RESUMO
Protein phosphatase 2A (PP2A) is a major phospho-Ser/Thr phosphatase and a key regulator of cellular signal transduction pathways. While PP2A dysfunction has been linked to human cancer and neurodegenerative disorders such as Alzheimer's disease (AD), PP2A regulation remains relatively poorly understood. It has been reported that the PP2A catalytic subunit (PP2Ac) is inactivated by a single phosphorylation at the Tyr307 residue by tyrosine kinases such as v-Src. However, multiple mass spectrometry studies have revealed the existence of other putative PP2Ac phosphorylation sites in response to activation of Src and Fyn, two major Src family kinases (SFKs). Here, using PP2Ac phosphomutants and novel phosphosite-specific PP2Ac antibodies, we show that cellular pools of PP2Ac are instead phosphorylated on both Tyr127 and Tyr284 upon Src activation, and on Tyr284 following Fyn activation. We found these phosphorylation events enhanced the interaction of PP2Ac with SFKs. In addition, we reveal SFK-mediated phosphorylation of PP2Ac at Y284 promotes dissociation of the regulatory Bα subunit, altering PP2A substrate specificity; the phosphodeficient Y127/284F and Y284F PP2Ac mutants prevented SFK-mediated phosphorylation of Tau at the CP13 (pSer202) epitope, a pathological hallmark of AD, and SFK-dependent activation of ERK, a major growth regulatory kinase upregulated in many cancers. Our findings demonstrate a novel PP2A regulatory mechanism that challenges the existing dogma on the inhibition of PP2A catalytic activity by Tyr307 phosphorylation. We propose dysregulation of SFK signaling in cancer and AD can lead to alterations in PP2A phosphorylation and subsequent deregulation of key PP2A substrates, including ERK and Tau.
Assuntos
Proteína Fosfatase 2 , Proteínas Proto-Oncogênicas c-fyn , Quinases da Família src , Doença de Alzheimer/metabolismo , Humanos , Fosfoproteínas Fosfatases , Fosforilação , Proteína Fosfatase 2/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Tirosina/metabolismo , Quinases da Família src/genética , Quinases da Família src/metabolismo , Proteínas tau/metabolismoRESUMO
The tyrosine kinase Src acts as a key regulator of cell motility by phosphorylating multiple protein substrates that control cytoskeletal and adhesion dynamics. In an earlier phosphotyrosine proteomics study, we identified a novel Rho-GTPase activating protein, now known as ARHGAP42, as a likely biologically relevant Src substrate. ARHGAP42 is a member of a family of RhoGAPs distinguished by tandem BAR-PH domains lying N-terminal to the GAP domain. Like other family members, ARHGAP42 acts preferentially as a GAP for RhoA. We show that Src principally phosphorylates ARHGAP42 on tyrosine 376 (Tyr-376) in the short linker between the BAR-PH and GAP domains. The expression of ARHGAP42 variants in mammalian cells was used to elucidate its regulation. We found that the BAR domain is inhibitory toward the GAP activity of ARHGAP42, such that BAR domain deletion resulted in decreased active GTP-bound RhoA and increased cell motility. With the BAR domain intact, ARHGAP42 GAP activity could be activated by phosphorylation of Tyr-376 to promote motile cell behavior. Thus, phosphorylation of ARHGAP42 Tyr-376 is revealed as a novel regulatory event by which Src can affect actin dynamics through RhoA inhibition.
Assuntos
Movimento Celular/fisiologia , Adesões Focais/metabolismo , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Tirosina/metabolismo , Quinases da Família src/metabolismo , Animais , Humanos , Camundongos , Fosforilação , Proteínas rho de Ligação ao GTP/antagonistas & inibidores , Proteínas rho de Ligação ao GTP/metabolismo , Proteína rhoA de Ligação ao GTP/antagonistas & inibidores , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The Cas family scaffolding protein p130Cas is a Src substrate localized in focal adhesions (FAs) and functions in integrin signaling to promote cell motility, invasion, proliferation, and survival. p130Cas targeting to FAs is essential for its tyrosine phosphorylation and downstream signaling. Although the N-terminal SH3 domain is important for p130Cas localization, it has also been reported that the C-terminal region is involved in p130Cas FA targeting. The C-terminal region of p130Cas or Cas family homology domain (CCHD) has been reported to adopt a structure similar to that of the focal adhesion kinase C-terminal focal adhesion-targeting domain. The mechanism by which the CCHD promotes FA targeting of p130Cas, however, remains unclear. In this study, using a calorimetry approach, we identified the first LD motif (LD1) of the FA-associated protein paxillin as the binding partner of the p130Cas CCHD (in a 1:1 stoichiometry with a Kd â¼4.2 µm) and elucidated the structure of the p130Cas CCHD in complex with the paxillin LD1 motif by X-ray crystallography. Of note, a comparison of the CCHD/LD1 complex with a previously solved structure of CCHD in complex with the SH2-containing protein NSP3 revealed that LD1 had almost identical positioning of key hydrophobic and acidic residues relative to NSP3. Because paxillin is one of the key scaffold molecules in FAs, we propose that the interaction between the p130Cas CCHD and the LD1 motif of paxillin plays an important role in p130Cas FA targeting.
Assuntos
Proteínas Aviárias/metabolismo , Proteína Substrato Associada a Crk/metabolismo , Modelos Moleculares , Paxilina/metabolismo , Motivos de Aminoácidos , Substituição de Aminoácidos , Animais , Proteínas Aviárias/química , Sítios de Ligação , Galinhas , Proteína Substrato Associada a Crk/química , Proteína Substrato Associada a Crk/genética , Cristalografia por Raios X , Interações Hidrofóbicas e Hidrofílicas , Cinética , Leucina , Camundongos , Mutação , Paxilina/química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/metabolismo , Conformação Proteica , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Estabilidade Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Homologia Estrutural de ProteínaRESUMO
The extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) pathway is a highly conserved signaling pathway that regulates diverse cellular processes including differentiation, proliferation, and survival. Kinase suppressor of Ras-1 (KSR1) binds each of the three ERK cascade components to facilitate pathway activation. Even though KSR1 contains a C-terminal kinase domain, evidence supporting the catalytic function of KSR1 remains controversial. In this study, we produced recombinant wild-type or kinase-inactive (D683A/D700A) KSR1 proteins in Escherichia coli to test the hypothesis that KSR1 is a functional protein kinase. Recombinant wild-type KSR1, but not recombinant kinase-inactive KSR1, underwent autophosphorylation on serine residue(s), phosphorylated myelin basic protein (MBP) as a generic substrate, and phosphorylated recombinant kinase-inactive MAPK/ERK kinase-1 (MEK1). Furthermore, FLAG immunoprecipitates from KSR1(-/-) colon epithelial cells stably expressing FLAG-tagged wild-type KSR1 (+KSR1), but not vector (+vector) or FLAG-tagged kinase-inactive KSR1 (+D683A/D700A), were able to phosphorylate kinase-inactive MEK1. Since TNF activates the ERK pathway in colon epithelial cells, we tested the biological effects of KSR1 in the survival response downstream of TNF. We found that +vector and +D683A/D700A cells underwent apoptosis when treated with TNF, whereas +KSR1 cells were resistant. However, +KSR1 cells were sensitized to TNF-induced cell loss in the absence of MEK kinase activity. These data provide clear evidence that KSR1 is a functional protein kinase, MEK1 is an in vitro substrate of KSR1, and the catalytic activities of both proteins are required for eliciting cell survival responses downstream of TNF.
Assuntos
MAP Quinase Quinase 1/metabolismo , Proteínas Quinases/metabolismo , Serina/metabolismo , Fator de Necrose Tumoral alfa/farmacologia , Animais , Apoptose/efeitos dos fármacos , Catálise , Colo/citologia , Células Epiteliais , Proteínas de Escherichia coli , Camundongos , Fosforilação , Especificidade por SubstratoRESUMO
The docking protein p130Cas is a major Src substrate involved in integrin signaling and mechanotransduction. Tyrosine phosphorylation of p130Cas in focal adhesions (FAs) has been linked to enhanced cell migration, invasion, proliferation, and survival. However, the mechanism of p130Cas targeting to FAs is uncertain, and dynamic aspects of its localization have not been explored. Using live cell microscopy, we show that fluorophore-tagged p130Cas is a component of FAs throughout the FA assembly and disassembly stages, although it resides transiently in FAs with a high mobile fraction. Deletion of either the N-terminal Src homology 3 (SH3) domain or the Cas-family C-terminal homology (CCH) domain significantly impaired p130Cas FA localization, and deletion of both domains resulted in full exclusion. Focal adhesion kinase was implicated in the FA targeting function of the p130Cas SH3 domain. Consistent with their roles in FA targeting, both the SH3 and CCH domains were found necessary for p130Cas to fully undergo tyrosine phosphorylation and promote cell migration. By revealing the capacity of p130Cas to function in FAs throughout their lifetime, clarifying FA targeting mechanism, and demonstrating the functional importance of the highly conserved CCH domain, our results advance the understanding of an important aspect of integrin signaling.
Assuntos
Proteína Substrato Associada a Crk/metabolismo , Adesões Focais/metabolismo , Animais , Anticorpos Monoclonais , Movimento Celular , Proteína Substrato Associada a Crk/análise , Proteína Substrato Associada a Crk/genética , Fibroblastos/metabolismo , Genes Reporter , Variação Genética , Immunoblotting , Proteínas Luminescentes/genética , Camundongos/embriologia , Paxilina/análise , Paxilina/genética , Fosforilação , Plasmídeos , Reação em Cadeia da Polimerase , Especificidade por Substrato , Cicatrização/fisiologia , Quinases da Família src/metabolismoRESUMO
BACKGROUND INFORMATION: FAK (focal adhesion kinase), an essential non-receptor tyrosine kinase, plays pivotal roles in migratory responses, adhesive signalling and mechanotransduction. FAK-dependent regulation of cell migration involves focal adhesion turnover dynamics as well as actin cytoskeleton polymerization and lamellipodia protrusion. Whereas roles for FAK in migratory and mechanosensing responses have been established, the contribution of FAK to the generation of adhesive forces is not well understood. RESULTS: Using FAK-null cells expressing wild-type and mutant FAK under an inducible tetracycline promoter, we analysed the role of FAK in the generation of steady-state adhesive forces using micropatterned substrates and a hydrodynamic adhesion assay. FAK expression reduced steady-state strength by 30% compared with FAK-null cells. FAK expression reduced VCL (vinculin) localization to focal adhesions by 35% independently of changes in integrin binding and localization of talin and paxillin. RNAi (RNA interference) knock-down of VCL abrogated the FAK-dependent differences in adhesive forces. FAK-dependent changes in VCL localization and adhesive forces were confirmed in human primary fibroblasts with FAK knocked down by RNAi. The autophosphorylation Tyr-397 and kinase domain Tyr-576/Tyr-577 sites were differentially required for FAK-mediated adhesive responses. CONCLUSIONS: We demonstrate that FAK reduces steady-state adhesion strength by modulating VCL recruitment to focal adhesions. These findings provide insights into the role of FAK in mechanical interactions between a cell and the extracellular matrix.
Assuntos
Adesão Celular , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Adesões Focais/metabolismo , Vinculina/metabolismo , Células Cultivadas , Regulação para Baixo , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Proteína-Tirosina Quinases de Adesão Focal/genética , Expressão Gênica , Humanos , Mutação , Vinculina/genéticaRESUMO
A recently developed stable isotope dilution liquid chromatography-multiple reaction/mass spectrometry method to quantify focal adhesion kinase (FAK) activation loop phosphorylation was used to study endogenous Src kinase activity. This revealed that bis-phosphorylated pTyr(576)/Tyr(577)-FAK was a biomarker of Src activity and inactivation in vitro and in cell culture. Mouse embryonic fibroblasts (MEFs) expressing endogenous Src family kinases contained 65% unmodified Tyr(576)/Tyr(577), 33% mono-phosphorylated-pTyr(576)-FAK, and 6% bis-phosphorylated-pTyr(576)/pTyr(577)-FAK. In contrast, MEFs expressing oncogenic Y(529)FSrc contained 38% unmodified Tyr(576)/Tyr(577)-FAK, 29% mono-phosphorylated-pTyr(576)-FAK, and 19% bis-phosphorylated-pTyr(576)/pTyr(577)-FAK. This new method has made it possible to accurately determine the absolute amounts of FAK phosphorylation that occur after Src inhibition in cell culture and in vitro with increasing concentrations of the Src inhibitor N-(5-chloro-1,3-benzodioxol-4-yl)-7-[2-(4-methylpiperazin-1-yl)ethoxy]-5-(tetrahydro-2H-pyran-4-yloxy)quinazolin-4-amine (AZD0530). Phosphorylation of FAK at Tyr(576)/Tyr(577) was inhibited by AZD0530 in a dose-dependent manner both in cell culture and in vitro. However, there was a substantial difference in the ability of AZD0530 to inhibit Src that was constitutively activated in a cellular context (IC(50) = 2.12 muM) compared with the isolated enzyme (IC(50) = 0.14 muM). When normal MEFs and Y(529)FSrc-expressing MEFs were treated with pervanadate (a global phosphatase inhibitor), pTyr(576)/pTyr(577)-FAK accounted for almost 60% of the total FAK present in the cells. This suggests that activation loop phosphorylation is regulated by tyrosine phosphatases. These results confirm that FAK phosphorylation is a useful biomarker of Src inhibition in vivo. The accuracy and specificity of stable isotope dilution liquid chromatography-mass spectrometry methodology offers significant advantages over current immunochemical approaches for monitoring Src activity.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/análise , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Quinases da Família src/análise , Quinases da Família src/metabolismo , Animais , Biomarcadores/análise , Biomarcadores/metabolismo , Linhagem Celular , Relação Dose-Resposta a Droga , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Proteína-Tirosina Quinases de Adesão Focal/antagonistas & inibidores , Humanos , Camundongos , Fosforilação/efeitos dos fármacos , Fosforilação/fisiologia , Inibidores de Proteases/farmacologia , Quinases da Família src/antagonistas & inibidoresRESUMO
The migration of epithelial layers requires specific and coordinated organization of the cells at the leading edge of the sheet. Mice that are conditionally deleted for the c-jun protooncogene in epidermis are born at expected frequencies, but with open eyes and with defects in epidermal wound healing. Keratinocytes lacking c-Jun are unable to migrate or elongate properly in culture at the border of scratch assays. Histological analyses in vitro and in vivo demonstrate an inability to activate EGF receptor at the leading edge of wounds, and we demonstrate that this can be rescued by supplementation with conditioned medium or the EGF receptor ligand HB-EGF. Lack of c-Jun prevents EGF-induced expression of HB-EGF, indicating that c-jun controls formation of the epidermal leading edge through its control of an EGF receptor autocrine loop.
Assuntos
Epiderme/crescimento & desenvolvimento , Receptores ErbB/metabolismo , Genes jun/fisiologia , Animais , Divisão Celular , Movimento Celular , Células Cultivadas , Células Epidérmicas , Fator de Crescimento Epidérmico/metabolismo , Epiderme/lesões , Pálpebras/anormalidades , Pálpebras/embriologia , Deleção de Genes , Fator de Crescimento Semelhante a EGF de Ligação à Heparina , Peptídeos e Proteínas de Sinalização Intercelular , Queratinócitos/citologia , Queratinócitos/fisiologia , Queratinas/metabolismo , Camundongos , Camundongos Transgênicos , Mutação , Fosfoproteínas Fosfatases , Antígeno Nuclear de Célula em Proliferação/metabolismo , Fatores de Tempo , Fator de Transcrição AP-1/metabolismoRESUMO
A vital point of convergence for many signaling pathways at cellular focal adhesions is the interaction of two nonreceptor tyrosine kinases, focal adhesion kinase (FAK) and Src. The binding of Src to FAK leads to the phosphorylation of Y(576) and Y(577), located within the activation loop domain of FAK. However, it has not been possible previously to determine the absolute quantitative relationship between phosphorylated and nonphosphorylated forms of this activation loop domain in cells undergoing normal metabolism. We have developed a stable isotope dilution liquid chromatography-multiple reaction monitoring/mass spectrometry (LC-MRM/MS) technique that allows such determinations to be made. Isotopically labeled and phosphorylated FAK protein standards were synthesized and used to control for loss during immunoprecipitation of FAK. A control tryptic peptide, representing an unmodified region of FAK, was employed to monitor the mass balance of post-translational modifications (PTMs) on the activation loop domain. Absolute quantification was conducted using stable isotope labeled peptide standards with four endogenous amino acid overhangs at the trypsin digestion sites of both the amino and carboxy terminus. The LC-MRM/MS method was rigorously validated using in vitro kinase assays and employed to conduct absolute quantification of FAK phosphorylation in normal mouse embryonic fibroblasts (MEFs). This methodology will have particular utility for biomarker studies of kinase-inhibiting anticancer drugs and for quantitative proteomic investigations that examine kinase- and phosphatase-mediated cellular signal transduction pathways.
Assuntos
Proteína-Tirosina Quinases de Adesão Focal/química , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Sequência de Aminoácidos , Cromatografia Líquida , Ativação Enzimática , Proteína-Tirosina Quinases de Adesão Focal/análise , Proteína-Tirosina Quinases de Adesão Focal/síntese química , Humanos , Imunoprecipitação , Marcação por Isótopo , Espectrometria de Massas , Dados de Sequência Molecular , Peptídeos/análise , Peptídeos/química , Peptídeos/metabolismo , Fosforilação , Reprodutibilidade dos Testes , Tripsina/metabolismoRESUMO
Colon cancer arises through a multistep process involving inactivation of tumor suppressor proteins and activation of oncogene-encoded proteins. Development of colon cancer frequently involves mutation of the adenomatous polyposis coli (APC) tumor suppressor. The activity of the proto-oncogene-encoded Src tyrosine kinase is commonly elevated in colon cancer, with higher activity observed as tumors progress and metastasize. Both APC and Src are multifunctional proteins that have been implicated in the control of cell proliferation, but also as regulators of cytoskeletal changes associated with cell motility and invasion. To investigate the potential for biological cooperativity between APC partial loss-of-function and Src gain-of-function, oncogenic Src was stably expressed in mouse colon epithelial cell lines IMCE (APC(+/min)) and YAMC (APC(+/+)). Under permissive growth conditions, these lines are conditionally immortalized through inactivation of p53. Irrespective of the APC genotype or p53 status, oncogenic Src expression led to morphologic transformation associated with loss of cell-cell junctions, cytoskeletal disorganization, and acquisition of invasive properties. However IMCE cells that carry one copy of the mutant APC(min) allele exhibited increased capacity for Src-mediated anchorage-independent proliferation as compared to the YAMC cells, and this property was enhanced under permissive growth conditions. beta-catenin levels and transcriptional activity were also elevated in the Src-transformed IMCE cells. The selective Src inhibitor, AZD0530, was found to be effective in blocking both cell invasion and anchorage-independent proliferation. These findings suggest that the combined effects of elevated Src activity and APC partial loss-of-function may contribute to the growth of colon tumors.
Assuntos
Adesão Celular/fisiologia , Divisão Celular/fisiologia , Transformação Celular Neoplásica/genética , Colo/patologia , Genes APC , Genes src , Animais , Células Epiteliais/patologia , CamundongosRESUMO
Integrins can intercommunicate with cadherins. Here, we examined their possible relationship by use of small interfering RNA-mediated protein knockdown in HeLa cells. We found that a subset of integrin signaling molecules, namely Fak and paxillin, but not p130 Crk-associated substrate or proline-rich tyrosine kinase 2, participate in processes regulating N-cadherin-based cell-cell adhesion. Paxillin was found to be required primarily for the recruitment of Fak to robust focal adhesions. Our results suggest that at least some signals involving Fak are linked to a mechanism down-regulating Rac1 activity at the cell periphery, which appears to be important for the formation of N-cadherin-based adhesions in motile cells. Our analyses simultaneously exemplified the essential role of Fak in the maintenance of cell-cell adhesions in collective cell migration, a type of migration occurring in embryonic development and carcinoma invasion.
Assuntos
Caderinas/fisiologia , Adesão Celular , Integrinas/fisiologia , Transdução de Sinais , Proteínas do Citoesqueleto/fisiologia , Quinase 1 de Adesão Focal , Quinase 2 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Células HeLa , Humanos , Paxilina , Fosfoproteínas/fisiologia , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-cbl , RNA Interferente Pequeno/farmacologia , Ubiquitina-Proteína Ligases/fisiologiaRESUMO
Netrins are an important family of axon guidance cues. Here, we report that netrin-1 induces tyrosine phosphorylation of p130(CAS) (Crk-associated substrate). Our biochemical studies indicate that p130(CAS) is downstream of the Src family kinases and upstream of the small GTPase Rac1 and Cdc42. Inhibition of p130(CAS) signaling blocks both the neurite outgrowth-promoting activity and the axon attraction activity of netrin-1. p130(CAS) RNA interference inhibits the attraction of commissural axons in the spinal cord by netrin-1 and causes defects in commissural axon projection in the embryo. These results demonstrate that p130(CAS) is a key component in the netrin signal transduction pathway and plays an important role in guiding commissural axons in vivo.
Assuntos
Axônios/metabolismo , Proteína Substrato Associada a Crk/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Fatores de Crescimento Neural/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Animais , Axônios/fisiologia , Linhagem Celular , Células Cultivadas , Embrião de Galinha , Feminino , Humanos , Camundongos , Netrina-1 , Gravidez , Proteínas rac1 de Ligação ao GTP/fisiologiaRESUMO
In 2001, The Joint Commission released new pain guidelines and attempted to address the problem of undertreated pain; however, the resulting focus on the pain score number has inadvertently led to some serious adverse drug events. In addition, the recent dramatic increase in opioid use has resulted in greater abuse of these prescribed drugs, often by teenagers.
RESUMO
Tumor cells resist the apoptotic stimuli associated with invasion and metastasis by activating survival signals that suppress apoptosis. Focal adhesion kinase (FAK), a tyrosine kinase that is overexpressed in a variety of human tumors, mediates one of these survival signals. Attenuation of FAK expression in tumor cells results in apoptosis that is mediated by caspase 8- and FADD-dependent pathways, suggesting that death receptor pathways are involved in the process. Here, we report a functional link between FAK and death receptors. We have demonstrated that FAK binds to the death domain kinase receptor-interacting protein (RIP). RIP is a major component of the death receptor complex and has been shown to interact with Fas and tumor necrosis factor receptor 1 through its binding to adapter proteins. We have shown that RIP provides proapoptotic signals that are suppressed by its binding to FAK. We thus propose that FAK overexpression in human tumors provides a survival signal function by binding to RIP and inhibiting its interaction with the death receptor complex.
Assuntos
Apoptose/fisiologia , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Animais , Antígenos CD/genética , Apoptose/efeitos dos fármacos , Sequência de Bases , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Feminino , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Camundongos , Camundongos Knockout , Ligação Proteica , Proteínas Tirosina Quinases/deficiência , Proteínas Tirosina Quinases/genética , Proteínas/genética , RNA Interferente Pequeno/genética , Proteína Serina-Treonina Quinases de Interação com Receptores , Receptores do Fator de Necrose Tumoral/deficiência , Receptores do Fator de Necrose Tumoral/genética , Receptores Tipo I de Fatores de Necrose Tumoral , Transdução de Sinais , Estaurosporina/farmacologiaRESUMO
Crk-associated substrate (CAS, p130Cas) is a major tyrosine phosphorylated protein in cells transformed by v-crk and v-src oncogenes. We recently reported that reexpression of CAS in CAS-deficient mouse embryo fibroblasts transformed by oncogenic Src promoted an invasive phenotype associated with enhanced cell migration through Matrigel, organization of actin into large podosome ring and belt structures, activation of matrix metalloproteinase-2, and elevated tyrosine phosphorylation of the focal adhesion proteins FAK and paxillin. We have now extended these studies to examine the mechanism by which CAS achieves these changes and to evaluate the potential role for CAS in promoting in vivo tumor growth and metastasis. Whereas the presence or absence of CAS did not alter the primary growth of subcutaneous-injected Src-transformed mouse embryo fibroblasts, CAS expression was required to promote lung metastasis following removal of the primary tumor. The substrate domain YxxP tyrosines, the major sites of CAS phosphorylation by Src that mediate interactions with Crk, were found to be critical for promoting both invasive and metastatic properties of the cells. The ability of CAS to promote Matrigel invasion, formation of large podosome structures, and tyrosine phosphorylation of Src substrates, including FAK, paxillin, and cortactin, was also strictly dependent on the YxxP tyrosines. In contrast, matrix metalloproteinase-2 activation was most dependent on the CAS SH3 domain, whereas the substrate domain YxxP sites also contributed to this property. Thus multiple CAS-mediated signaling events are implicated in promoting invasive and metastatic properties of Src-transformed cells.
Assuntos
Movimento Celular , Genes src , Invasividade Neoplásica , Proteínas Oncogênicas de Retroviridae/metabolismo , Tirosina/química , Animais , Sítios de Ligação , Linhagem Celular Transformada , Células Cultivadas , Colágeno/metabolismo , Proteínas do Citoesqueleto/metabolismo , Combinação de Medicamentos , Ativação Enzimática , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Injeções Subcutâneas , Laminina/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Metaloproteinase 2 da Matriz/metabolismo , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteína Oncogênica v-crk , Paxilina , Fosfoproteínas/metabolismo , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteoglicanas/metabolismo , Proteínas Oncogênicas de Retroviridae/genética , Tirosina/metabolismo , Domínios de Homologia de srcRESUMO
Previously we reported that the rapid activation of the Fak/Src multicomponent signaling complex mediates load-induced activation of growth and survival signaling pathways in adult rat heart. In this study, we report that 5% to 20% (10-minute) cyclic stretch (1 Hz) of neonatal rat ventricular myocytes (NRVMs) was paralleled by increases of Fak phosphorylation at Tyr-397 (from 1.5- to 2.8-fold), as detected by anti-Fak-pY397 phosphospecific antibody. Moreover, 15% cyclic stretch lasting from 10 to 120 minutes increased Fak phosphorylation at Tyr-397 by 2.5- to 3.5-fold. This activation was accompanied by a dramatic change in Fak localization in NRVMs from densely concentrated in the perinuclear regions in nonstretched cells to aggregates regularly distributed along the myofilaments in stretched cells. Furthermore, a 4-hour cyclic stretch enhanced the activity of an atrial natriuretic factor (ANF) promoter-luciferase reporter gene by 2.7-fold. Disrupting endogenous Fak/Src signaling either by expression of a dominant-negative Fak mutant with phenylalanine substituted for Tyr-397 or by treatment with a c-Src pharmacological inhibitor (PP-2) markedly attenuated stretch-induced Fak activation and clustering at myofilaments and inhibited stretch-induced ANF gene activation. On the other hand, overexpression of wild-type Fak potentiated the stretch-induced Fak phosphorylation but did not enhance either baseline or stretch-induced ANF promoter-luciferase reporter gene activity compared with the responses of nontransfected NRVMs. These findings identify Fak as an important element in the early responses induced by stretch in cardiac myocytes, indicating that it may coordinate the cellular signaling machinery that controls gene expression program associated with load-induced cardiac myocyte hypertrophy.
Assuntos
Cardiomegalia/enzimologia , Miócitos Cardíacos/fisiologia , Proteínas Tirosina Quinases/metabolismo , Citoesqueleto de Actina/metabolismo , Animais , Animais Recém-Nascidos , Cardiomegalia/etiologia , Células Cultivadas , Ativação Enzimática/fisiologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Genes Reporter , Miócitos Cardíacos/citologia , Miócitos Cardíacos/enzimologia , Fosforilação , Transporte Proteico , Ratos , Ratos Wistar , Transdução de Sinais/fisiologia , Estresse Mecânico , Transfecção , Quinases da Família src/metabolismoRESUMO
In addition to tyrosine sites, FAK (focal adhesion kinase) is phosphorylated on multiple serine residues. In the present study, the regulation of two of these sites, Ser-722 (S1) and Ser-911 (S4), was investigated. Phosphorylation of S1 (but not S4) decreased in resuspended cells, and recovered during spreading on fibronectin, indicating adhesion-dependent regulation. GSK3 (glycogen synthase kinase 3) inhibitors decreased S1 phosphorylation, and siRNA (short interfering RNA) silencing indicated further the involvement of GSK3beta. Furthermore, GSK3beta was found to become activated during cell spreading on fibronectin, and to physically associate with FAK. S1 phosphorylation was observed to decrease in wounded cell monolayers, while GSK3beta underwent inactivation and later was observed to increase to the original level within 24 h. Direct phosphorylation of S1, requiring pre-phosphorylation of Ser-726 in the +4 position, was demonstrated using purified GSK3 and a synthetic peptide containing FAK residues 714-730. An inhibitory role for S1 phosphorylation in FAK signalling was indicated by findings that both alanine substitution for S1 and dephosphorylation of S1 by PP1 (serine/threonine protein phosphatase type-1) resulted in an increase in FAK kinase activity; likewise, this role was also shown by cell treatment with the GSK3 inhibitor LiCl. The inhibitory role was confirmed by the finding that cells expressing FAK with alanine substitution for S1 displayed improved cell spreading and faster migration in wound-healing and trans-well assays. Finally, the finding that S1 phosphorylation increased in cells treated with the PP1 inhibitor okadaic acid indicated targeting of this site by PP1. These results indicate an additional mechanism for regulation of FAK activity during cell spreading and migration, involving Ser-722 phosphorylation modulated through the competing actions of GSK3beta and PP1.
Assuntos
Movimento Celular , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Quinase 3 da Glicogênio Sintase/metabolismo , Fosfoproteínas Fosfatases/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular , Forma Celular , Tamanho Celular , Células Cultivadas , Galinhas , Ativação Enzimática , Fibroblastos/citologia , Fibroblastos/metabolismo , Deleção de Genes , Regulação Enzimológica da Expressão Gênica , Inativação Gênica , Glicogênio Sintase Quinase 3 beta , Humanos , Mutação , Fosforilação , RNA Interferente Pequeno , Coelhos , RatosRESUMO
Extracellular matrix (ECM) remodeling during physiological processes is mediated by invasive protrusions called podosomes. Positioning and dynamics of podosomes define the extent of ECM degradation. Microtubules are known to be involved in podosome regulation, but the role of microtubule (MT) network configuration in podosome dynamics and positioning is not well understood. Here, we show that the arrangement of the microtubule network defines the pattern of podosome formation and relocation in vascular smooth muscle cells (VSMCs). We show that microtubule plus-end targeting facilitates de novo formation of podosomes, in addition to podosome remodeling. Moreover, specialized bent microtubules with plus ends reversed towards the cell center promote relocation of podosomes from the cell edge to the cell center, resulting in an evenly distributed podosome pattern. Microtubule bending is induced downstream of protein kinase C (PKC) activation and requires microtubule-stabilizing proteins known as cytoplasmic linker associated proteins (CLASPs) and retrograde actin flow. Similar to microtubule depolymerization, CLASP depletion by siRNA blocks microtubule bending and eliminates centripetal relocation of podosomes. Podosome relocation also coincides with translocation of podosome-stimulating kinesin KIF1C, which is known to move preferentially along CLASP-associated microtubules. These findings indicate that CLASP-dependent microtubule network configuration is critical to the cellular location and distribution of KIF1C-dependent podosomes. © 2016 Wiley Periodicals, Inc.
Assuntos
Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Podossomos/metabolismo , Actinas/genética , Actinas/metabolismo , Animais , Linhagem Celular , Cinesinas/genética , Cinesinas/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Microtúbulos/genética , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/citologia , Podossomos/genética , RatosRESUMO
CAS ('Crk-associated substrate') is an Src substrate found at sites of integrin-mediated cell adhesion and linked to cell motility and survival. In this study, the involvement of CAS in oncogenic transformation was evaluated through analysis of mouse embryo fibroblast populations expressing an activated Src mutant, either in the presence or absence of CAS expression. CAS was not found to be a critical determinant of either Src-mediated morphologic transformation or anchorage-independent growth. However, CAS had a profound effect on other aspects of oncogenic Src function. CAS expression led to a substantial increase in the phosphotyrosine content of FAK and paxillin, supporting a role for CAS as a positive regulator of Src activity at integrin adhesion sites. Importantly, CAS expression resulted in a striking enhancement of the capacity of Src-transformed cells to invade through Matrigel. The increased invasiveness was associated with increased activation of matrix metalloproteinase MMP-2 and formation of large actin-rich podosomal aggregates appearing as ring and belt structures. Thus, elevated CAS-associated tyrosine phosphorylation signaling events occurring at sites of integrin-mediated cell adhesion can have a major role in the development of an invasive cell phenotype.
Assuntos
Proteína de Suscetibilidade a Apoptose Celular/deficiência , Proteína de Suscetibilidade a Apoptose Celular/genética , Genes src/genética , Invasividade Neoplásica/genética , Animais , Adesão Celular , Divisão Celular , Transformação Celular Neoplásica/genética , Fibroblastos/citologia , Fibroblastos/patologia , Fibroblastos/fisiologia , Integrinas/fisiologia , Camundongos , Camundongos KnockoutRESUMO
Focal adhesion kinase (FAK) signaling may be mediated through the modulation of Ras activity. We have shown previously that grade III malignant astrocytoma biopsy samples exhibit elevated levels of FAK, and that overexpression of FAK in U-251MG malignant astrocytoma cells promotes the phosphorylation of Shc, a potential upstream mediator of Ras activity. Here, we report that overexpression of FAK promotes Ras activity in U-251MG malignant astrocytoma cells cultured in aggregate suspension or as monolayers adherent to vitronectin. The overexpression of FAK also promoted the association of FAK with p120RasGAP, which is a negative regulator of Ras activity, in the U-251MG cells cultured in aggregate suspension, with this association being abrogated upon plating of the cells onto vitronectin. An association of FAK with p120RasGAP also was observed in malignant astrocytoma biopsy samples, but not in normal brain samples. As overexpression of FAK in U-251MG cells in aggregate suspension culture reduced the amount of p120RasGAP complexed with active Ras, we hypothesize that the association of FAK with p120 RasGAP may facilitate Ras activity. The overexpression of a mutated FAK in which the Y397 had been mutated to F did not result in the formation of the FAK/p120RasGAP complex and did not promote Ras activity, indicating that the Y397 residue of FAK plays a role in the formation of this complex and in the activation of Ras. Moreover, the overexpression of mutated FAK (397F) was found to inhibit anchorage-independent growth. These data provide the basis for a previously undescribed mechanism in which the elevated expression of FAK can promote Ras activity through its competitive recruitment of p120RasGAP, thereby diminishing the association of p120RasGAP with active Ras.