RESUMO
The circular waveguide aperture or open-end radiator, one of the canonical antenna elements, can be filled with a dielectric material for miniaturization. With dielectric filling, the aperture reflection increases and impedance matching is necessary. This paper presents a simple but innovative simulation-based approach to the aperture matching of a dielectric-filled circular waveguide aperture. By properly loading the aperture with two- or three-section dielectric rings, the impedance matching is possible over a wide frequency range starting slightly above the TE11-mode cutoff and continuing upward. The material for the aperture matching is the same as that filling the waveguide. The proposed matching structure is analyzed and optimized using a simulation tool for the dielectric constant εr of the filling material ranging from 1.8 to 10. For εr ≥ 5, the unmatched reflection coefficient ranges from -6.0 dB to -0.9 dB while the matched reflection coefficient is from -20.4 dB to -10.0 dB. The impedance matching has been achieved over more than an octave bandwidth.
RESUMO
The maximum reflection at an open end of a standard rectangular waveguide is about -10 dB in its operating frequency range. It is often used without matching. For critical applications, it is desirable to further reduce the reflection coefficient. In this paper, a new technique is presented for the broadband impedance matching of an open-ended rectangular waveguide. The proposed technique employs three thin capacitive matching elements placed at proper intervals via a low-loss dielectric material. The capacitance of, and distance between, the matching elements are optimized for broadband impedance matching using a simulation tool. Based on the proposed technique, two design examples are presented for the matching of a WR75 waveguide radiator. A reflection coefficient of less than -16 dB and -20 dB has been achieved over a ratio bandwidth of 2.13:1 and 1.62:1, respectively.
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This paper presents a design for a monopulse reflector antenna with asymmetric beamwidths for radar applications at the Ku band. The proposed design features a rectangular waveguide monopulse feed and a truncated parabolic reflector. An array of four open-ended rectangular waveguides were employed to realize a compact monopulse feed. The reflector is cut in the H plane of the feed producing a wider beam in the azimuth plane. This type of pattern is useful in applications such as projectile tracking and airport surveillance. The design parameters for optimum performances are chosen at all stages of the design. The design and analysis have been carried out using the commercial simulation tool CST Studio Suite 2022. The directivity of the sum, elevation difference and azimuth difference channels of the reflector antenna are 32.1, 28.1, and 26.4 dB at 14 GHz; 30.9, 29, and 27.3 dB at 15 GHz; 31.7, 29.6, and 27.6 dB at 16 GHz; 31.6, 29.9, and 27.8 dB at 17 GHz.
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Although deformation and aging treatments of Cu-3 wt%Ti alloys dramatically enhance their mechanical properties, the corrosion behavior of ultra-fine grained (UFG) Cu-3 wt%Ti alloys produced by a combination of hot rolling and artificial aging has not been extensively explored yet. To bridge this gap, we herein probe the corrosion behavior of an UFG Cu-3 wt%Ti alloy produced by cold rolling and artificial aging, revealing that cast sample corrosion preferentially occurs around the ß-Cu4Ti phase. Compared to that of the coarse-grained Cu-3 wt%Ti alloy, the corrosion resistance of its UFG counterpart is remarkably higher, which is ascribed to the effects of grain refinement and enveloping between the α-Cu matrix and ß-Cu4Ti in the absence of pitting corrosion. The development of ultra-fine microstructure upon the introduction of severe deformation is shown to dramatically improve the corrosion resistance of aging-hardened Cu-3 wt%Ti alloys without sacrificing their mechanical properties. Finally, we demonstrate that solid solution treatment of the Cu-3 wt%Ti alloy results in serious mechanical property deterioration, even though the thus treated samples feature the lowest corrosion current density.
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ß-Catenin, a component of Wnt signaling, plays a key role in colorectal carcinogenesis. The phosphorylation status of ß-catenin determines its fate and affects its cellular function, and serine 675 (S675) was previously identified as a common target of p21-activated kinase 1 (PAK1) and protein kinase A. In the present study, we explored the PAK1-specific phosphorylation site(s) in ß-catenin. Active PAK1 T423E but not inactive PAK1 K299R interacted with and phosphorylated ß-catenin. Mutagenesis followed by a kinase assay revealed that PAK1 phosphorylated S663 in addition to S675, and an anti-phospho-ß-catenin(S663) antibody detected the phosphorylation of S663 downstream of PAK1 in various human colon cancer cells. Furthermore, the Wnt3a-stimulated S663 phosphorylation was inhibited by the PAK1-specific inhibitor, IPA-3, but not by H-89 or LY294002. The non-phosphorylatable mutant forms of ß-catenin, S663A, S675A and S663/675A, showed similar defects in their PAK1-induced TCF/LEF transactivation, whereas the phosphomimetic form of ß-catenin, S663D, demonstrated a transcriptional activity that was comparable to that of ß-catenin S675D and ß-catenin S663D/S675D. Taken together, these results provide evidence that PAK1 specifically phosphorylates ß-catenin at S663 and that this phosphorylation is essential for the PAK1-mediated transcriptional activation of ß-catenin.
Assuntos
Serina/metabolismo , Ativação Transcricional , beta Catenina/metabolismo , Quinases Ativadas por p21/metabolismo , Neoplasias Colorretais/genética , Neoplasias Colorretais/metabolismo , Células HCT116 , Células HEK293 , Humanos , Fosforilação , Serina/genética , Fatores de Transcrição TCF/metabolismo , Transcrição Gênica , beta Catenina/genética , Quinases Ativadas por p21/genéticaRESUMO
Proper targeting of the ßPAK-interacting exchange factor (ßPIX)/G protein-coupled receptor kinase-interacting target protein (GIT) complex into distinct cellular compartments is essential for its diverse functions including neurite extension and synaptogenesis. However, the mechanism for translocation of this complex is still unknown. In the present study, we reported that the conventional kinesin, called kinesin-1, can transport the ßPIX/GIT complex. Additionally, ßPIX bind to KIF5A, a neuronal isoform of kinesin-1 heavy chain, but not KIF1 and KIF3. Mapping analysis revealed that the tail of KIF5s and LZ domain of ßPIX were the respective binding domains. Silencing KIF5A or the expression of a variety of mutant forms of KIF5A inhibited ßPIX targeting the neurite tips in PC12 cells. Furthermore, truncated mutants of ßPIX without LZ domain did not interact with KIF5A, and were unable to target the neurite tips in PC12 cells. These results defined kinesin-1 as a motor protein of ßPIX, and may provide new insights into ßPIX/GIT complex-dependent neuronal pathophysiology. [BMB Reports 2021; 54(7): 380-385].
Assuntos
Proteínas de Ciclo Celular/metabolismo , Cinesinas/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Animais , Proteínas de Ciclo Celular/fisiologia , Neurônios/metabolismo , Células PC12 , Isoformas de Proteínas/metabolismo , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho/fisiologiaRESUMO
PURPOSE: This study intended to explore the clinical outcomes of PFNA-II, one of the commonly used fixation devices for intertrochanteric fractures and the association of clinical results with the extent of proximal nail protrusion. MATERIALS AND METHODS: Of 315 cases that underwent internal fixation using PFNA-II between September 2010 and June 2018 among intertrochanteric fracture patients aged over 65 years, a total of 86 patients with an ability to communicate clearly and a minimum follow-up of 6 months were retrospectively reviewed. We classified the subjects according to PFNA-II protrusion over the greater trochanter area on anteroposterior radiographs. Differences between the two groups were examined by comparing demographic characteristics including gender, age, height, weight and BMI, instrumental characteristics including PFNA nail size, nail diameter, blade length and blade position, radiologic characteristics including reduction quality, Dorr type and bone union, and clinical characteristics including GT pain,VAS score and Harris Hip Score (HHS). RESULTS: A total of 86 cases were divided into 30 (34.9%) in the protrusion group (group A) and 56 (65.1%) in the non-protrusion group (group B). No significant difference was found in demographic characteristics such as gender, age, height, weight and BMI between the two groups. Two groups had no statistically significant difference in PFNA nail length, nail diameter and blade length, but showed a statistically significant difference in blade position. At the latest follow-up, the mean HHS shows no statistically significant difference between the two groups. On the contrary, the number of patients complaining of GT pain and VAS score were statistically significantly higher in group A. Removal of metal implants was performed in two patients in the protrusion group due to a complaint of persistent GT pain. CONCLUSION: Nail protrusion over the greater trochanter area occurs frequently after the surgical treatment of intertrochanteric fracture using PFNA-II. When the nail protruded into the greater trochanter, the number of patients who clinically complained of pain was statistically significantly high. We recommend a modification to the PFNA-II that would further shorten the proximal nail end suitable for the Asian population to achieve better clinical results in the fixation of intertrochanteric fractures.
Assuntos
Pinos Ortopédicos/efeitos adversos , Fixação Interna de Fraturas/instrumentação , Fraturas do Quadril/cirurgia , Idoso , Idoso de 80 Anos ou mais , Feminino , Fêmur/diagnóstico por imagem , Seguimentos , Fraturas do Quadril/etnologia , Humanos , Masculino , Duração da Cirurgia , Radiografia/métodos , Estudos Retrospectivos , Resultado do TratamentoRESUMO
Bioinformatic and functional data link integrin-mediated cell adhesion to cellular senescence; however, the significance of and molecular mechanisms behind these connections are unknown. We now report that the focal adhesion-localized ßPAK-interacting exchange factor (ßPIX)-G protein-coupled receptor kinase interacting protein (GIT) complex controls cellular senescence in vitro and in vivo. ßPIX and GIT levels decline with age. ßPIX knockdown induces cellular senescence, which was prevented by reexpression. Loss of ßPIX induced calpain cleavage of the endocytic adapter amphiphysin 1 to suppress clathrin-mediated endocytosis (CME); direct competition of GIT1/2 for the calpain-binding site on paxillin mediates this effect. Decreased CME and thus integrin endocytosis induced abnormal integrin signaling, with elevated reactive oxygen species production. Blocking integrin signaling inhibited senescence in human fibroblasts and mouse lungs in vivo. These results reveal a central role for integrin signaling in cellular senescence, potentially identifying a new therapeutic direction.
Assuntos
Calpaína , Integrinas , Animais , Senescência Celular , Adesões Focais/metabolismo , Integrinas/metabolismo , Camundongos , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismoRESUMO
We previously showed that p21-activated kinase 2 (PAK2), a major PAK isoform expressed in PC12 cells, mediates neurite outgrowth via Rac1 GTPase. RhoGDI1 forms a complex with Rac1, resulting in its inhibition. Rac1 activation requires dissociation from RhoGDI1. Here, we show that PAK2 mediates basic fibroblast growth factor (bFGF)-stimulated neurite outgrowth via phosphorylation of RhoGDI1. RhoGDI1 was shown to be associated with PAK2, with phosphorylation of Ser34 and Ser101 by active PAK2 evident in vitro and in vivo. A RhoGDI1 phosphomimetic mutant (S34E/S101E) was dissociated from Rac1/Cdc42, whereas the wild-type or a nonphosphorylatable mutant (S34A/S101A) formed a tight complex. Consistent with this, PC12 cells expressing the phosphomimetic mutant displayed Rac1/Cdc42 activation in response to bFGF stimulation. Neurite outgrowth was also enhanced in PC12 cells expressing the phosphomimetic mutant. These results suggest that PAK2-mediated RhoGDI1 phosphorylation stimulates dissociation of RhoGDI1-Rac1/Cdc42 complex accompanied by relief of inhibitory effect on Rac1/Cdc42, which promotes neuronal differentiation.
Assuntos
Diferenciação Celular , Inibidores de Dissociação do Nucleotídeo Guanina/metabolismo , Neuritos/fisiologia , Neurônios/citologia , Quinases Ativadas por p21/metabolismo , Animais , Fatores de Crescimento de Fibroblastos/farmacologia , Inibidores de Dissociação do Nucleotídeo Guanina/genética , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/metabolismo , Neurônios/fisiologia , Células PC12 , Fosforilação , Ratos , Serina/genética , Serina/metabolismo , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Inibidores da Dissociação do Nucleotídeo Guanina rho-EspecíficoRESUMO
Angiotensin II (Ang II) stimulates migration of vascular smooth muscle cell (VSMC) in addition to its contribution to contraction and hypertrophy. It is well established that Rho GTPases regulate cellular contractility and migration by reorganizing the actin cytoskeleton. Ang II activates Rac1 GTPase, but its upstream guanine nucleotide exchange factor (GEF) remains elusive. Here, we show that Ang II-induced VSMC migration occurs in a betaPIX GEF-dependent manner. betaPIX-specific siRNA treatment significantly inhibited Ang II-induced VSMC migration. Ang II activated the catalytic activity of betaPIX towards Rac1 in dose- and time-dependent manners. Activity reached a peak at 10 min and declined close to a basal level by 30 min following stimulation. Pharmacological inhibition with specific kinase inhibitors revealed the participation of protein kinase C, Src family kinase, and phosphatidylinositol 3-kinase (PI3-K) upstream of betaPIX. Both p21-activated kinase and reactive oxygen species played key roles in cytoskeletal reorganization downstream of betaPIX-Rac1. Taken together, our results suggest that betaPIX is involved in Ang II-induced VSMC migration.
Assuntos
Angiotensina II/metabolismo , Movimento Celular , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Miócitos de Músculo Liso/citologia , Animais , Células Cultivadas , Fatores de Troca do Nucleotídeo Guanina/genética , Músculo Liso Vascular/citologia , NADPH Oxidases/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteína Quinase C/metabolismo , RNA Interferente Pequeno/genética , Ratos , Ratos Sprague-Dawley , Fatores de Troca de Nucleotídeo Guanina Rho , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismo , Quinases da Família src/metabolismoRESUMO
BACKGROUND: Aberrant gene expression in the gut mucosa might contribute to the initiation and progression of Crohn's disease (CD). RNA sequencing (RNA-seq) provides precise measurements of expression levels of transcripts and their isoforms. The aim of this study was to use RNA-seq to investigate transcriptomic differences and identify significantly differentially expressed transcripts in inflamed and noninflamed intestinal mucosa of CD patients. METHODS: RNA-seq was performed on 13 pairs of inflamed and noninflamed intestinal mucosa from 13 CD patients and on sex-matched normal mucosa of 13 healthy controls. Significantly differentially expressed transcripts were validated by immunohistochemistry, quantitative reverse transcriptase polymerase chain reaction, and enzyme-linked immunosorbent assay. RESULTS: RNA-seq revealed genome-wide transcriptomic differences between normal mucosa, noninflamed, and inflamed CD mucosa. Among 950 differentially expressed genes, 19 were up- or downregulated (upregulation: ANGPT2, CHN1, CPXM1, CPZ, CXCL1, FCN3, GJC1, HSD11B1, LZTS1, MEOX1, MMP12, PLA1A, SERPINE1, SGIP1, and TRPC4; downregulation: FAM189A1, PDE6A, SLC38A4, and HMGCS2) with statistical significance (p < 0.01 and q < 0.05). Among them, CXCL1 exhibited the highest fold change between groups. Immunohistochemistry for CXCL1 revealed no expression in normal mucosa, slightly increased expression in noninflamed CD mucosa, and highly increased expression in inflamed CD mucosa. Quantitative reverse transcriptase polymerase chain reaction showed that CXCL1 expression was significantly associated with epithelial damage, increased infiltration of polymorphonuclear leukocytes, and submucosal fibrosis. Serum CXCL1 concentration measured by enzyme-linked immunosorbent assay was better correlated with CD activity index (r = 0.660) than with C-reactive protein (r = 0.204). CONCLUSIONS: RNA-seq revealed transcriptomic differences between normal mucosa, noninflamed CD mucosa, and inflamed CD mucosa. Intestinal and serum CXCL1 was substantially increased with CD activity and can be used as a potential biomarker of CD.
Assuntos
Doença de Crohn/genética , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Mediadores da Inflamação/metabolismo , Inflamação/diagnóstico , Mucosa Intestinal/metabolismo , Transcriptoma , Adulto , Estudos de Casos e Controles , Doença de Crohn/imunologia , Doença de Crohn/patologia , Feminino , Humanos , Inflamação/etiologia , Mucosa Intestinal/imunologia , Mucosa Intestinal/patologia , MasculinoRESUMO
p21-activated kinases (PAKs) are key regulators of actin dynamics, cell proliferation and cell survival. Deregulation of PAK activity contributes to the pathogenesis of various human diseases, including cancer and neurological disorders. Using an ELISA-based screening protocol, we identified naphtho(hydro)quinone-based small molecules that allosterically inhibit PAK activity. These molecules interfere with the interactions between the p21-binding domain (PBD) of PAK1 and Rho GTPases by binding to the PBD. Importantly, they inhibit the activity of full-length PAKs and are selective for PAK1 and PAK3 in vitro and in living cells. These compounds may potentially be useful for determining the details of the PAK signaling pathway and may also be used as lead molecules in the development of more selective and potent PAK inhibitors.
Assuntos
Domínios e Motivos de Interação entre Proteínas , Inibidores de Proteínas Quinases/farmacologia , Quinases Ativadas por p21/antagonistas & inibidores , Quinases Ativadas por p21/metabolismo , Regulação Alostérica/efeitos dos fármacos , Linhagem Celular , Descoberta de Drogas/métodos , Avaliação Pré-Clínica de Medicamentos/métodos , Ativação Enzimática/efeitos dos fármacos , Humanos , Isoenzimas , Ligação Proteica , Inibidores de Proteínas Quinases/química , Bibliotecas de Moléculas Pequenas , Relação Estrutura-Atividade , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/químicaRESUMO
p21-activated kinase (PAK) targeting to the plasma membrane is essential for PC12 cell neurite outgrowth. Phospholipase C-gamma1 (PLC-gamma1) can mediate the PAK translocation in response to growth factors, since PLC-gamma1 binds to both tyrosine-phosphorylated receptor tyrosine kinases and PAK through its SH2 and SH3 domain, respectively. In the present study, we examined a potential role for PLC-gamma1 in the basic fibroblast growth factor (bFGF)-induced PAK translocation using stable PC12 cell lines that overexpress in a tetracycline-inducible manner either the wild-type FGFR-1 or the Y766F FGFR-1 mutant. Phosphatidylinositol hydrolysis was increased 6.5-fold in response to bFGF in the wild type cells but negligible in the mutant cells. The recombinant GST-PLC-gamma1 SH3 was able to bind to PAK1 but not GST alone. However, examination of PLC-gamma1 as an adaptor for translocation of PAK1 in cells showed that both cells transfected with pEGFP-PAK1 was able to differentiate for 24 h, as visualized by laser confocal microscopy. Translocation of PAK1 to growth cones occurs at similar levels in both wild and mutant cells. These results suggest that a protein(s) other than PLC-gamma1 is functionally relevant for PAK targeting.
Assuntos
Membrana Celular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Fosfolipases Tipo C/metabolismo , Animais , Diferenciação Celular/fisiologia , Células PC12 , Fosfolipase C gama , Transporte Proteico , Ratos , Quinases Ativadas por p21 , Domínios de Homologia de srcRESUMO
p21-activated kinase (PAK)-interacting exchange factor (PIX) is known to be involved in regulation of Cdc42/Rac GTPases and PAK activity. PIX binds to the proline-rich region of PAK, and regulates biological events through activation of Cdc42/Rac GTPase. To further investigate the role of PIX we produced monoclonal antibodies (Mab) against bPIX. Three clones; N-C6 against N-terminal half and C-A3 and C-B7 against C- terminal half of bPIX were generated and characterized. N-C6 Mab detected bPIX as a major band in most cell lines. C-A3 Mab recognizes GIT-binding domain (GBD), but it does not interfere with GIT binding to bPIX. Using C-A3 Mab possible bPIX interaction with actin in PC12 cells was examined. bPIX Mab (C-A3) specifically precipitated actin of the PC12 cell lysates whereas actin Mab failed to immunoprecipitate bPIX. Co-sedimentation of PC12 cell lysates with the polymerized F-actin resulted in the recovery of most of bPIX in the cell lysates. These results suggest that bPIX may not interact with soluble actin but with polymerized F-actin and revealed that bPIX constitutes a functional complex with actin. These data indicate real usefulness of the bPIX Mab in the study of bPIX role(s) in regulation of actin cyoskeleton.
Assuntos
Citoesqueleto de Actina/fisiologia , Actinas/metabolismo , Proteínas de Ciclo Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Animais , Anticorpos Monoclonais/imunologia , Proteínas de Ciclo Celular/imunologia , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/metabolismo , Mapeamento de Epitopos , Fatores de Troca do Nucleotídeo Guanina/imunologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Imunoprecipitação , Camundongos , Estrutura Terciária de Proteína , Ratos , Fatores de Troca de Nucleotídeo Guanina RhoRESUMO
BACKGROUND: Non-muscle myosin II (NM II) regulates a wide range of cellular functions, including neuronal differentiation, which requires precise spatio-temporal activation of Rho GTPases. The molecular mechanism underlying the NM II-mediated activation of Rho GTPases is poorly understood. The present study explored the possibility that NM II regulates neuronal differentiation, particularly morphological changes in growth cones and the distal axon, through guanine nucleotide exchange factors (GEFs) of the Dbl family. PRINCIPAL FINDINGS: NM II colocalized with GEFs, such as ßPIX, kalirin and intersectin, in growth cones. Inactivation of NM II by blebbistatin (BBS) led to the increased formation of short and thick filopodial actin structures at the periphery of growth cones. In line with these observations, FRET analysis revealed enhanced Cdc42 activity in BBS-treated growth cones. BBS treatment also induced aberrant targeting of various GEFs to the distal axon where GEFs were seldom observed under physiological conditions. As a result, numerous protrusions and branches were generated on the shaft of the distal axon. The disruption of the NM II-GEF interactions by overexpression of the DH domains of ßPIX or Tiam1, or by ßPIX depletion with specific siRNAs inhibited growth cone formation and induced slender axons concomitant with multiple branches in cultured hippocampal neurons. Finally, stimulation with nerve growth factor induced transient dissociation of the NM II-GEF complex, which was closely correlated with the kinetics of Cdc42 and Rac1 activation. CONCLUSION: Our results suggest that NM II maintains proper morphology of neuronal growth cones and the distal axon by regulating actin dynamics through the GEF-Rho GTPase signaling pathway.
Assuntos
Actinas/metabolismo , Neurônios/metabolismo , Miosina não Muscular Tipo IIB/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho/metabolismo , Animais , Axônios/efeitos dos fármacos , Axônios/metabolismo , Células Cultivadas , Feminino , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Hipocampo/citologia , Modelos Biológicos , Fatores de Crescimento Neural/metabolismo , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Estrutura Terciária de Proteína , Pseudópodes/efeitos dos fármacos , Pseudópodes/metabolismo , Ratos Sprague-Dawley , Fatores de Troca de Nucleotídeo Guanina Rho/química , Transdução de Sinais/efeitos dos fármacos , Proteína cdc42 de Ligação ao GTP/metabolismo , Quinases Ativadas por p21/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Cell migration requires the coordinated spatiotemporal regulation of actomyosin contraction and cell protrusion/adhesion. Nonmuscle myosin II (MII) controls Rac1 and Cdc42 activation, and cell protrusion and focal complex formation in migrating cells. However, these mechanisms are poorly understood. Here, we show that MII interacts specifically with multiple Dbl family guanine nucleotide exchange factors (GEFs). Binding is mediated by the conserved tandem Dbl homology-pleckstrin homology module, the catalytic site of these GEFs, with dissociation constants of approximately 0.3 microM. Binding to the GEFs required assembly of the MII into filaments and actin-stimulated ATPase activity. Binding of MII suppressed GEF activity. Accordingly, inhibition of MII ATPase activity caused release of GEFs and activation of Rho GTPases. Depletion of betaPIX GEF in migrating NIH3T3 fibroblasts suppressed lamellipodial protrusions and focal complex formation induced by MII inhibition. The results elucidate a functional link between MII and Rac1/Cdc42 GTPases, which may regulate protrusion/adhesion dynamics in migrating cells.
Assuntos
Movimento Celular/fisiologia , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Miosina Tipo II/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Actomiosina/metabolismo , Animais , Sítios de Ligação , Adesão Celular , Ativação Enzimática , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Células Jurkat , Camundongos , Miosina Tipo II/genética , Células NIH 3T3 , Fator de Crescimento Derivado de Plaquetas/metabolismo , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Fatores de Troca de Nucleotídeo Guanina Rho , Proteína cdc42 de Ligação ao GTP/genética , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/genética , Proteínas rac1 de Ligação ao GTP/metabolismo , Proteínas rho de Ligação ao GTP/genéticaRESUMO
Neuritogenesis requires active actin cytoskeleton rearrangement in which Rho GTPases play a pivotal role. In a previous study (Shin, E. Y., Woo, K. N., Lee, C. S., Koo, S. H., Kim, Y. G., Kim, W. J., Bae, C. D., Chang, S. I., and Kim, E. G. (2004) J. Biol. Chem. 279, 1994-2004), we demonstrated that betaPak-interacting exchange factor (betaPIX) guanine nucleotide exchange factor (GEF) mediates basic fibroblast growth factor (bFGF)-stimulated Rac1 activation through phosphorylation of Ser-525 and Thr-526 at the GIT-binding domain (GBD). However, the mechanism by which this phosphorylation event regulates the Rac1-GEF activity remained elusive. We show here that betaPIX binds to Rac1 via the GBD and also activates the GTPase via an associated GEF, smgGDS, in a phosphorylation-dependent manner. Notably, the Rac1-GEF activity of betaPIX persisted for an extended period of time following bFGF stimulation, unlike other Rho GEFs containing the Dbl homology domain. We demonstrate that C-PIX, containing proline-rich, GBD, and leucine zipper domains can interact with Rac1 via the GBD in vitro and in vivo and also mediated bFGF-stimulated Rac1 activation, as determined by a modified GEF assay and fluorescence resonance energy transfer analysis. However, nonphosphorylatable C-PIX (S525A/T526A) failed to generate Rac1-GTP. Finally, betaPIX is shown to form a trimeric complex with smgGDS and Rac1; down-regulation of smgGDS expression by short interfering RNA causing significant inhibition of betaPIX-mediated Rac1 activation and neurite outgrowth. These results provide evidence for a new and unexpected mechanism whereby betaPIX can regulate Rac1 activity.
Assuntos
Proteínas de Ciclo Celular/fisiologia , Fator 2 de Crescimento de Fibroblastos/química , Fatores de Troca do Nucleotídeo Guanina/química , Neuritos/metabolismo , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas rac1 de Ligação ao GTP/metabolismo , Animais , Proteínas de Ciclo Celular/química , Proliferação de Células , Ativação Enzimática , Proteínas de Fluorescência Verde/química , Fatores de Troca do Nucleotídeo Guanina/fisiologia , Humanos , Modelos Biológicos , Células PC12 , Proteínas Serina-Treonina Quinases/química , Ratos , Proteínas Recombinantes/química , Fatores de Troca de Nucleotídeo Guanina Rho , Quinases Ativadas por p21RESUMO
Guanine nucleotide exchange factors (GEFs) have been implicated in growth factor-induced neuronal differentiation through the activation of small GTPases. Although phosphorylation of these GEFs is considered an activation mechanism, little is known about the upstream of PAK-interacting exchange factor (PIX), a member of the Dbl family of GEFs. We report here that phosphorylation of p85 betaPIX/Cool/p85SPR is mediated via the Ras/ERK/PAK2 pathway. To understand the role of p85 betaPIX in basic fibroblast growth factor (bFGF)-induced neurite outgrowth, we established PC12 cell lines that overexpress the fibroblast growth factor receptor-1 in a tetracycline-inducible manner. Treatment with bFGF induces the phosphorylation of p85 betaPIX, as determined by metabolic labeling and mobility shift upon gel electrophoresis. Interestingly, phosphorylation of p85 betaPIX is inhibited by PD98059, a specific MEK inhibitor, suggesting the involvement of the ERK cascade. PAK2, a major PAK isoform in PC12 cells as well as a binding partner of p85 betaPIX, also functions upstream of p85 betaPIX phosphorylation. Surprisingly, PAK2 directly binds to ERK, and its activation is dependent on ERK. p85 betaPIX specifically localizes to the lamellipodia at neuronal growth cones in response to bFGF. A mutant form of p85 betaPIX (S525A/T526A), in which the major phosphorylation sites are replaced by alanine, shows significant defect in targeting. Moreover, expression of the mutant p85 betaPIX efficiently blocks PC12 cell neurite outgrowth. Our study defines a novel signaling pathway for bFGF-induced neurite outgrowth that involves activation of the PAK2-p85 betaPIX complex via the ERK cascade and subsequent translocation of this complex.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fator 2 de Crescimento de Fibroblastos/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Neurônios/fisiologia , Transdução de Sinais , Animais , Diferenciação Celular , Divisão Celular , Células Cultivadas , Ativação Enzimática , Immunoblotting , Microscopia de Fluorescência , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Modelos Biológicos , Neurônios/citologia , Células PC12 , Fosforilação , Plasmídeos/metabolismo , Testes de Precipitina , Ligação Proteica , Isoformas de Proteínas , Transporte Proteico , Ratos , Fatores de Troca de Nucleotídeo Guanina Rho , Serina/metabolismo , Tetraciclina/farmacologia , Treonina/metabolismo , Fatores de Tempo , TransfecçãoRESUMO
In a previous study (Shin, E. Y., Shin, K. S., Lee, C. S., Woo, K. N., Quan, S. H., Soung, N. K., Kim, Y. G., Cha, C. I., Kim, S. R., Park, D., Bokoch, G. M., and Kim, E. G. (2002) J. Biol. Chem. 277, 44417-44430) we reported that phosphorylation of p85 betaPIX, a guanine nucleotide exchange factor (GEF) for Rac1/Cdc42, is a signal for translocation of the PIX complex to neuronal growth cones and is associated with basic fibroblast growth factor (bFGF)-induced neurite outgrowth. However, the issue of whether p85 betaPIX phosphorylation affects GEF activity on Rac1/Cdc42 is yet to be explored. Here we show that Rac1 activation occurs in a p85 betaPIX phosphorylation-dependent manner. A GST-PBD binding assay reveals that Rac1 is activated in a dose- and time-dependent manner in PC12 cells in response to bFGF. Inhibition of ERK or PAK2, the kinases upstream of p85 betaPIX in the bFGF signaling, prevents Rac1 activation, suggesting that phosphorylation of p85 betaPIX functions upstream of Rac1 activation. To directly address this issue, transfection studies with wild-type and mutant p85 betaPIX (S525A/T526A, a non-phosphorylatable form) were performed. Expression of mutant PIX markedly inhibits both bFGF- and nerve growth factor (NGF)-induced activation of Rac1, indicating that phosphorylation of p85 betaPIX is responsible for activation of this G protein. Both wild-type and mutant p85 betaPIX displaying negative GEF activity (L238R/L239S) are similarly recruited to growth cones, suggesting that Rac1 activation is not essential for translocation of the PIX complex (PAK2-p85 betaPIX-Rac1). However, expression of mutant p85 betaPIX (L238R/L239S) results in retraction of the pre-existing neurites. Our results provide evidence that bFGF- and NGF-induced phosphorylation of p85 betaPIX mediates Rac1 activation, which in turn regulates cytoskeletal reorganization at growth cones, but not translocation of the PIX complex.