fibronectin leaves more of the membrane susceptible to Rac1 activation and protrusion. Indeed, we demonstrate that fibronectin-stimulated activation of Rac1 is accelerated in the absence of caveolin and that, when caveolin is knocked down, polarization of active Rac1 is lost in FRET experiments and culminates in shunting migration in a fibrous fibronectin matrix. Although the concept of polarized Rac1 activity in response to chemoattractants has always been apparent, our understanding of the balance between recycling and degradation explains how polarity can be maintained when the chemotactic gradient has faded.     

MAVS Protein Is Attenuated by Rotavirus Nonstructural Protein 1

Rotavirus is the single, most important agent of infantile gastroenteritis in many animal species, including humans. In developing countries, rotavirus infection attributes approximately 500,000 deaths annually. Like other viruses it establishes an intimate and complex interaction with the host cell to counteract the antiviral responses elicited by the cell. Among various pattern recognition receptors (PAMPs) of the host, the cytosolic RNA helicases interact with viral RNA to activate the Mitochondrial Antiviral Signaling protein (MAVS), which regulates cellular interferon response. With an aim to identify the role of different PAMPs in rotavirus infected cell, MAVS was found to degrade in a time dependent and strain independent manner. Rotavirus non-structural protein 1 (NSP1) which is a known IFN antagonist, interacted with MAVS and degraded it in a strain independent manner, resulting in a complete loss of RNA sensing machinery in the infected cell. To best of our knowledge, this is the first report on NSP1 functionality where a signaling protein is targeted unanimously in all strains. In addition NSP1 inhibited the formation of detergent resistant MAVS aggregates, thereby averting the antiviral signaling cascade. The present study highlights the multifunctional role of rotavirus NSP1 and reinforces the fact that the virus orchestrates the cellular antiviral response to its own benefit by various back up strategies.     

Protein Phosphatase PPM1G Regulates Protein Translation and Cell Growth by Dephosphorylating 4E Binding Protein 1 (4E-BP1)

Protein translation initiation is a tightly controlled process responding to nutrient availability and mitogen stimulation. Serving as one of the most important negative regulators of protein translation, 4E binding protein 1 (4E-BP1) binds to translation initiation factor 4E and inhibits cap-dependent translation in a phosphorylation-dependent manner. Although it has been demonstrated previously that the phosphorylation of 4E-BP1 is controlled by mammalian target of rapamycin in the mammalian target of rapamycin complex 1, the mechanism underlying the dephosphorylation of 4E-BP1 remains elusive. Here, we report the identification of PPM1G as the phosphatase of 4E-BP1. A coimmunoprecipitation experiment reveals that PPM1G binds to 4E-BP1 in cells and that purified PPM1G dephosphorylates 4E-BP1 in vitro. Knockdown of PPM1G in 293E and colon cancer HCT116 cells results in an increase in the phosphorylation of 4E-BP1 at both the Thr-37/46 and Ser-65 sites. Furthermore, the time course of 4E-BP1 dephosphorylation induced by amino acid starvation or mammalian target of rapamycin inhibition is slowed down significantly in PPM1G knockdown cells. Functionally, the amount of 4E-BP1 bound to the cap-dependent translation initiation complex is decreased when the expression of PPM1G is depleted. As a result, the rate of cap-dependent translation, cell size, and protein content are increased in PPM1G knockdown cells. Taken together, our study has identified protein phosphatase PPM1G as a novel regulator of cap-dependent protein translation by negatively controlling the phosphorylation of 4E-BP1.     

AtRGS1-GFP融合蛋白对AtRGS1细胞定位的研究

应用Gateway克隆技术构建了以CaMV35S为启动子,含AtRGS1-GFP融合基因的植物表达载体,并分别用根癌农杆菌介导法和PEG介导法转化拟南芥野生型（C01）悬浮细胞系和幼苗叶片原生质体,利用荧光显微镜观察AtRGS1-GFP融合基因在转化受体系统中的表达与定位。结果显示,在含AtRGS1-GFP融合基因的转化细胞系中,GFP绿色荧光在细胞膜（壁）上特异表达;原生质体瞬时表达系统中,GFP绿色荧光在细胞膜上强烈表达,表明AtRGS1蛋白定位于细胞质膜上。     

IEX-1相互作用蛋白的筛选与鉴定

骨肉瘤即原发于骨的恶性肿瘤,易发生早期肺转移且预后差,恶性程度高.本研究小组前期研究发现,IEX-1在骨肉瘤中具有重要作用.为了阐明其作用机制,本研究运用酵母双杂交技术筛选其相互作用蛋白,共鉴定出12个IEX-1相互作用蛋白,包括生物氧化相关酶类、分子伴侣、信号转导相关蛋白等.并首次证实clusterin(CLU,又名载脂蛋白J)与IEX-1存在相互作用,两者在细胞中具有很好的共定位.采用RNAi干扰减低骨肉瘤细胞中内源性CLU表达水平,显著抑制了细胞增殖与细胞侵袭能力.为阐明IEX-1在骨肉瘤发生发展中的作用机制提供了重要的线索,为骨肉瘤的早期诊治及预后监测提供了新的靶点.     

The N-1-Naphthylphthalamic Acid-Binding Protein Is an Integral Membrane Protein

N-1-Naphthylphthalmic acid (NPA)-binding protein is a plasmalemma (PM) protein involved in the control of cellular auxin efflux. We re-evaluated the spatial relationship of this protein with the PM of zucchini (Cucurbita pepo L.) hypocotyls. First, Triton X-114 partitioning indicated that the NPA-binding protein was more hydrophobic than most PM proteins. Second, the NPA-binding activity was found to be resistant to proteolytic digestion in membranes. Maximum concentrations of binding sites for NPA were virtually identical in untreated and proteinase K-treated PMs: 19.2 and 20.6 pmol [3H]NPA bound/mg protein, respectively. The insensitivity of the NPA-binding protein was not due to its presence inside tightly sealed vesicles or due to lack of protease activity in the conditions tested. This protein could be made sensitive to proteolytic degradation upon solubilization by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate in the presence of sodium molybdate. Proteinase K treatment decreased the concentration of binding sites to 0.84 pmol [3H]NPA bound/mg protein from 9.2 for untreated, solubilized PM. Third, this activity could not be solubilized by chaotropic agents or sodium carbonate treatment of intact PM. This study indicates that the NPA-binding protein may be an integral membrane protein and contradicts previously reported findings that suggested that this protein was peripheral to the PM.     

The Nuclear Envelope Protein,LAP1B,Is a Novel Protein Phosphatase 1 Substrate

Protein phosphatase 1 (PP1) binding proteins are quintessential regulators, determining substrate specificity and defining subcellular localization and activity of the latter. Here, we describe a novel PP1 binding protein, the nuclear membrane protein lamina associated polypeptide 1B (LAP1B), which interacts with the DYT1 dystonia protein torsinA. The PP1 binding domain in LAP1B was here identified as the REVRF motif at amino acids 55-59. The LAP1B:PP1 complex can be immunoprecipitated from cells in culture and rat cortex and the complex was further validated by yeast co-transformations and blot overlay assays. PP1, which is enriched in the nucleus, binds to the N-terminal nuclear domain of LAP1B, as shown by immunocolocalization and domain specific binding studies. PP1 dephosphorylates LAP1B, confirming the physiological relevance of this interaction. These findings place PP1 at a key position to participate in the pathogenesis of DYT1 dystonia and related nuclear envelope-based diseases.     

Neuregulin Mediates F-actin-driven Cell Migration through Inhibition of Protein Kinase D1 via Rac1 Protein

Neuregulin (NRG; heregulin) is overexpressed in ∼30% of breast cancers and mediates various processes involved in tumor progression, including tumor cell migration and invasion. Here, we show that NRG mediates its effects on tumor cell migration via PKD1. Downstream of RhoA, PKD1 can prevent directed cell migration through phosphorylation of its substrate SSH1L. NRG exerts its inhibitory effects on PKD1 through Rac1/NADPH oxidase, leading to decreased PKD1 activation loop phosphorylation and decreased activity toward SSH1L. The consequence of PKD1 inhibition by NRG is decreased binding of 14-3-3 to SSH1L, localization of SSH1L to F-actin at the leading edge, and increased cofilin activity, resulting in increased reorganization of the actin cytoskeleton and cell motility. Our data provide a mechanism through which the Rho GTPase Rac1 cross-talks with PKD1 signaling pathways to facilitate directed cell migration.     

Banana Ovate Family Protein MaOFP1 and MADS-Box Protein MuMADS1 Antagonistically Regulated Banana Fruit Ripening

The ovate family protein named MaOFP1 was identified in banana (Musa acuminata L.AAA) fruit by a yeast two-hybrid (Y2H) method using the banana MADS-box gene MuMADS1 as bait and a 2 day postharvest (DPH) banana fruit cDNA library as prey. The interaction between MuMADS1 and MaOFP1 was further confirmed by Y2H and Bimolecular Fluorescence Complementation (BiFC) methods, which showed that the MuMADS1 K domain interacted with MaOFP1. Real-time quantitative PCR evaluation of MuMADS1 and MaOFP1 expression patterns in banana showed that they are highly expressed in 0 DPH fruit, but present in low levels in the stem, which suggests that simultaneous but different expression patterns exist for both MuMADS1 and MaOFP1 in different tissues and developing fruits. Meanwhile, MuMADS1 and MaOFP1 expression was highly stimulated and greatly suppressed, respectively, by exogenous ethylene. In contrast, MaOFP1 expression was highly stimulated while MuMADS1 was greatly suppressed by the ethylene competitor 1-methylcyclopropene (1-MCP). These results indicate that MuMADS1 and MaOFP1 are antagonistically regulated by ethylene and might play important roles in postharvest banana fruit ripening.     

蛋白磷酸酯酶2A调节微管相关蛋白1b的磷酸化

微管相关蛋白MAP1b的生物学活性受其磷酸化修饰的调节,后者则受相应的蛋白激酶和蛋白磷酸酯酶(PP)调控.为研究蛋白磷酸酯酶在脑内对MAP1b磷酸化的调控作用,采用有代谢活性的大鼠脑片作为模型,分别应用冈田酸(okadaic acid)和cyclosporin A选择性地抑制PP2A 和PP2B活性,来研究其对脑内蛋白磷酸酯酶MAP1b磷酸化的调控.采用特异性的MAP1bⅠ型磷酸化依赖性抗体522和免疫印迹技术检测MAP1bⅠ型磷酸化.结果表明,当PP2A被okadaic acid选择性抑制后,MAP1bⅠ型磷酸化明显增加.而PP2B被选择性地抑制后,MAP1b磷酸化的变化不大.免疫组化染色显示,MAP1b广泛分布于鼠大脑神经元和突起中,与对照组相比,在PP2A抑制的脑片中抗体522的免疫活性在神经元中明显升高.上述结果表明,PP2A是脑中调控MAP1bⅠ型磷酸化的主要蛋白磷酸酯酶.     

Protein Targeting into Secondary Plastids1

ABSTRACT. Most of the coding capacity of primary plastids is reserved for expressing some central components of the photosynthesis machinery and the translation apparatus. Thus, for the bulk of biochemical and cell biological reactions performed within the primary plastids, many nucleus‐encoded components have to be transported posttranslationally into the organelle. The same is true for plastids surrounded by more than two membranes, where additional cellular compartments have to be supplied with nucleus‐encoded proteins, leading to a corresponding increase in complexity of topogenic signals, transport and sorting machineries. In this review, we summarize recent progress in elucidating protein transport across up to five plastid membranes in plastids evolved in secondary endosymbiosis. Current data indicate that the mechanisms for protein transport across multiple membranes have evolved by altering pre‐existing ones to new requirements in secondary plastids.     

Protein phosphatase-1 and insulin action

Protein Phosphatase-1 (PP-1) appears to be the key component of the insulin signalling pathway which is responsible for bridging the initial insulin-simulated phosphorylation cascade with the ultimate dephosphorylation of insulin sensitive substrates. Dephosphorylations catalyzed by PP-1 activate glycogen synthase (GS) and simultaneously inactivate phosphorylase a and phosphorylase kinase promoting glycogen synthesis. Our in vivo studies using L6 rat skeletal muscle cells and freshly isolated adipocytes indicate that insulin stimulates PP-1 by increasing the phosphorylation status of its regulatory subunit (PP-1_G). PP-1 activation is accompanied by an inactivation of Protein Phosphatase-2A (PP-2A) activity. To gain insight into the upstream kinases that mediate insulin-stimulated PP-1_G phosphorylation, we employed inhibitors of the ras/MAPK, PI3-kinase, and PKC signalling pathways. These inhibitor studies suggest that PP-1_G phosphorylation is mediated via a complex, cell type specific mechanism involving PI3-kinase/PKC/PKB and/or the ras/MAP kinase/Rsk kinase cascade. cAMP agonists such as SpcAMP (via PKA) and TNF- (recently identified as endogenous inhibitor of insulin action via ceramide) block insulin-stimulated PP-1_G phosphorylation with a parallel decrease of PP-1 activity, presumably due to the dissociation of the PP-1 catalytic subunit from the regulatory G-subunit. It appears that any agent or condition which interferes with the insulin-induced phosphorylation and activation of PP-1, will decrease the magnitude of insulin's effect on downstream metabolic processes. Therefore, regulation of the PP-1_G subunit by site-specific phosphorylation plays an important role in insulin signal transduction in target cells. Mechanistic and functional studies with cell lines expressing PP-1_G subunit site-specific mutations will help clarify the exact role and regulation of PP-1_G site-specific phosphorylations on PP-1 catalytic function.     

胶质瘤发病相关蛋白1与肿瘤

胶质瘤发病相关蛋白1（glioma pathogenesis-related protein 1,GLIPR1）隶属于CAP蛋白超家族,由1个指导分泌的信号肽、1个保守的富含半胱氨酸的结构域及1个跨膜结构域组成CAP。近年研究表明,胶质瘤发病相关蛋白1启动子甲基化水平决定了其在胶质瘤和黑色素瘤中的表达上调。而在前列腺癌、肺癌、骨肉瘤以及白血病等肿瘤中表达下调。在不同类型的肿瘤中,GLIPR1通过不同的信号通路参与调控肿瘤的发展进程。本文针对胶质瘤发病相关蛋白1参与调控多种肿瘤发生及发展的机制进行综述,以期为上述肿瘤的诊断与治疗提供重要的线索。     

Protein phosphatase 1 dephosphorylates profilin-1 at Ser-137

Profilin-1 (PFN1) plays an important role in the control of actin dynamics, and could represent an important therapeutic target in several diseases. We previously identified PFN1 as a huntingtin aggregation inhibitor, and others have implicated it as a tumor-suppressor. Rho-associated kinase (ROCK) directly phosphorylates PFN1 at Ser-137 to prevent its binding to polyproline sequences. This negatively regulates its anti-aggregation activity. However, the phosphatase that dephosphorylates PFN1 at Ser-137, and thus activates it, is unknown. Using a phospho-specific antibody against Ser-137 of PFN1, we characterized PFN1 dephosphorylation in cultured cells based on immunocytochemistry and a quantitative plate reader-based assay. Both okadaic acid and endothall increased pS137-PFN1 levels at concentrations more consistent with their known IC(50)s for protein phosphatase 1 (PP1) than protein phosphatase 2A (PP2A). Knockdown of the catalytic subunit of PP1 (PP1Cα), but not PP2A (PP2ACα), increased pS137-PFN1 levels. PP1Cα binds PFN1 in cultured cells, and this interaction was increased by a phosphomimetic mutation of PFN1 at Ser-137 (S137D). Together, these data define PP1 as the principal phosphatase for Ser-137 of PFN1, and provide mechanistic insights into PFN1 regulation by phosphorylation.     

Rotavirus NSP1 Protein Inhibits Interferon-Mediated STAT1 Activation

Rotavirus (RV) replicates efficiently in intestinal epithelial cells (IECs) in vivo despite the activation of a local host interferon (IFN) response. Previously, we demonstrated that homologous RV efficiently inhibits IFN induction in single infected and bystander villous IECs in vivo. Paradoxically, RV also induces significant type I IFN expression in the intestinal hematopoietic cell compartment in a relatively replication-independent manner. This suggests that RV replication and spread in IECs must occur despite exogenous stimulation of the STAT1-mediated IFN signaling pathway. Here we report that RV inhibits IFN-mediated STAT1 tyrosine 701 phosphorylation in human IECs in vitro and identify RV NSP1 as a direct inhibitor of the pathway. Infection of human HT29 IECs with simian (RRV) or porcine (SB1A or OSU) RV strains, which inhibit IFN induction by targeting either IFN regulatory factor 3 (IRF3) or NF-κB, respectively, resulted in similar regulation of IFN secretion. By flow cytometric analysis at early times during infection, neither RRV nor SB1A effectively inhibited the activation of Y701-STAT1 in response to exogenously added IFN. However, at later times during infection, both RV strains efficiently inhibited IFN-mediated STAT1 activation within virus-infected cells, indicating that RV encodes inhibitors of IFN signaling targeting STAT1 phosphorylation. Expression of RV NSP1 in the absence of other viral proteins resulted in blockage of exogenous IFN-mediated STAT1 phosphorylation, and this function was conserved in NSP1 from simian, bovine, and murine RV strains. Analysis of NSP1 determinants responsible for the inhibition of IFN induction and signaling pathways revealed that these determinants are encoded on discrete domains of NSP1. Finally, we observed that at later times during infection with SB1A, there was almost complete inhibition of IFN-mediated Y701-STAT1 in bystander cells staining negative for viral antigen. This property segregated with the NSP1 gene and was observed in a simian SA11 monoreassortant that encoded porcine OSU NSP1 but not in wild-type SA11 or a reassortant encoding simian RRV NSP1.     

Protein phosphatase 1 dephosphorylates Orc2

Phosphorylation of Thr¹¹⁶ and Thr²²⁶ on Orc2, one of the six subunits of the origin recognition complex (ORC), by cyclin A/CDK2 during S phase leads to the dissociation of Orc2, Orc3, Orc4, and Orc5 subunits (Orc2-5) from human chromatin and replication origins. The phosphorylated Orc2 becomes dephosphorylated in the late M phase of the cell cycle. Here we show that protein phosphatase 1 (PP1) dephosphorylates Orc2. Dephosphorylation of Orc2 was accompanied by associating the dissociated Orc subunits with chromatin. Inhibitors of PP1 preferentially inhibited the dephosphorylation of Orc2. The overexpression of the α, β and γ PP1 isoforms decreased the amount of phosphorylated Orc2, and the depletion of these isoforms by RNA interference increased the amount of phosphorylated Orc2. These results suggest that PP1 dephosphorylates Orc2 to promote the binding of ORC to chromatin.