RESUMO
Asgard archaea genomes contain potential eukaryotic-like genes that provide intriguing insight for the evolution of eukaryotes. The eukaryotic actin polymerization/depolymerization cycle is critical for providing force and structure in many processes, including membrane remodeling. In general, Asgard genomes encode two classes of actin-regulating proteins from sequence analysis, profilins and gelsolins. Asgard profilins were demonstrated to regulate actin filament nucleation. Here, we identify actin filament severing, capping, annealing and bundling, and monomer sequestration activities by gelsolin proteins from Thorarchaeota (Thor), which complete a eukaryotic-like actin depolymerization cycle, and indicate complex actin cytoskeleton regulation in Asgard organisms. Thor gelsolins have homologs in other Asgard archaea and comprise one or two copies of the prototypical gelsolin domain. This appears to be a record of an initial preeukaryotic gene duplication event, since eukaryotic gelsolins are generally comprise three to six domains. X-ray structures of these proteins in complex with mammalian actin revealed similar interactions to the first domain of human gelsolin or cofilin with actin. Asgard two-domain, but not one-domain, gelsolins contain calcium-binding sites, which is manifested in calcium-controlled activities. Expression of two-domain gelsolins in mammalian cells enhanced actin filament disassembly on ionomycin-triggered calcium release. This functional demonstration, at the cellular level, provides evidence for a calcium-controlled Asgard actin cytoskeleton, indicating that the calcium-regulated actin cytoskeleton predates eukaryotes. In eukaryotes, dynamic bundled actin filaments are responsible for shaping filopodia and microvilli. By correlation, we hypothesize that the formation of the protrusions observed from Lokiarchaeota cell bodies may involve the gelsolin-regulated actin structures.
Assuntos
Fatores de Despolimerização de Actina/metabolismo , Actinas/metabolismo , Archaea/metabolismo , Proteínas Arqueais/metabolismo , Gelsolina/metabolismo , Fatores de Despolimerização de Actina/química , Fatores de Despolimerização de Actina/genética , Actinas/química , Actinas/genética , Sequência de Aminoácidos , Archaea/química , Archaea/genética , Proteínas Arqueais/química , Proteínas Arqueais/genética , Citoesqueleto/química , Citoesqueleto/genética , Citoesqueleto/metabolismo , Evolução Molecular , Gelsolina/química , Gelsolina/genética , Genoma Arqueal , Polimerização , Conformação Proteica em alfa-Hélice , Alinhamento de SequênciaRESUMO
Dorsal closure (DC) of the Drosophila embryo is a model for the study of wound healing and developmental epithelial fusions, and involves the sealing of a hole in the epidermis through the migration of the epidermal flanks over the tissue occupying the hole, the amnioserosa. During DC, the cells at the edge of the migrating epidermis extend Rac- and Cdc42-dependent actin-based lamellipodia and filopodia from their leading edge (LE), which exhibits a breakdown in apicobasal polarity as adhesions are severed with the neighbouring amnioserosa cells. Studies using mammalian cells have demonstrated that Scribble (Scrib), an important determinant of apicobasal polarity that functions in a protein complex, controls polarized cell migration through recruitment of Rac, Cdc42 and the serine/threonine kinase Pak, an effector for Rac and Cdc42, to the LE. We have used DC and the follicular epithelium to study the relationship between Pak and the Scrib complex at epithelial membranes undergoing changes in apicobasal polarity and adhesion during development. We propose that, during DC, the LE membrane undergoes an epithelial-to-mesenchymal-like transition to initiate epithelial sheet migration, followed by a mesenchymal-to-epithelial-like transition as the epithelial sheets meet up and restore cell-cell adhesion. This latter event requires integrin-localized Pak, which recruits the Scrib complex in septate junction formation. We conclude that there are bidirectional interactions between Pak and the Scrib complex modulating epithelial plasticity. Scrib can recruit Pak to the LE for polarized cell migration but, as migratory cells meet up, Pak can recruit the Scrib complex to restore apicobasal polarity and cell-cell adhesion.
Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Drosophila/metabolismo , Epitélio/metabolismo , Quinases Ativadas por p21/metabolismo , Actinas/metabolismo , Animais , Adesão Celular , Membrana Celular/metabolismo , Polaridade Celular , Drosophila/metabolismo , Epiderme/metabolismo , Integrinas/metabolismo , Pseudópodes/metabolismo , Junções Íntimas/metabolismoRESUMO
Charting the emergence of eukaryotic traits is important for understanding the characteristics of organisms that contributed to eukaryogenesis. Asgard archaea and eukaryotes are the only organisms known to possess regulated actin cytoskeletons. Here, we determined that gelsolins (2DGels) from Lokiarchaeota (Loki) and Heimdallarchaeota (Heim) are capable of regulating eukaryotic actin dynamics in vitro and when expressed in eukaryotic cells. The actin filament severing and capping, and actin monomer sequestering, functionalities of 2DGels are strictly calcium controlled. We determined the X-ray structures of Heim and Loki 2DGels bound actin monomers. Each structure possesses common and distinct calcium-binding sites. Loki2DGel has an unusual WH2-like motif (LVDV) between its two gelsolin domains, in which the aspartic acid coordinates a calcium ion at the interface with actin. We conclude that the calcium-regulated actin cytoskeleton predates eukaryogenesis and emerged in the predecessors of the last common ancestor of Loki, Heim and Thorarchaeota.
Assuntos
Actinas , Cálcio , Citoesqueleto de Actina/metabolismo , Actinas/metabolismo , Archaea/metabolismo , Cálcio/metabolismo , Gelsolina/química , Gelsolina/metabolismoRESUMO
The formation of healthy tissue involves continuous remodeling of the extracellular matrix (ECM). Whilst it is known that this requires integrin-associated cell-ECM adhesion sites (CMAs) and actomyosin-mediated forces, the underlying mechanisms remain unclear. Here, we examine how tensin3 contributes to the formation of fibrillar adhesions (FBs) and fibronectin fibrillogenesis. Using BioID mass spectrometry and a mitochondrial targeting assay, we establish that tensin3 associates with the mechanosensors such as talin and vinculin. We show that the talin R11 rod domain binds directly to a helical motif within the central intrinsically disordered region (IDR) of tensin3, whilst vinculin binds indirectly to tensin3 via talin. Using CRISPR knock-out cells in combination with defined tensin3 mutations, we show (i) that tensin3 is critical for the formation of α5ß1-integrin FBs and for fibronectin fibrillogenesis, and (ii) the talin/tensin3 interaction drives this process, with vinculin acting to potentiate it.
Assuntos
Fibronectinas , Adesões Focais , Talina , Tensinas , Adesão Celular , Matriz Extracelular/metabolismo , Fibronectinas/genética , Fibronectinas/metabolismo , Adesões Focais/genética , Adesões Focais/metabolismo , Integrinas/metabolismo , Talina/genética , Talina/metabolismo , Tensinas/genética , Tensinas/metabolismo , Vinculina/genética , Vinculina/metabolismoRESUMO
Uncapping of actin filaments is essential for driving polymerization and depolymerization dynamics from capping protein-associated filaments; however, the mechanisms of uncapping leading to rapid disassembly are unknown. Here, we elucidated the x-ray crystal structure of the actin/twinfilin/capping protein complex to address the mechanisms of twinfilin uncapping of actin filaments. The twinfilin/capping protein complex binds to two G-actin subunits in an orientation that resembles the actin filament barbed end. This suggests an unanticipated mechanism by which twinfilin disrupts the stable capping of actin filaments by inducing a G-actin conformation in the two terminal actin subunits. Furthermore, twinfilin disorders critical actin-capping protein interactions, which will assist in the dissociation of capping protein, and may promote filament uncapping through a second mechanism involving V-1 competition for an actin-binding surface on capping protein. The extensive interactions with capping protein indicate that the evolutionary conserved role of twinfilin is to uncap actin filaments.
RESUMO
Human PAK4 is an ubiquitously expressed p21-activated kinase which acts downstream of Cdc42. Since PAK4 is enriched in cell-cell junctions, we probed the local protein environment around the kinase with a view to understanding its location and substrates. We report that U2OS cells expressing PAK4-BirA-GFP identify a subset of 27 PAK4-proximal proteins that are primarily cell-cell junction components. Afadin/AF6 showed the highest relative biotin labelling and links to the nectin family of homophilic junctional proteins. Reciprocally >50% of the PAK4-proximal proteins were identified by Afadin BioID. Co-precipitation experiments failed to identify junctional proteins, emphasizing the advantage of the BioID method. Mechanistically PAK4 depended on Afadin for its junctional localization, which is similar to the situation in Drosophila. A highly ranked PAK4-proximal protein LZTS2 was immuno-localized with Afadin at cell-cell junctions. Though PAK4 and Cdc42 are junctional, BioID analysis did not yield conventional cadherins, indicating their spatial segregation. To identify cellular PAK4 substrates we then assessed rapid changes (12') in phospho-proteome after treatment with two PAK inhibitors. Among the PAK4-proximal junctional proteins seventeen PAK4 sites were identified. We anticipate mammalian group II PAKs are selective for the Afadin/nectin sub-compartment, with a demonstrably distinct localization from tight and cadherin junctions.
Assuntos
Junções Intercelulares/metabolismo , Proteínas dos Microfilamentos/genética , Nectinas/genética , Proteômica/métodos , Proteína cdc42 de Ligação ao GTP/genética , Quinases Ativadas por p21/genética , Biotina/química , Carbono-Nitrogênio Ligases/genética , Carbono-Nitrogênio Ligases/metabolismo , Linhagem Celular Tumoral , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Junções Intercelulares/genética , Junções Intercelulares/ultraestrutura , Marcação por Isótopo , Espectrometria de Massas , Proteínas dos Microfilamentos/metabolismo , Nectinas/metabolismo , Osteoblastos/metabolismo , Osteoblastos/ultraestrutura , Ligação Proteica , Mapeamento de Interação de Proteínas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismoRESUMO
GIT1-like proteins are GTPase-activating proteins (GAPs) for Arfs and interact with a variety of signaling molecules to function as integrators of pathways controlling cytoskeletal organization and cell motility. In this report, we describe the characterization of a Drosophila homologue of GIT1, dGIT, and show that it is required for proper muscle morphogenesis and myotube guidance in the fly embryo. The dGIT protein is concentrated at the termini of growing myotubes and localizes to muscle attachment sites in late stage embryos. dgit mutant embryos show muscle patterning defects and aberrant targeting in subsets of their muscles. dgit mutant muscles fail to localize the p21-activated kinase, dPak, to their termini. dPak and dGIT form a complex in the presence of dPIX and dpak mutant embryos show similar muscle morphogenesis and targeting phenotypes to that of dgit. We propose that dGIT and dPak are part of a complex that promotes proper muscle morphogenesis and myotube targeting during embryogenesis.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Desenvolvimento Embrionário , Proteínas Ativadoras de GTPase/metabolismo , Morfogênese , Músculos/embriologia , Homologia de Sequência de Aminoácidos , Alelos , Animais , Adesão Celular , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Embrião não Mamífero/anormalidades , Embrião não Mamífero/enzimologia , Fibras Musculares Esqueléticas/citologia , Fibras Musculares Esqueléticas/metabolismo , Músculos/citologia , Músculos/enzimologia , Mutação/genética , Penetrância , Fenótipo , Transporte Proteico , Transfecção , Asas de Animais/anormalidades , Quinases Ativadas por p21/metabolismoRESUMO
PRL-3 is a metastasis-associated phosphatase. We and others have shown that its overexpression increases cell motility and invasiveness. These phenotypic changes are reminiscent of the epithelial-mesenchymal transition (EMT) that occurs during embryonic development and oncogenesis. The EMT is a complex process that converts epithelia into migratory mesenchymal cells. We here attempt to unravel the underlying mechanistic basis of these phenomena. HeLa cells transiently expressing EGFP-PRL-3 (HeLa-PRL-3) exhibit reduced levels of paxillin. Similarly, Chinese hamster ovary cells stably expressing myc-PRL-3 (CHO-PRL-3) also show marked reductions in paxillin, phosphorylated paxillin-Tyr(31), and vinculin at focal adhesion complexes and notable reductions in the levels of RhoA-GTP, Rac1-GTP, and filamentous-actin filaments. DLD-1 human colorectal cancer cells engineered to express EGFP-PRL-3 (DLD-1-PRL-3) underwent changes consistent with EMT. In these cells, PRL-3 activates Akt and inactivates glycogen synthase kinase-3beta as assessed by phosphospecific antibodies. PRL-3 up-regulates mesenchymal markers fibronectin and Snail and down-regulates epithelial markers E-cadherin, gamma-catenin (plakoglobin), and integrin beta(3), which are major effectors in the EMT pathway. The changes in these EMT characteristics brought about by PRL-3 can be abrogated by the phosphoinositide 3-kinase (PI3K) inhibitor LY294002, implying that PRL-3 acts upstream of PI3K and could play an initiating role to trigger the EMT switch during cancer metastasis. In addition, PRL-3 can down-regulate phosphatase and tensin homologue deleted on chromosome 10, which is an important antagonist of PI3K, further reinforcing PI3K/Akt function in PRL-3-triggered EMT. Catalytically inactive PRL-3 (C104S) was impaired in the above PRL-3-mediated events, indicating that these properties require phosphatase activity. Targeting PRL-3 may thus be a useful strategy to impede cancer cell invasion and metastasis.
Assuntos
Proteínas de Neoplasias/metabolismo , PTEN Fosfo-Hidrolase/biossíntese , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Tirosina Fosfatases/metabolismo , Actinas/metabolismo , Animais , Células CHO , Neoplasias Colorretais/enzimologia , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Cricetinae , Cricetulus , Regulação para Baixo , Células Epiteliais/patologia , Células HeLa , Humanos , Mesoderma/patologia , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , PTEN Fosfo-Hidrolase/genética , Paxilina/metabolismo , Fosforilação , Proteínas Tirosina Fosfatases/biossíntese , Proteínas Tirosina Fosfatases/genética , Transdução de Sinais , Transfecção , Vinculina/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The Paks (p21-activated kinases) Pak1, Pak2 and Pak3 are among the most studied effectors of the Rho-family GTPases, Rac, Cdc42 (cell division cycle 42) and Chp (Cdc42 homologous protein). Pak kinases influence a variety of cellular functions, but the process of Pak down-regulation, following activation, is poorly understood. In the present study, we describe for the first time a negative-inhibitory loop generated by the small Rho-GTPases Cdc42 and Chp, resulting in Pak1 inhibition. Upon overexpression of Chp, we unexpectedly observed a T-cell migration phenotype consistent with Paks inhibition. In line with this observation, overexpression of either Chp or Cdc42 caused a marked reduction in the level of Pak1 protein in a number of different cell lines. Chp-induced degradation was accompanied by ubiquitination of Pak1, and was dependent on the proteasome. The susceptibility of Pak1 to Chp-induced degradation depended on its p21-binding domain, kinase activity and a number of Pak1 autophosphorylation sites, whereas the PIX- (Pak-interacting exchange factor) and Nck-binding sites were not required. Together, these results implicate Chp-induced kinase autophosphorylation in the degradation of Pak1. The N-terminal domain of Chp was found to be required for Chp-induced degradation, although not for Pak1 activation, suggesting that Chp provides a second function, distinct from kinase activation, to trigger Pak degradation. Collectively, our results demonstrate a novel mechanism of signal termination mediated by the Rho-family GTPases Chp and Cdc42, which results in ubiquitin-mediated degradation of one of their direct effectors, Pak1.
Assuntos
Proteínas de Ligação ao GTP/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/fisiologia , Proteínas rho de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Movimento Celular/fisiologia , Proteínas de Ligação ao GTP/genética , Regulação da Expressão Gênica , Humanos , Proteínas de Neoplasias/genética , Fosforilação , Complexo de Endopeptidases do Proteassoma/metabolismo , Ligação Proteica , Proteínas Serina-Treonina Quinases/genética , Linfócitos T/citologia , Linfócitos T/fisiologia , Ubiquitina/metabolismo , Quinases Ativadas por p21 , Proteínas rho de Ligação ao GTP/genéticaRESUMO
The regulation of Rac1 by HACE1-mediated ubiquitination and proteasomal degradation is emerging as an essential element in the maintenance of cell homeostasis. However, how the E3 ubiquitin ligase activity of HACE1 is regulated remains undetermined. Using a proteomic approach, we identified serine 385 as a target of group-I PAK kinases downstream Rac1 activation by CNF1 toxin from pathogenic E. coli. Moreover, cell treatment with VEGF also promotes Ser-385 phosphorylation of HACE1. We have established in vitro that HACE1 is a direct target of PAK1 kinase activity. Mechanistically, we found that the phospho-mimetic mutant HACE1(S385E), as opposed to HACE1(S385A), displays a lower capacity to ubiquitinate Rac1 in cells. Concomitantly, phosphorylation of Ser-385 plays a pivotal role in controlling the oligomerization state of HACE1. Finally, Ser-385 phosphorylated form of HACE1 localizes in the cytosol away from its target Rac1. Together, our data point to a feedback inhibition of HACE1 ubiquitination activity on Rac1 by group-I PAK kinases.
Assuntos
Serina/metabolismo , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Toxinas Bacterianas/farmacologia , Linhagem Celular , Proteínas de Escherichia coli/farmacologia , Células Endoteliais da Veia Umbilical Humana , Humanos , Fosforilação , Multimerização Proteica , Proteômica , Ubiquitinação , Fator A de Crescimento do Endotélio Vascular/farmacologiaRESUMO
The Rho GTPases are involved in many signaling pathways and cellular functions, including the organization of the actin cytoskeleton, regulation of transcription, cell motility, and cell division. The p21 (Cdc42/Rac)-activated kinase PAK mediates a number of biological effects downstream of these Rho GTPases (reviewed by [1]). The phosphorylation state of mammalian PAK is highly regulated: upon binding of GTPases, PAK is potently activated by autophosphorylation at multiple sites, although the mechanisms of PAK downregulation are not known. We now report two PP2C-like serine/threonine phosphatases (POPX1 and POPX2) that efficiently inactivate PAK. POPX1 was isolated as a binding partner for the PAK interacting guanine nucleotide exchange factor PIX. The dephosphorylating activity of POPX correlates with an ability to block the in vivo effects of active PAK. Consonant with these effects on PAK, POPX can also inhibit actin stress fiber breakdown and morphological changes driven by active Cdc42(V12). The association of the POPX phosphatases with PAK complexes may allow PAK to cycle rapidly between active and inactive states; it represents a unique regulatory component of the signaling pathways of the PAK kinase family.
Assuntos
Fosfoproteínas Fosfatases/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae , Sequência de Aminoácidos , Animais , Células COS , Proteínas de Ciclo Celular/metabolismo , Regulação para Baixo , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Dados de Sequência Molecular , Família Multigênica , Fenótipo , Fosfoproteínas Fosfatases/química , Fosfoproteínas Fosfatases/genética , Fosforilação , Ligação Proteica , Proteína Fosfatase 2 , Proteína Fosfatase 2C , Proteínas Serina-Treonina Quinases/genética , Proteínas Recombinantes de Fusão , Fatores de Troca de Nucleotídeo Guanina Rho , Transdução de Sinais , Técnicas do Sistema de Duplo-Híbrido , Proteína cdc42 de Ligação ao GTP/antagonistas & inibidores , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21RESUMO
We demonstrate here that Chinese hamster ovary cells stably expressing PRL-3, a M(r) 20000 prenylated protein tyrosine phosphatase, or its relative, PRL-1, exhibit enhanced motility and invasive activity. A catalytically inactive PRL-3 mutant has significantly reduced migration-promoting activity. We observe that PRL-3 is associated with diverse membrane structures involved in cell movement. Furthermore, we show that PRL-3- and -1-expressing cells, but not control cells, induce metastatic tumor formation in mice. Thus, our results deliver the first evidence for a causative role of PRL-3 and -1 in promoting cell motility, invasion activity, and metastasis.
Assuntos
Movimento Celular/fisiologia , Proteínas Imediatamente Precoces/fisiologia , Neoplasias Experimentais/enzimologia , Neoplasias Experimentais/patologia , Proteínas Tirosina Fosfatases/fisiologia , Animais , Células CHO , Membrana Celular/enzimologia , Membrana Celular/fisiologia , Cricetinae , Feminino , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Camundongos , Camundongos Nus , Invasividade Neoplásica , Metástase Neoplásica , Proteínas Tirosina Fosfatases/genética , Proteínas Tirosina Fosfatases/metabolismo , Proteínas Proto-Oncogênicas c-myc/biossíntese , Proteínas Proto-Oncogênicas c-myc/genéticaRESUMO
Rho GTPases play pivotal roles in regulating cell morphology. We previously showed that RhoA acts via ROKalpha to counteract the effects of the classical second messenger cyclic AMP on cell shape changes. Here we show that active Cdc42V12 also competes against the cAMP-induced stellate morphology in SH-EP cells. This Cdc42 effect is not mediated by the RhoA/ ROK pathway but rather the related MRCKalpha, a myotonic dystrophy kinase-related Cdc42-binding kinase. Co-expression of a dominant inhibitory MRCKalpha mutant with Cdc42V12 blocks the ability of the GTPase to counteract cAMP, suggesting that MRCK acts downstream of Cdc42 in this process. Cdc42V12 enhances the phosphorylation of myosin light chain (MLC) at the cell periphery and sustains focal adhesion complexes, while MLC kinase inhibitors destroy focal adhesion complexes and impair the Cdc42V12 protective effect. The data suggest that the maintenance of focal adhesion complexes via the regulation of myosin II activity underlies the ability of Cdc42 to protect against the effect of elevated cAMP.
Assuntos
AMP Cíclico/metabolismo , Cadeias Leves de Miosina/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , ADP Ribose Transferases/metabolismo , Animais , Toxinas Botulínicas/metabolismo , Linhagem Celular , Tamanho Celular , Colforsina/metabolismo , Adesões Focais/metabolismo , Humanos , Imuno-Histoquímica , Microinjeções , Mutação , Quinase de Cadeia Leve de Miosina/antagonistas & inibidores , Quinase de Cadeia Leve de Miosina/metabolismo , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína cdc42 de Ligação ao GTP/genética , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
PURPOSE: Epithelial cell migration is required for regeneration of tissues and can be defective in a number of ocular surface diseases. This study aimed to determine the expression pattern of Rho family small G-proteins in human corneal epithelial cells to test their requirement in directional cell migration. METHODS: Rho family small G-protein expression was assessed by reverse transcription-polymerase chain reaction. Dominant-inhibitory constructs encoding Rho proteins or Rho protein targeting small interfering RNA were transfected into human corneal epithelial large T antigen cells, and wound closure rate were evaluated by scratch wounding assay, and a complementary non-traumatic cell migration assay. Immunofluorescence staining was performed to study cell polarization and to assess Cdc42 downstream effector. RESULTS: Cdc42, Chp, Rac1, RhoA, TC10 and TCL were expressed in human corneal epithelial cells. Among them, Cdc42 and TCL were found to significantly affect cell migration in monolayer scratch assays. These results were confirmed through the use of validated siRNAs directed to Cdc42 and TCL. Scramble siRNA transfected cells had high percentage of polarized cells than Cdc42 or TCL siRNA transfected cells at the wound edge. We showed that the Cdc42-specific effector p21-activated kinase 4 localized predominantly to cell-cell junctions in cell monolayers, but failed to translocate to the leading edge in Cdc42 siRNA transfected cells after monolayer wounding. CONCLUSION: Rho proteins expressed in cultured human corneal epithelial cells, and Cdc42, TCL facilitate two-dimensional cell migration in-vitro. Although silencing of Cdc42 and TCL did not noticeably affect the appearance of cell adhesions at the leading edge, the slower migration of these cells indicates both GTP-binding proteins play important roles in promoting cell movement of human corneal epithelial cells.
Assuntos
Movimento Celular , Polaridade Celular , Epitélio Corneano/citologia , Proteínas rho de Ligação ao GTP/metabolismo , Inativação Gênica , Humanos , Transporte Proteico , RNA Interferente Pequeno/genética , Proteína cdc42 de Ligação ao GTP/deficiência , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rho de Ligação ao GTP/deficiência , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.
Assuntos
Coanoflagelados/citologia , Coanoflagelados/enzimologia , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Ligantes , Dados de Sequência Molecular , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Ligação Proteica , Estrutura Terciária de Proteína , Especificidade por SubstratoRESUMO
Fractionation of brain extracts and functional biochemical assays identified PP2Calpha, a serine/threonine phosphatase, as the major biochemical activity inhibiting PAK1. PP2Calpha dephosphorylated PAK1 and p38, both of which were activated upon hyperosmotic shock with the same kinetics. In comparison to growth factors, hyperosmolality was a more potent activator of PAK1. Therefore we characterize the PAK signaling pathway in the hyperosmotic shock response. Endogenous PAKs were recruited to the p38 kinase complex in a phosphorylation-dependent manner. Overexpression of a PAK inhibitory peptide or dominant negative Cdc42 revealed that p38 activation was dependent on PAK and Cdc42 activities. PAK mutants deficient in binding to Cdc42 or PAK-interacting exchange factor were not activated. Using a panel of kinase inhibitors, we identified PI3K acting upstream of PAK, which correlated with PAK repression by pTEN overexpression. RNA interference knockdown of PAK expression reduced stress-induced p38 activation and conversely, PP2Calpha knockdown increased its activation. Hyperosmotic stress-induced PAK translocation away from focal adhesions to the perinuclear compartment and resulted in disassembly of focal adhesions, which are hallmarks of PAK activation. Inhibition of PAK by overexpression of PP2Calpha or the kinase inhibitory domain prevented sorbitol-induced focal adhesion dissolution. Inhibition of MAPK pathways showed that MEK-ERK signaling but not p38 is required for full PAK activation and focal adhesion turnover. We conclude that 1) PAK plays a required role in hyperosmotic signaling through the PI3K/pTEN/Cdc42/PP2Calpha/p38 pathway, and 2) PAK and PP2Calpha modulate the effects of this pathway on focal adhesion dynamics.
Assuntos
Adesões Focais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Sistema de Sinalização das MAP Quinases/fisiologia , PTEN Fosfo-Hidrolase/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Fosfatase 2/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Animais , Células COS , Chlorocebus aethiops , Ativação Enzimática/fisiologia , Adesões Focais/genética , Fatores de Troca do Nucleotídeo Guanina/genética , Células HeLa , Humanos , Pressão Osmótica , PTEN Fosfo-Hidrolase/genética , Fosfatidilinositol 3-Quinases/genética , Proteína Fosfatase 2/genética , Estrutura Terciária de Proteína/fisiologia , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho , Proteína cdc42 de Ligação ao GTP/genética , Quinases Ativadas por p21/genética , Proteínas Quinases p38 Ativadas por Mitógeno/genéticaRESUMO
Beclin1 has a key regulatory role in the initiation of autophagy and is a tumor suppressor. We have examined the interplay between viral or human Bcl-2-like proteins and UVRAG and their opposite effects on Beclin1. We show that Beclin1 forms a dimer in solution via its coiled-coil domain both in vivo and in vitro. Viral Bcl-2 binds independently to two sites on the Beclin1 dimer, one with high affinity and one with lower affinity, whereas human Bcl-x(L) binds both sites equally with relatively low affinity. UVRAG disrupts the Beclin1-dimer interface, forming a heterodimer with Beclin1, suggesting that this is how UVRAG causes its effects on Beclin1 to activate autophagy. Both Bcl-2-like proteins reduce the affinity of UVRAG for Beclin1 approximately 4-fold, suggesting that they stabilize the Beclin1 dimer. Moreover, coimmunoprecipitation assays show that UVRAG substantially reduces Beclin1 dimerization in vivo. These data explain the concentration-dependent interplay between Bcl-2, UVRAG, and Beclin1, as both tumor suppressors, UVRAG and Beclin1, have single-copy mutations in human cancers. Furthermore, our data suggest that an alternative strategy for developing anti-cancer compounds would be to disrupt the Beclin1-dimer interface.
Assuntos
Proteínas Reguladoras de Apoptose/química , Proteínas de Membrana/química , Proteínas Supressoras de Tumor/metabolismo , Proteína bcl-X/metabolismo , Animais , Proteína Beclina-1 , Sítios de Ligação , Células COS , Calorimetria , Chlorocebus aethiops , Dimerização , Humanos , Modelos Biológicos , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , TermodinâmicaRESUMO
Rho GTPases and their downstream effectors regulate changes in the actin cytoskeleton that underlie cell motility and adhesion. They also participate, with RhoA, in the regulation of gene transcription by activating serum response factor (SRF)-mediated transcription from the serum response element (SRE). SRF-mediated transcription is also promoted by several proteins that regulate the polymerization or stability of actin. We have previously identified a family of PP2C phosphatases, POPXs, which can dephosphorylate the CDC42/RAC-activated kinase PAK and downregulate its enzymatic and actin cytoskeletal activity. We now report that POPX2 interacts with the formin protein mDia1 (DIAPH1). This interaction is enhanced when mDia1 is activated by RhoA. The binding of POPX2 to mDia1 or to an mDia-containing complex greatly decreases the ability of mDia1 to activate transcription from the SRE. We propose that the interaction between mDia1 and POPX2 (PPM1F) serves to regulate both the actin cytoskeleton and SRF-mediated transcription, and to link the CDC42/RAC1 pathways with those of RhoA.
Assuntos
Proteínas de Transporte/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Transdução de Sinais , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Transporte Biológico , Proteínas de Transporte/genética , Forminas , Imunoprecipitação , Camundongos , Modelos Biológicos , Células NIH 3T3 , Fosfoproteínas Fosfatases/genética , Ligação Proteica , RNA Interferente Pequeno/genética , Fator de Resposta Sérica/genética , Fator de Resposta Sérica/metabolismo , Fibras de Estresse/metabolismo , Transcrição GênicaRESUMO
BACKGROUND: Intermediate filament (IF) is one of the three major cytoskeletal filaments. Vimentin is the most widely expressed IF protein component. The Rho family of small GTPases, such as Cdc42, Rac and Rho, are thought to control the organization of actin filaments as well as other cytoskeletal filaments. RESULTS: We determined if the vimentin filaments can be regulated by p21-activated kinase (PAK), one of targets downstream of Cdc42 or Rac. In vitro analyses revealed that vimentin served as an excellent substrate for PAK. This phosphorylated vimentin lost the potential to form 10 nm filaments. We identified Ser25, Ser38, Ser50, Ser65 and Ser72 in the amino-terminal head domain as the major phosphorylation sites on vimentin for PAK. The ectopic expression of constitutively active PAK in COS-7 cells induced vimentin phosphorylation. Fibre bundles or granulates of vimentin were frequent in these transfected cells. However, the kinase-inactive mutant induced neither vimentin phosphorylation nor filament reorganization. CONCLUSION: Our observations suggest that PAK may regulate the reorganization of vimentin filaments through direct vimentin phosphorylation.