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1.
Mol Biol Cell ; 18(8): 3214-23, 2007 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-17553930

RESUMO

Cadherin-based cell-cell contacts are prominent sites for phosphotyrosine signaling, being enriched in tyrosine-phosphorylated proteins and tyrosine kinases and phosphatases. The functional interplay between cadherin adhesion and tyrosine kinase signaling, however, is complex and incompletely understood. In this report we tested the hypothesis that cadherin adhesion activates c-Src signaling and sought to assess its impact on cadherin function. We identified c-Src as part of a cadherin-activated cell signaling pathway that is stimulated by ligation of the adhesion receptor. However, c-Src has a biphasic impact on cadherin function, exerting a positive supportive role at lower signal strengths, but inhibiting function at high signal strengths. Inhibiting c-Src under circumstances when it is activated by cadherin adhesion decreased several measures of cadherin function. This suggests that the cadherin-activated c-Src signaling pathway serves positively to support cadherin function. Finally, our data implicate PI3-kinase signaling as a target for cadherin-activated c-Src signaling that contributes to its positive impact on cadherin function. We conclude that E-cadherin signaling is an important activator of c-Src at cell-cell contacts, providing a key input into a signaling pathway where quantitative changes in signal strength may result in qualitative differences in functional outcome.


Assuntos
Caderinas/metabolismo , Comunicação Celular , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Transdução de Sinais , Animais , Células CHO , Adesão Celular , Linhagem Celular Tumoral , Cricetinae , Cricetulus , Ativação Enzimática , Humanos , Fosfatidilinositol 3-Quinases/metabolismo
2.
Mol Biol Cell ; 16(10): 4531-42, 2005 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16030252

RESUMO

Classical cadherins accumulate at cell-cell contacts as a characteristic response to productive adhesive ligation. Such local accumulation of cadherins is a developmentally regulated process that supports cell adhesiveness and cell-cell cohesion. Yet the molecular effectors responsible for cadherin accumulation remain incompletely understood. We now report that Myosin 2 is critical for cells to concentrate E-cadherin at cell-cell contacts. Myosin 2 is found at cadherin-based cell-cell contacts and its recruitment requires E-cadherin activity. Indeed, both Myosin 2 recruitment and its activation were stimulated by E-cadherin homophilic ligation alone. Inhibition of Myosin 2 activity by blebbistatin or ML-7 rapidly impaired the ability of cells to concentrate E-cadherin at adhesive contacts, accompanied by decreased cadherin-based cell adhesiveness. The total surface expression of cadherins was unaffected, suggesting that Myosin 2 principally regulates the regional distribution of cadherins at the cell surface. The recruitment of Myosin 2 to cadherin contacts, and its activation, required Rho kinase; furthermore, inhibition of Rho kinase signaling effectively phenocopied the effects of Myosin 2 inhibition. We propose that Myosin 2 is a key effector of Rho-Rho kinase signaling that regulates cell-cell adhesion by determining the ability of cells to concentrate cadherins at contacts in response to homophilic ligation.


Assuntos
Caderinas/fisiologia , Membrana Celular/fisiologia , Junções Intercelulares/metabolismo , Miosina Tipo II/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Azepinas/farmacologia , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Cricetinae , Cricetulus , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Miosina Tipo II/antagonistas & inibidores , Naftalenos/farmacologia , Miosina não Muscular Tipo IIA/metabolismo , Transdução de Sinais
3.
Genome Announc ; 6(26)2018 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-29954890

RESUMO

Maize bacterial leaf streak disease has spread across maize crops in South Africa and therefore potentially poses a threat to maize production and food security. Until recently, this pathogen was identified as a Xanthomonas campestris pathovar, whereas our South African genomes seem to be more divergent and create their own subclade.

4.
Mol Cell Biol ; 23(22): 8124-36, 2003 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-14585972

RESUMO

RanBP type proteins have been reported to increase the catalytic efficiency of the RanGAP-mediated GTPase reaction on Ran. Since the structure of the Ran-RanBP1-RanGAP complex showed RanBP1 to be located away from the active site, we reinvestigated the reaction using fluorescence spectroscopy under pre-steady-state conditions. We can show that RanBP1 indeed does not influence the rate-limiting step of the reaction, which is the cleavage of GTP and/or the release of product P(i). It does, however, influence the dynamics of the Ran-RanGAP interaction, its most dramatic effect being the 20-fold stimulation of the already very fast association reaction such that it is under diffusion control (4.5 x 10(8) M(-1) s(-1)). Having established a valuable kinetic system for the interaction analysis, we also found, in contrast to previous findings, that the highly conserved acidic C-terminal end of RanGAP is not required for the switch-off reaction. Rather, genetic experiments in Saccharomyces cerevisiae demonstrate a profound effect of the acidic tail on microtubule organization during mitosis. We propose that the acidic tail of RanGAP is required for a process during mitosis.


Assuntos
Proteínas Ativadoras de GTPase/química , Proteínas Ativadoras de GTPase/metabolismo , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Proteína ran de Ligação ao GTP/química , Proteína ran de Ligação ao GTP/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Proteínas Ativadoras de GTPase/genética , Humanos , Técnicas In Vitro , Cinética , Substâncias Macromoleculares , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Nucleares/genética , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Schizosaccharomyces/genética , Schizosaccharomyces/metabolismo , Proteínas de Schizosaccharomyces pombe/química , Proteínas de Schizosaccharomyces pombe/genética , Proteínas de Schizosaccharomyces pombe/metabolismo , Deleção de Sequência , Homologia de Sequência de Aminoácidos , Espectrometria de Fluorescência , Proteína ran de Ligação ao GTP/genética
5.
J Mol Biol ; 324(4): 763-74, 2002 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-12460576

RESUMO

Downregulation of small guanine nucleotide-binding proteins (GNBPs) requires the interaction with their corresponding GTPase-activating proteins (GAPs), which increase the slow intrinsic GTPase reaction by several orders of magnitude. On the basis of the structure of H-Ras in complex with the catalytic domain of p120-GAP, we have developed a set of site-specifically labelled Ras-variants, one of which turned out to be particularly sensitive for studying the interaction with Ras-specific GAPs. This specific fluorescent reporter group and the use of manganese to increase the rate of the chemical reaction step allowed us to identify differences in the rate-limiting step of either the GAP-334 or NF1-333 catalyzed reaction. The assay was also applied to study the interaction of the Ras-related protein Rap1B with Rap1GAP, for which no detailed kinetic analysis was available. Single-turnover experiments of this reaction show that the low affinity of the complex (50 microM) is due to a slow association rate as well as a fast dissociation rate. RapGAP promotes AlFx binding to Rap1B, even though it does not contain a catalytic arginine. The rate-limiting step of the RapGAP catalysed reaction is release of inorganic phosphate, which is about five times slower than the chemical cleavage step. Our data reveal marked differences in GAP/target interactions even between closely related systems and suggest that the fluorescent reporter group method might be generally applicable to many other GNBPs and their cognate GAPs.


Assuntos
Corantes Fluorescentes , Proteínas de Ligação ao GTP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Substituição de Aminoácidos , Sítios de Ligação , Catálise , Ativação Enzimática , Escherichia coli/metabolismo , GTP Fosfo-Hidrolases/metabolismo , Guanosina Difosfato/metabolismo , Manganês/química , Conformação Molecular , Mutagênese Sítio-Dirigida , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas p21(ras)/química , Sensibilidade e Especificidade , Espectrometria de Fluorescência , Proteína p120 Ativadora de GTPase , Proteínas rap de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Proteínas ras/metabolismo
6.
Trends Cell Biol ; 25(4): 190-7, 2015 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-25759176

RESUMO

Ras is a major intracellular signaling hub. This elevated position comes at a precarious cost: a single point mutation can cause aberrant signaling. The capacity of Ras for signaling is inextricably linked to its enrichment at the plasma membrane (PM). This PM localization is dynamically maintained by three essential elements: alteration of membrane affinities via lipidation and membrane-interaction motifs; trapping on specific membranes coupled with unidirectional vesicular transport to the PM; and regulation of diffusion via interaction with a solubilization factor. This system constitutes a cycle that primarily corrects for the entropic equilibration of Ras to all membranes that dilutes its signaling capacity. We illuminate how this reaction-diffusion system maintains an out-of-equilibrium localization of Ras GTPases and thereby confers signaling functionality to the PM.


Assuntos
Membrana Celular/fisiologia , Transdução de Sinais , Proteínas ras/fisiologia , Humanos , Mutação , Estrutura Terciária de Proteína , Transporte Proteico
7.
Am J Physiol Cell Physiol ; 292(3): C1061-9, 2007 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-17020937

RESUMO

Classic cadherins function as adhesion-activated cell signaling receptors. On adhesive ligation, cadherins induce signaling cascades leading to actin cytoskeletal reorganization that is imperative for cadherin function. In particular, cadherin ligation activates actin assembly by the actin-related protein (Arp)2/3 complex, a process that critically affects the ability of cells to form and extend cadherin-based contacts. However, the signaling pathway(s) that activate Arp2/3 downstream of cadherin adhesion remain poorly understood. In this report we focused on the Rho family GTPases Rac and Cdc42, which can signal to Arp2/3. We found that homophilic engagement of E-cadherin simultaneously activates both Rac1 and Cdc42. However, by comparing the impact of dominant-negative Rac1 and Cdc42 mutants, we show that Rac1 is the dominant regulator of cadherin-directed actin assembly and homophilic contact formation. To pursue upstream elements of the Rac1 signaling pathway, we focused on the potential contribution of Tiam1 to cadherin-activated Rac signaling. We found that Tiam1 or the closely-related Tiam2/STEF1 was recruited to cell-cell contacts in an E-cadherin-dependent fashion. Moreover, a dominant-negative Tiam1 mutant perturbed cell spreading on cadherin-coated substrata. However, disruption of Tiam1 activity with dominant-negative mutants or RNA interference did not affect the ability of E-cadherin ligation to activate Rac1. We conclude that Rac1 critically influences cadherin-directed actin assembly as part of a signaling pathway independent of Tiam1.


Assuntos
Actinas/metabolismo , Caderinas/metabolismo , Adesão Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Transdução de Sinais/fisiologia , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac de Ligação ao GTP/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Proteína 1 Indutora de Invasão e Metástase de Linfoma de Células T
8.
J Biol Chem ; 281(10): 6471-81, 2006 Mar 10.
Artigo em Inglês | MEDLINE | ID: mdl-16373333

RESUMO

Forced expression of HOXA1 is sufficient to stimulate oncogenic transformation of immortalized human mammary epithelial cells and subsequent tumor formation. We report here that the expression and transcriptional activity of HOXA1 are increased in mammary carcinoma cells at full confluence. This confluence-dependent expression of HOXA1 was abrogated by incubation of cells with EGTA to produce loss of intercellular contact and rescued by extracellular addition of Ca2+. Increased HOXA1 expression at full confluence was prevented by an E-cadherin function-blocking antibody and attachment of non-confluent cells to a substrate by homophilic ligation of E-cadherin increased HOXA1 expression. E-cadherin-directed signaling increased HOXA1 expression through Rac1. Increased HOXA1 expression consequent to E-cadherin-activated signaling decreased apoptotic cell death and was required for E-cadherin-dependent anchorage-independent proliferation of human mammary carcinoma cells. HOXA1 is therefore a downstream effector of E-cadherin-directed signaling required for anchorage-independent proliferation of mammary carcinoma cells.


Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Caderinas/fisiologia , Proteínas de Homeodomínio/fisiologia , Fatores de Transcrição/fisiologia , Adesão Celular/fisiologia , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular/fisiologia , Feminino , Regulação Neoplásica da Expressão Gênica/fisiologia , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Humanos , Microscopia Confocal , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , RNA Mensageiro/metabolismo , Transdução de Sinais/fisiologia , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genética , Proteínas rac1 de Ligação ao GTP/fisiologia
9.
J Biol Chem ; 280(4): 3043-50, 2005 Jan 28.
Artigo em Inglês | MEDLINE | ID: mdl-15556934

RESUMO

Classical cadherin adhesion molecules can function as adhesion-activated cell-signaling receptors. One key target for cadherin signaling is the lipid kinase phosphoinositide (PI) 3-kinase, which is recruited to cell-cell contacts and activated by E-cadherin. In this study, we sought to identify upstream factors necessary for E-cadherin to activate PI 3-kinase signaling. We found that inhibition of tyrosine kinase signaling blocked recruitment of PI 3-kinase to E-cadherin contacts and abolished the ability of E-cadherin to activate PI 3-kinase signaling. Tyrosine kinase inhibitors further perturbed several parameters of cadherin function, including cell adhesion and the ability of cells to productively extend nascent cadherin-adhesive contacts. Notably, the functional effects of tyrosine kinase blockade were rescued by expression of a constitutively active form of PI 3-kinase that restores PI 3-kinase signaling. Finally, using dominant negative Src mutants and Src-null cells, we identified Src as one key upstream kinase in the E-cadherin/PI 3-kinase-signaling pathway. Taken together, our findings indicate that tyrosine kinase activity, notably Src signaling, can contribute positively to cadherin function by supporting E-cadherin signaling to PI 3-kinase.


Assuntos
Caderinas/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Animais , Células CHO , Cricetinae , Ativação Enzimática , Genes Dominantes , Immunoblotting , Imunoprecipitação , Microscopia de Fluorescência , Mutação , Plasmídeos/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Transdução de Sinais , Fatores de Tempo , Tirosina/metabolismo
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