mrck与cdc42的一些资料

关于 MRCKbata 和 Cdc42 两者之间关系（来自丁香通）： Protein kinases comprise a large group of encoded factors that regulate cellular processes by catalyzing the transfer of a phosphate group to a hydroxyl（羟基，氢氧基） acceptor in serine, threonine or tyrosine residues. Myotonic dystrophy kinaserelated Cdc42-binding (DMPK-like) kinases- and (MRCK-, ) contain a cysteine-rich motif and a putative pleckstrin homology domain. MRCKs can phosphorylate nonmuscle Myosin light chain and influences Actin-Myosin contractility（收缩性， 伸缩力）. MRCK- can phosphorylate and activate LIM kinases downstream of Cdc42, which leads to inactivation of ADF/Cofilin and to Actin cytoskeletal reorganization. MRCK- can also influence neurite outgrowth promoted by Cdc42 and Rac. Myotonic Dystrophy（肌强制性营养不良） Kinase-Related cdc42-binding kinase beta (MRCKb) belongs to the DMPK subfamily. The myotonic dystrophy kinase-related kinases and myotonic dystrophy kinase-related Cdc42 binding kinase (MRCK) are effectors of RhoA and Cdc42, respectively, where they are involved in actin cytoskeletal reorganization and neurite（神 经突） outgrowth. Effects of the repeat expansion on the DMPK gene may be responsible for muscle and heart features of Myotonic Dystrophy. Characterization of a monoclonal antibody panel shows that the myotonic dystrophy protein kinase, DMPK, is expressed almost exclusively in muscle and heart. Hum. Mol. Genet. 9 , 2167-2173, (2000) Abstract Tan, I. et al., Phosphorylation of a novel myosin binding subunit of protein phosphatase 1 reveals a conserved mechanism in the regulation of actin cytoskeleton. J. Biol. Chem. 276 , 21209-21216, (2001) Abstract 细胞体前移的过程受到小 Rho GTP 酶 Cdc42, Rac 和 RhoA 的调控。而这些 蛋白相互之间却是拮抗的关系。RhoA 激活 Rho-激酶（又名 ROCK），后者在 激活状态下会将肌球蛋白轻链磷酸酶（myosin light chain phosphatase，简称 MLC 磷酸酶。该酶为 MLC 去掉磷酸基团）磷酸化，就是使它失活了，导致细 胞的收缩增强。Cdc42 也是通过 MRCK 起到类似的作用。与它们相反的是， Rac 会激活 PAK，PAK 能磷酸化 MLC 激酶，使之失活，后果是细胞收缩力减 退，扩展受阻。但是 PAK 也能直接磷酸化 MLC，增加细胞收缩能力。究竟是 哪种作用占优，取决于 PAK 的空间分布和它的活性调节水平 在肌球蛋白介导的胞体收缩过程中，RhoA 和 Rac 通过 Rho-激酶和 PAK 可 以调节 MLC 的磷酸化，这也为尾部粘着斑解聚做出贡献 MRCK 和 ROCK（Rho 相关激酶）共同作用，在 BE 细胞的移动中起作用,维 持细胞形态伸长，在细胞中肌球蛋白收缩中起作用；在肌动蛋白轻链上磷酸化 从而使 Cdc42 在细胞形状改变时对抗 cAMP 的作用； MRCK alpha 和 beta 是新 近鉴定出的 Cdc42 结合 serine/threonine 激酶，在希拉细胞肌动蛋白识别和丝状 伪足形成中起作用 Screening of Aptamers and selective inhibitors for myotonic dystrophy kinase-related CDC42-Binding Kinase (MRCK) by CE-SELEX and Chemical inhibition ABSTRACT: MRCK, a Rac/Cdc42-binding kinase, regulates lamellar actomyosin retrograde flow during membrane protrusion and cell migration. MRCK inhibition therefore affects cell motility. As no known selective MRCK inhibitor is available, screening for MRCK inhibitors is thus required. In this project, aptamers and inhibitors for MRCK were screened. Aptamers, short DNA/RNA sequences which able to bind different targets, can be used as molecular probe for protein detection. Systematic evolution of ligands by exponential enrichment(SELEX) combined with capillary electrophoresis(CE) was used for aptamers selection. MRCK aptamers were selected from and compared between an original method and a modified approach. Concurrently, around 170 chemicals were also screened for MRCK inhibition. Chelerythrine chloride shown to give MRCK inhibition with IC50=0.86 B5M. Weaker inhibition was observed with ROK(IC50=8.5 B5M) and Citron Kinase(IC50=6.5 B5M). Chelerythrine(5 B5M) was able to perturb MRCK in vivo localization. Reduced cell migration was also observed in chelerythrine treatment in wound healing assay. 多克隆 ZO-1 抗体 紧密连接是上皮细胞的细胞间粘附结构，与黏着连接和桥粒组成了上皮连接复 合物。 紧密连接将细胞封闭起来，从而形成阻止溶质透过细胞表面扩散的主要 屏障，而且还起到分隔顶端膜区和基侧膜区以产生细胞极性的作用。 1. 紧密连接绞链 (tignt junction strands) 主要由 claudin、occludin 和 JAM 组成。 2. 支架蛋白 ZO-1、ZO-2 和 ZO-3 在连接复合物的细胞质面聚集，决定着 这些连接的特化和定位。 The zona occludens (ZO) proteins form the plaque structures underlying plasma membranes together with proteins such as cingulin, symplekin, the Par-3/Par-6/atypical protein kinase C complex, ZONAB, and guanine nucleotide exchange factor-H1/Lfc. 3. 三种 ZO 蛋白都具有三个 PDZ 结构域、一个 Src 同源 3 结构域、和一 个表明这三种蛋白为膜相关鸟苷酸激酶样同源体 (MAGUK) 的特征性鸟苷 酸激酶样同源结构域。 4. 最近有研究表明，ZO-1 的低表达与乳腺癌患者的不良预后相关。 5. 另一项研究发现，滑膜肉瘤标本中存在 ZO-1 异常表达。 6. ZO-1 抗体是细胞间紧密连接的有效标志物，因为 ZO-1 仅仅存在于细胞 间紧密连接处。 Immunofluorescent co-localization (yellow) of ZO-1 (green) and Occludin (red) in Caco-2 (top row) and MDCK II (bottom row) cells using Zymeds Rb anti-ZO-1 (Mid, Cat. No. 40-2200). 此图片由 University of North Carolina (Chapel Hill) 的 Jacey Bennis 和 James Anderson 博士惠赠。 现在就试用 Zymed 的新型多克隆抗体，对紧密连接进行前所未有的高特异性标 记！ 使用 Zymed 的 Rb anti-ZO-1 抗体 （N 端，目录号 40-2300）对小鼠心 脏组织中的血管进行免疫荧光染色。 图片提供：Dr. J Western blot analysis of (A) MDCKII, (B) A431, (C) Caco-2, (D) Rat-1, and (E) NRK-52E cell lysates using Zymeds Rb anti-ZO- 1 (Mid, Cat. No. 40-2200) ZO-1 and ZO-2 are major PDZ-domain-containing TJ proteins and bind directly to claudins, 关于 cdc 42 的 （乱七八糟） 研究发现 Cdc42在肝脏再生过程中调控的新机制 近期，国际著名期刊 肝脏病学 （Hepatology. 2009 Jan;49(1):240-9）发表了上海生科院生化与细胞所陈正军组关于 Cdc42影响肝脏再生过程的最新研究成果。 Cdc42蛋白是 Rho GTP 酶家族中的一员，它是细胞内一种十分重要的蛋白质，担负着调节细胞骨架结构、 细胞生长、细胞极性以及细胞内运输等多种功能。然而目前对 Cdc42在哺乳动物肝脏中的作用仍然知之甚少。陈正军 组的袁海心等利用肝脏2/3切除手术模型，研究了 Cdc42基因敲除对肝损伤后再生过程的影响。 Cdc42在对照小鼠肝脏 部分切除后的3 小时-24小时显著激活，证明其参与了肝脏再生过程。而在缺失 Cdc42蛋白后，小鼠肝脏的再生能力明 显下降，主要表现为肝重回复的延迟。对分子机理的研究进一步显示在肝再生过程中，缺失 Cdc42的小鼠肝脏 DNA 合 成水平显著下降，并且细胞周期因子的表达以及数条与 Cdc42相关的重要生长信号通路（如 ERK、JNK 和 p70S6K） 的激活发生延迟。此外，Cdc42的缺失还影响了脂肪转运蛋白 ABCA1的细胞内定位，造成肝再生过程中脂肪代谢的异 常。该项研究揭示了 Cdc42在肝脏再生过程中担负的重要功能，Cdc42在肝细胞中的功能调控是一个复杂的网络，可 能涉及到多种不同的细胞内功能和复杂的信号分子通路，对这些问题的阐释对于我们更深入理解肝脏的生理功能和发 病机理具有重要的指导意义。 Cdc42 Regulates Adherens Junction Stability and Endothelial Permeability by Inducing -Catenin Interaction With the Vascular Endothelial Cadherin Complex Abstract The endothelial adherens junctions (AJs) consist of trans-oligomers of membrane spanning vascular endothelial (VE)-cadherin proteins, which bind -catenin through their cytoplasmic domain. -Catenin in turn binds -catenin and connects the AJ complex with the actin cytoskeleton. We addressed the in vivo effects of loss of VE-cadherin interactions on lung vascular endothelial permeability and the role of specific Rho GTPase effectors in regulating the increase in permeability induced by AJ destabilization. We used cationic liposomes encapsulating the mutant of VE-cadherin lacking the extracellular domain (EXD) to interfere with AJ assembly in mouse lung endothelial cells. We observed that lung vascular permeability (quantified as microvessel filtration coefficient Kf,c) was increased 5-fold in lungs expressing EXD. This did not occur to the same degree on expression of the VE- cadherin mutant, EXD, lacking the -catenineCbinding site. The increased vascular permeability was the result of destabilization of VE-cadherin homotypic interaction induced by a shift in the binding of -catenin from wild-type VE- cadherin to the expressed EXD mutant. Because EXD expression in endothelial cells activated the Rho GTPase Cdc42, we addressed its role in the mechanism of increased endothelial permeability induced by AJ destabilization. Coexpression of dominant-negative Cdc42 (N17Cdc42) prevented the increase in Kf,c induced by EXD. This was attributed to inhibition of the association of -catenin with the EXDeC-catenin complex. The results demonstrate that Cdc42 regulates AJ permeability by controlling the binding of -catenin with -catenin and the consequent interaction of the VE-cadherin/catenin complex with the actin cytoskeleton. Key Words: adhesion molecules gene transfer catenins Cdc42 VE-cadherin Introduction Adherens junctions (AJs) are dynamic structures mediating endothelial celleCcell adhesion1 and thereby regulate endothelial barrier function and tissue fluid homeostasis.2,3 Endothelial cell adhesion to contiguous cells, forming the endothelial monolayer, is determined in part by the transmembrane protein vascular endothelial (VE)-cadherin, which forms homotypic adhesive interactions2,4 and binds 2 members of the catenin family of cytosolic proteins ( and catenins).5 -Catenin binds -catenin and connects junctional cadherins with the actin cytoskeleton. Permeability-increasing mediators such as histamine and thrombin increase junctional permeability in part by inducing the disassembly of AJs.6eC8 The end result is a shift in fluid and plasma proteins to the extravascular space and development of protein-rich tissue edema. Phosphorylation of AJ proteins contributes to the mechanism of destabilization of AJs.7 Studies showed that histamine induced the phosphorylation of VE-cadherin and - and -catenins within 60 sec consistent with the rapidly increased microvascular permeability secondary to the formation of 100 to 400 nm-wide interendothelial GAPs.8 It was also shown that thrombin-induced activation of PKC regulated the phosphorylation of VE-cadherin and catenins and thereby contributed to disruption of AJs and the increase endothelial permeability.9 Phosphatases such as protein tyrosine phosphatase VE-PTP may also regulate the phosphorylation of VE-cadherin/catenin components10 and thus AJ integrity. In addition, Rho GTPases RhoA, Rac1, and Cdc42 are important in regulating AJ assembly.11,12 Inhibition of RhoA prevented both thrombin- and histamine-induced disassembly of AJs13 and the increase in endothelial permeability. RhoA is also directly linked to AJs through p120 catenin, a Src substrate that binds to the juxta-membrane domain of VE-cadherin.14 Overexpression of p120 catenin led to inhibition of RhoA in a GDP dissociation inhibitoreClike manner.15 Up- or downregulation of p120 catenin in endothelial cells influenced VE- cadherin levels in endothelial cells.16 In addition, activation of Cdc42 and Rac1 regulated the post-Golgi transport of endothelial cadherin to the AJs.17 Other studies showed that Cdc42 can signal the activation of actin polymerization at the plasma membrane leading to formation of filopodia18 and thus contributing to assembly of AJs.19 Cdc42 and Rac1 may also affect AJ function through their interactions with the scaffold protein IQGAP1.20,21 Although the role of IQGAP1 in regulating AJ assembly has not been studied in endothelial cells, it was shown to colocalize with members of the AJ complex in other cell types21 and thus may be an important determinant of junctional permeability downstream of Cdc42 and Rac1. In the present study, we addressed the role of Cdc42 in the mechanism of increased endothelial permeability. Studies were made in the mouse lung microcirculation, so that inferences could be drawn concerning the in vivo regulation of endothelial permeability. We used the VE-cadherin mutant lacking the extracellular domain (EXD) and another mutant lacking both extracellular domain and cytosolic distal -catenineCbinding domain (EXD).22 We tested the hypothesis that expression of EXD promotes a shift in the VE-cadherineCbound -catenin as well as -catenin toward the expressed mutant and thus would destabilize AJs. Because studies have shown that expression of EXD in endothelial cells induced Cdc42 activation,22 we also addressed whether destabilization of AJs secondary to the loss of VE-cadherin homotypic interaction increases endothelial permeability through a Cdc42-dependent mechanism. We observed an increase in lung microvascular permeability on EXD expression, which did not occur to the same degree with EXD expression. The increased endothelial permeability was the result of loss of normal VE-cadherin homotypic interaction induced by the binding of -catenin and -catenin to the expressed EXD mutant. We observed that coexpression of the dominant-negative (dn) Cdc42 mutant (N17Cdc42) significantly reduced the increase in lung vascular permeability induced by EXD. This was the result of preventing the association of -catenin with the EXDeC- catenin complex. Thus, activation of Cdc42 plays an important role in the mechanism of EXD-induced AJ disruption and increased endothelial permeability by promoting the interaction of -catenin with the EXDeC-catenin complex. Materials and Methods VE-Cadherin and Rho GTPase Mutants The plasmid vector pcDNA3 was cleaved at its multicloning site and specific cDNAs corresponding to truncated sequences of VE-cadherin were inserted downstream of cytomegalovirus promoter. We constructed, as described,22 a VE-cadherin mutant lacking the extracellular domain (EXD) and a mutant lacking both the extracellular domain and cytosolic distal -catenineCbinding domain (EXD) (Figure 1A). Vector-control studies were performed with empty pcDNA3 plasmid vector (Invitrogen, Carlsbad, Calif). Cytomegalovirus-driven dominant active Cdc42 (V12), dnCdc42 (N17), Rac1 (N17), and RhoA (N19) plasmid DNAs were obtained from Drs Tohru Kozasa and Tatyana Voyno- Yasenetskaya (University of Illinois, Chicago). Cell Culture and Transfection HMEC-1 (Human Microvascular Endothelial Cells) and HPAEC (Human Pulmonary Artery Endothelial Cells) were grown22 and transfected by electroporation as described in the online data supplement, available at . Immunofluorescence and Confocal Microscopy Endothelial cells were either seeded onto glass coverslips posttransfection and allowed to grow for 24 hours in complete medium or freshly obtained from collagenase-digested lungs and plated onto coverslips for 1.5 hour (described below). Cells were immunostained and visualized as described23 in the online data supplement. Preparation of Cationic Liposomes for In Vivo Studies Liposomes composed of dimethyldioctadecyl ammonium bromide (Sigma) in a 1:1 molar ratio with cholesterol (Sigma) were prepared as described,24eC28 except that the dried lipid film was resuspended in 5% dextrose in water and then sonicated for 20 minutes, followed by incubation at 42C for 20 minutes and 0.45-e filtration. Liposomes were extruded through a 50-nm pore polycarbonate filter (Avestin). CD1 mice (Charles River, Wilmington, Mass), weighing 20 to 25 g, were injected IV with 50 e of plasmid DNA, which was mixed with 100 e 蘈 of liposome suspension and allowed to equilibrate for 20 minutes before injection. Protein expression constructs was assessed 24 hours thereafter. All experimental procedures complied with Institutional and National Institutes of Health guidelines for animal use, and approvals were obtained from the Institutional Animal Care Committee. Lung Endothelial Fractionization CD1 mice were anesthetized29 and excised lungs were perfused with Hanks balanced salt solution through the pulmonary artery for 5 minutes, then with endothelial fractionation buffer (0.2% Triton X-100, 50 mmol/L Tris-Cl pH 7.9, 1x Protease Inhibitor Cocktail, Phosphatase Inhibitor Cocktails 1 and 2, Sigma) at 0.5 mL/min for 30 sec. Eluate was collected from the left atrial cannula (200 e 蘈) for Western blotting, and remaining lung tissue was homogenized in tissue lysis buffer (1.5% Triton X-100, 0.1% sodium dodecyl sulfate, 0.5% dideoxycholate, 100 mmol/L phenylmethylsulfonyl fluoride, 1x Protease Inhibitor Cocktail in PBS), followed by centrifugation at 3000g at 4C for 10 minutes to remove insoluble material. Remaining lung tissue was compared with endothelial-rich lysate by Western blotting using endothelial cell markers (eg, angiotensin-converting enzyme, VE- cadherin). Endothelial-rich lysate stained strongly positive for endothelial antigens (data not shown); the purity of this lysate was 90%. Isolation of Endothelial Cells by Collagenase Digestion of Mouse Lungs To assess the expression of transfected proteins in endothelial cells, mice were euthanized 20 hours postinjection of liposomeeCDNA mixture. Lungs were perfused as above, diced into 2-mm cubes and transferred to 1% collagenase A (Roche)/Hanks balanced salt solution (Gibco) and mixed at 37C for 1 hour. Lung tissue was aspirated 10x through a serological pipette and allowed to settle at room temperature. The supernatant was removed and centrifuged at 3000g for 1 minute. The supernatant was discarded, and cells were resuspended in modified EGM-2 endothelial cell medium (penicillin/streptomycin instead of gentamicin/amphotericin B; Clonetics), and allowed to adhere to gelatinized coverslips for 1.5 hour. Cells were subsequently analyzed by immunofluorescence. Pulmonary Microvascular Filtration Coefficient and Isogravimetric Lung Water Determinations CD1 mice were anesthetized and lungs were removed, ventilated, and perfused ex vivo using our methods2,29 to obtain stable pulmonary artery pressure (7 cm H2O) and lung wet weight over a minimum of a 90-minute period. Microvessel filtration coefficient (Kf,c) was measured by applying a 10 cm H2O brief step increase in left atrial pressure at 20 minutes postextraction (during the isogravimetric period).2,29 Immunoprecipitation and Western Blotting Cell and tissue lysates were subjected to immunoprecipitation and Western blotting as described in the online data supplement. Cdc42 Activation Assay Cdc42 activity was measured using the Cdc42 activation assay Biochem Kit (BK034, Cytoskeleton, Denver, Colo). Whole cell lysates were also analyzed for Cdc42 expression and total protein. Data Analysis Data were analyzed using the 2-tailed Students t test as well as ANOVA. Values are reported as meanSEM. Values were considered significant at P0.05. Densitometry measurements of Western blots were performed using the ImageJ program (NIH). Results Expression of EXD in Junctions of Lung Endothelial Cells In Situ Figure 1B shows the expression of FLAG-EXD as well as myc-N17Cdc42 in mouse lung endothelial cells. Endothelial cell-enriched lysates from mouse lungs were analyzed for expression of the constructs. The second and third lanes show the expression of FLAG-EXD (36 kDa), and the third lane shows the coexpression of myc- N17Cdc42 (25 kDa) (Figure 1B). Expression of FLAG-EXD construct did not affect the expression of wild-type VE- cadherin and Cdc42. Figure 1C shows the expression of EXD in lung endothelial cells isolated from the liposomeeCDNAeCinjected mice. Lung endothelial cells were obtained 24 hours postinjection of con