RESUMO
The development and homeostasis of multicellular organisms rely on coordinated regulation of cell migration. Cell migration is an essential event in the construction and regeneration of tissues, and is critical in embryonic development, immunological responses, and wound healing. Dysregulation of cell motility contributes to pathological disorders, such as chronic inflammation and cancer metastasis. Cell migration, tissue invasion, axon, and dendrite outgrowth all initiate with actin polymerization-mediated cell-edge protrusions. Here, we describe a simple, efficient, time-saving method for the imaging and quantitative analysis of cell-edge protrusion dynamics during spreading. This method measures discrete features of cell-edge membrane dynamics, such as protrusions, retractions, and ruffles, and can be used to assess how manipulations of key actin regulators impact cell-edge protrusions in diverse contexts.
Assuntos
Extensões da Superfície Celular , Microscopia , Actinas/metabolismo , Membrana Celular/metabolismo , Movimento Celular/fisiologiaRESUMO
The well-studied members of the Trio family of proteins are Trio and kalirin in vertebrates, UNC-73 in Caenorhabditis elegans and Trio in Drosophila Trio proteins are key regulators of cell morphogenesis and migration, tissue organization, and secretion and protein trafficking in many biological contexts. Recent discoveries have linked Trio and kalirin to human disease, including neurological disorders and cancer. The genes for Trio family proteins encode a series of large multidomain proteins with up to three catalytic activities and multiple scaffolding and protein-protein interaction domains. As such, Trio family proteins engage a wide array of cell surface receptors, substrates and interaction partners to coordinate changes in cytoskeletal regulatory and protein trafficking pathways. We provide a comprehensive review of the specific mechanisms by which Trio family proteins carry out their functions in cells, highlight the biological and cellular contexts in which they occur, and relate how alterations in these functions contribute to human disease.
Assuntos
Caenorhabditis elegans , Transdução de Sinais , Animais , Caenorhabditis elegans/genética , Movimento Celular/genética , Citoesqueleto , Humanos , Morfogênese/genéticaRESUMO
BACKGROUND & AIMS: We investigated whether ABL proto-oncogene 1, non-receptor tyrosine kinase (ABL1) is involved in development of hepatocellular carcinoma (HCC). METHODS: We analyzed clinical and gene expression data from The Cancer Genome Atlas. Albumin-Cre (HepWT) mice and mice with hepatocyte-specific disruption of Abl1 (HepAbl-/- mice) were given hydrodynamic injections of plasmids encoding the Sleeping Beauty transposase and transposons with the MET gene and a catenin ß1 gene with an N-terminal truncation, which induces development of liver tumors. Some mice were then gavaged with the ABL1 inhibitor nilotinib or vehicle (control) daily for 4 weeks. We knocked down ABL1 with short hairpin RNAs in Hep3B and Huh7 HCC cells and analyzed their proliferation and growth as xenograft tumors in mice. We performed RNA sequencing and gene set enrichment analysis of tumors. We knocked down or overexpressed NOTCH1 and MYC in HCC cells and analyzed proliferation. We measured levels of phosphorylated ABL1, MYC, and NOTCH1 by immunohistochemical analysis of an HCC tissue microarray. RESULTS: HCC tissues had higher levels of ABL1 than non-tumor liver tissues, which correlated with shorter survival times of patients. HepWT mice with the MET and catenin ß1 transposons developed liver tumors and survived a median 64 days; HepAbl-/- mice with these transposons developed tumors that were 50% smaller and survived a median 81 days. Knockdown of ABL1 in human HCC cells reduced proliferation, growth as xenograft tumors in mice, and expression of MYC, which reduced expression of NOTCH1. Knockdown of NOTCH1 or MYC in HCC cells significantly reduced cell growth. NOTCH1 or MYC overexpression in human HCC cells promoted proliferation and rescued the phenotype caused by ABL1 knockdown. The level of phosphorylated (activated) ABL1 correlated with levels of MYC and NOTCH1 in human HCC specimens. Nilotinib decreased expression of MYC and NOTCH1 in HCC cell lines, reduced the growth of xenograft tumors in mice, and slowed growth of liver tumors in mice with MET and catenin ß1 transposons, reducing tumor levels of MYC and NOTCH1. CONCLUSIONS: HCC samples have increased levels of ABL1 compared with nontumor liver tissues, and increased levels of ABL1 correlate with shorter survival times of patients. Loss or inhibition of ABL1 reduces proliferation of HCC cells and slows growth of liver tumors in mice. Inhibitors of ABL1 might be used for treatment of HCC.
Assuntos
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Receptor Notch1/metabolismo , Animais , Carcinoma Hepatocelular/tratamento farmacológico , Carcinoma Hepatocelular/mortalidade , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Conjuntos de Dados como Assunto , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Estimativa de Kaplan-Meier , Fígado/patologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/mortalidade , Neoplasias Hepáticas/patologia , Masculino , Camundongos , Fosforilação , Prognóstico , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-abl/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Pirimidinas/farmacologia , Pirimidinas/uso terapêutico , Receptor Notch1/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Fibronectin in the vascular wall promotes inflammatory activation of the endothelium during vascular remodeling and atherosclerosis. These effects are mediated in part by fibronectin binding to integrin α5, which recruits and activates phosphodiesterase 4D5 (PDE4D5) by inducing its dephosphorylation on an inhibitory site Ser651. Active PDE then hydrolyzes anti-inflammatory cAMP to facilitate inflammatory signaling. To test this model in vivo, we mutated the integrin binding site in PDE4D5 in mice. This mutation reduced endothelial inflammatory activation in athero-prone regions of arteries, and, in a hyperlipidemia model, reduced atherosclerotic plaque size while increasing markers of plaque stability. We then investigated the mechanism of PDE4D5 activation. Proteomics identified the PP2A regulatory subunit B55α as the factor recruiting PP2A to PDE4D5. The B55α-PP2A complex localized to adhesions and directly dephosphorylated PDE4D5. This interaction also unexpectedly stabilized the PP2A-B55α complex. The integrin-regulated, pro-atherosclerotic transcription factor Yap is also dephosphorylated and activated through this pathway. PDE4D5 therefore mediates matrix-specific regulation of EC phenotype via an unconventional adapter role, assembling and anchoring a multifunctional PP2A complex with other targets. These results are likely to have widespread consequences for control of cell function by integrins.
Assuntos
Aterosclerose/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/metabolismo , Integrina alfa5beta1/metabolismo , Proteína Fosfatase 2/metabolismo , Sistemas do Segundo Mensageiro , Animais , Aterosclerose/genética , Aterosclerose/patologia , AMP Cíclico/genética , AMP Cíclico/metabolismo , Nucleotídeo Cíclico Fosfodiesterase do Tipo 4/genética , Integrina alfa5beta1/genética , Camundongos , Camundongos Mutantes , Proteína Fosfatase 2/genéticaRESUMO
Platinum-based chemotherapies can result in ovarian insufficiency by reducing the ovarian reserve, a reduction believed to result from apoptosis of immature oocytes via activation/phosphorylation of TAp63α by multiple kinases including CHEK2, CK1, and ABL1. Here we demonstrate that cisplatin (CDDP) induces oocyte apoptosis through a novel pathway and that temporary repression of this pathway fully preserves ovarian function in vivo. Although ABL kinase inhibitors effectively block CDDP-induced apoptosis of oocytes, oocytic ABL1, and ABL2 are dispensable for damage-induced apoptosis. Instead, CDDP activates TAp63α through the ATR > CHEK1 pathway independent of TAp63α hyper-phosphorylation, whereas X-irradiation activates the ATM > CHEK2 > TAp63α-hyper-phosphorylation pathway. Furthermore, oocyte-specific deletion of Trp73 partially protects oocytes from CDDP but not from X-ray, highlighting the fundamental differences of two pathways. Nevertheless, temporary repression of DNA damage response by a kinase inhibitor that attenuates phosphorylation of ATM, ATR, CHEK1, and CHEK2 fully preserves fertility in female mice against CDDP as well as X-ray. Our current study establishes the molecular basis and feasibility of adjuvant therapies to protect ovarian function against two distinctive gonadotoxic therapeutics, CDDP, and ionizing radiation.
Assuntos
Antineoplásicos/efeitos adversos , Ovário/patologia , Insuficiência Ovariana Primária/etiologia , Proteína Supressora de Tumor p53/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose , Feminino , Humanos , Camundongos , Insuficiência Ovariana Primária/patologiaRESUMO
The actin-binding protein cortactin promotes the formation and maintenance of actin-rich structures, including lamellipodial protrusions in fibroblasts and neuronal dendritic spines. Cortactin cellular functions have been attributed to its activation of the Arp2/3 complex, which stimulates actin branch nucleation, and to its recruitment of Rho family GTPase regulators. Cortactin also binds actin filaments and significantly slows filament depolymerization, but the mechanism by which it does so and the relationship between actin binding and stabilization are unclear. Here we elucidated the cortactin regions that are necessary and sufficient for actin filament binding and stabilization. Using actin cosedimentation assays, we found that the cortactin repeat region binds actin but that the adjacent linker region is required for binding with the same affinity as full-length cortactin. Using total internal reflection fluorescence microscopy to measure the rates of single filament actin depolymerization, we observed that cortactin-actin interactions are sufficient to stabilize actin filaments. Moreover, conserved charged residues in repeat 4 were necessary for high-affinity actin binding, and substitution of these residues significantly impaired cortactin-mediated actin stabilization. Cortactin bound actin with higher affinity than did its paralog, hematopoietic cell-specific Lyn substrate 1 (HS1), and the effects on actin stability were specific to cortactin. Finally, cortactin stabilized ADP-actin filaments, indicating that the stabilization mechanism does not depend on the actin nucleotide state. Together, these results indicate that cortactin binding to actin is necessary and sufficient to stabilize filaments in a concentration-dependent manner, specific to conserved residues in the cortactin repeats, and independent of the actin nucleotide state.
Assuntos
Citoesqueleto de Actina/metabolismo , Actinas/química , Actinas/metabolismo , Difosfato de Adenosina/metabolismo , Cortactina/metabolismo , Mutação , Domínios e Motivos de Interação entre Proteínas , Substituição de Aminoácidos , Animais , Cortactina/química , Cortactina/genética , Camundongos , Ligação ProteicaRESUMO
Identifying direct substrates targeted by protein kinases is important in understanding cellular physiology and intracellular signal transduction. Mass spectrometry-based quantitative proteomics provides a powerful tool for comprehensively characterizing the downstream substrates of protein kinases. This approach is efficiently applied to receptor kinases that can be precisely, directly, and rapidly activated by some agent, such as a growth factor. However, nonreceptor tyrosine kinase Abl lacks the experimental advantage of extracellular growth factors as immediate and direct stimuli. To circumvent this limitation, we combine a chemical rescue approach with quantitative phosphoproteomics to identify targets of Abl and their phosphorylation sites with enhanced temporal resolution. Both known and novel putative substrates are identified, presenting opportunities for studying unanticipated functions of Abl under physiological and pathological conditions.
Assuntos
Proteínas Proto-Oncogênicas c-abl/metabolismo , Tirosina/metabolismo , Células 3T3 , Animais , Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Ontologia Genética , Células HEK293 , Humanos , Camundongos , Fosforilação , Mutação Puntual , Proteômica , Proteínas Proto-Oncogênicas c-abl/genética , Tirosina/genéticaRESUMO
BACKGROUND: Severe IgE-mediated, food-induced anaphylactic reactions are characterized by pulmonary venous vasodilatation and fluid extravasation, which are thought to lead to the life-threatening anaphylactic phenotype. The underlying immunologic and cellular processes involved in driving fluid extravasation and the severe anaphylactic phenotype are not fully elucidated. OBJECTIVE: We sought to define the interaction and requirement of IL-4 and vascular endothelial (VE) IL-4 receptor α chain (IL-4Rα) signaling in histamine-abelson murine leukemia viral oncogene homology 1 (ABL1)-mediated VE dysfunction and fluid extravasation in the severity of IgE-mediated anaphylactic reactions in mice. METHODS: Mice deficient in VE IL-4Rα and models of passive and active oral antigen- and IgE-induced anaphylaxis were used to define the requirements of the VE IL-4Rα and ABL1 pathway in severe anaphylactic reactions. The human VE cell line (EA.hy926 cells) and pharmacologic (imatinib) and genetic (short hairpin RNA knockdown of IL4RA and ABL1) approaches were used to define the requirement of this pathway in VE barrier dysfunction. RESULTS: IL-4 exacerbation of histamine-induced hypovolemic shock in mice was dependent on VE expression of IL-4Rα. IL-4- and histamine-induced ABL1 activation in human VE cells and VE barrier dysfunction was ABL1-dependent. Development of severe IgE-mediated hypovolemia and shock required VE-restricted ABL1 expression. Treatment of mice with a history of food-induced anaphylaxis with the ABL kinase inhibitor imatinib protected the mice from severe IgE-mediated anaphylaxis. CONCLUSION: IL-4 amplifies IgE- and histamine-induced VE dysfunction, fluid extravasation, and the severity of anaphylaxis through a VE IL-4Rα/ABL1-dependent mechanism. These studies implicate an important contribution by the VE compartment in the severity of anaphylaxis and identify a new pathway for therapeutic intervention of IgE-mediated reactions.
Assuntos
Anafilaxia/imunologia , Endotélio Vascular/imunologia , Imunoglobulina E/imunologia , Interleucina-4/administração & dosagem , Proteínas Proto-Oncogênicas c-abl/imunologia , Receptores de Interleucina-4/imunologia , Alérgenos/administração & dosagem , Alérgenos/imunologia , Animais , Anticorpos/administração & dosagem , Linhagem Celular , Feminino , Histamina/administração & dosagem , Humanos , Mesilato de Imatinib/farmacologia , Masculino , Camundongos Endogâmicos BALB C , Camundongos Transgênicos , Ovalbumina/administração & dosagem , Receptores de Interleucina-4/genética , Choque/imunologiaRESUMO
Super-resolution imaging of live cells over extended time periods with high temporal resolution requires high-density labeling and extraordinary fluorophore photostability. Herein, we achieve this goal by combining the attributes of the high-density plasma membrane probe DiI-TCO and the photostable STED dye SiR-Tz. These components undergo rapid tetrazine ligation within the plasma membrane to generate the HIDE probe DiI-SiR. Using DiI-SiR, we visualized filopodia dynamics in HeLa cells over 25â min at 0.5â s temporal resolution, and visualized dynamic contact-mediated repulsion events in primary mouse hippocampal neurons over 9â min at 2â s temporal resolution. HIDE probes such as DiI-SiR are non-toxic and do not require transfection, and their apparent photostability significantly improves the ability to monitor dynamic processes in live cells at super-resolution over biologically relevant timescales.
Assuntos
Membrana Celular/química , Corantes Fluorescentes/química , Nanotecnologia , Imagem Óptica , Células HeLa , Humanos , Microscopia de Fluorescência , Estrutura Molecular , Células Tumorais CultivadasRESUMO
Breast carcinoma cells use specialized, actin-rich protrusions called invadopodia to degrade and invade through the extracellular matrix. Phosphorylation of the actin nucleation-promoting factor and actin-stabilizing protein cortactin downstream of the epidermal growth factor receptor-Src-Arg kinase cascade is known to be a critical trigger for invadopodium maturation and subsequent cell invasion in breast cancer cells. The functions of cortactin phosphorylation in this process, however, are not completely understood. We identify the Rho-family guanine nucleotide exchange factor Vav2 in a comprehensive screen for human SH2 domains that bind selectively to phosphorylated cortactin. We demonstrate that the Vav2 SH2 domain binds selectively to phosphotyrosine-containing peptides corresponding to cortactin tyrosines Y421 and Y466 but not to Y482. Mutation of the Vav2 SH2 domain disrupts its recruitment to invadopodia, and an SH2-domain mutant form of Vav2 cannot support efficient matrix degradation in invasive MDA-MB-231 breast cancer cells. We show that Vav2 function is required for promoting invadopodium maturation and consequent actin polymerization, matrix degradation, and invasive migratory behavior. Using biochemical assays and a novel Rac3 biosensor, we show that Vav2 promotes Rac3 activation at invadopodia. Rac3 knockdown reduces matrix degradation by invadopodia, whereas a constitutively active Rac3 can rescue the deficits in invadopodium function in Vav2-knockdown cells. Together these data indicate that phosphorylated cortactin recruits Vav2 to activate Rac3 and promote invadopodial maturation in invasive breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Cortactina/metabolismo , Podossomos/metabolismo , Proteínas Proto-Oncogênicas c-vav/metabolismo , Actinas/metabolismo , Linhagem Celular Tumoral , Matriz Extracelular/metabolismo , Fatores de Troca do Nucleotídeo Guanina/metabolismo , Humanos , Invasividade Neoplásica , Fosforilação , Fosfotirosina/metabolismo , Podossomos/fisiologia , Proteínas Tirosina Quinases/metabolismo , Pseudópodes/metabolismo , Proteínas rac de Ligação ao GTP/metabolismoRESUMO
Cellular identity in metazoan organisms is frequently established through lineage-specifying transcription factors, which control their own expression through transcriptional positive feedback, while antagonizing the developmental networks of competing lineages. Here, we have uncovered a distinct positive feedback loop that arises from the reciprocal stabilization of the tyrosine kinase ABL and the transcriptional coactivator TAZ. Moreover, we determined that this loop is required for osteoblast differentiation and embryonic skeletal formation. ABL potentiated the assembly and activation of the RUNX2-TAZ master transcription factor complex that is required for osteoblastogenesis, while antagonizing PPARγ-mediated adipogenesis. ABL also enhanced TAZ nuclear localization and the formation of the TAZ-TEAD complex that is required for osteoblast expansion. Last, we have provided genetic data showing that regulation of the ABL-TAZ amplification loop lies downstream of the adaptor protein 3BP2, which is mutated in the craniofacial dysmorphia syndrome cherubism. Our study demonstrates an interplay between ABL and TAZ that controls the mesenchymal maturation program toward the osteoblast lineage and is mechanistically distinct from the established model of lineage-specific maturation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Núcleo Celular/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/metabolismo , Osteoblastos/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Núcleo Celular/genética , Querubismo/genética , Querubismo/metabolismo , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Células HEK293 , Humanos , Camundongos , Camundongos Knockout , PPAR gama/genética , PPAR gama/metabolismo , Proteínas Proto-Oncogênicas c-abl/genética , TransativadoresRESUMO
Leishmaniasis is a devastating disease that disfigures or kills nearly two million people each year. Establishment and persistence of infection by the obligate intracellular parasite Leishmania requires repeated uptake by macrophages and other phagocytes. Therefore, preventing uptake could be a novel therapeutic strategy for leishmaniasis. Amastigotes, the life cycle stage found in the human host, bind Fc receptors and enter macrophages primarily through immunoglobulin-mediated phagocytosis. However, the host machinery that mediates amastigote uptake is poorly understood. We have previously shown that the Arg (also known as Abl2) non-receptor tyrosine kinase facilitates L. amazonensis amastigote uptake by macrophages. Using small-molecule inhibitors and primary macrophages lacking specific Src family kinases, we now demonstrate that the Hck, Fgr and Lyn kinases are also necessary for amastigote uptake by macrophages. Src-mediated Arg activation is required for efficient uptake. Interestingly, the dual Arg and Src kinase inhibitor bosutinib, which is approved to treat cancer, not only decreases amastigote uptake, but also significantly reduces disease severity and parasite burden in Leishmania-infected mice. Our results suggest that leishmaniasis could potentially be treated with host-cell-active agents such as kinase inhibitors.
Assuntos
Imunoglobulina G/metabolismo , Leishmania/patogenicidade , Leishmaniose/parasitologia , Fagocitose , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-hck/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Quinases da Família src/metabolismo , Compostos de Anilina/farmacologia , Animais , Citocinas/metabolismo , Modelos Animais de Doenças , Mesilato de Imatinib/farmacologia , Leishmania/efeitos dos fármacos , Leishmaniose/enzimologia , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Macrófagos/parasitologia , Camundongos , Modelos Biológicos , Nitrilas/farmacologia , Parasitos/efeitos dos fármacos , Parasitos/parasitologia , Fagocitose/efeitos dos fármacos , Fosforilação/efeitos dos fármacos , Pirimidinas/farmacologia , Quinolinas/farmacologia , Células RAW 264.7 , Transdução de Sinais/efeitos dos fármacosRESUMO
ABL2/ARG (ABL-related gene) belongs to the ABL (Abelson tyrosine-protein kinase) family of tyrosine kinases. ARG plays important roles in cell morphogenesis, motility, growth and survival, and many of these biological roles overlap with the cellular functions of the ABL kinase. Chronic myeloid leukemia (CML) is associated with constitutive ABL kinase activation resulting from fusion between parts of the breakpoint cluster region (BCR) and ABL1 genes. Similarly, fusion of the ETV6 (Tel) and ARG genes drives some forms of T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Dasatinib is a tyrosine kinase inhibitor used for the treatment of CML by inhibiting ABL, and while it also inhibits ARG, there is currently no structure of ARG in complex with dasatinib. Here, the co-crystal structure of the mouse ARG catalytic domain with dasatinib at 2.5â Å resolution is reported. Dasatinib-bound ARG is found in the DFG-in conformation although it is nonphosphorylated on the activation-loop tyrosine. In this structure the glycine-rich P-loop is found in a relatively open conformation compared with other known ABL family-inhibitor complex structures.
Assuntos
Dasatinibe/química , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/química , Animais , Dasatinibe/metabolismo , Humanos , Camundongos , Estrutura Secundária de Proteína , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/metabolismoRESUMO
Integrins are heterodimeric α/ß extracellular matrix adhesion receptors that couple physically to the actin cytoskeleton and regulate kinase signaling pathways to control cytoskeletal remodeling and adhesion complex formation and disassembly. ß1 integrins signal through the Abl2/Arg (Abl-related gene) nonreceptor tyrosine kinase to control fibroblast cell motility, neuronal dendrite morphogenesis and stability, and cancer cell invasiveness, but the molecular mechanisms by which integrin ß1 activates Arg are unknown. We report here that the Arg kinase domain interacts directly with a lysine-rich membrane-proximal segment in the integrin ß1 cytoplasmic tail, that Arg phosphorylates the membrane-proximal Tyr-783 in the ß1 tail, and that the Arg Src homology domain then engages this phosphorylated region in the tail. We show that these interactions mediate direct binding between integrin ß1 and Arg in vitro and in cells and activate Arg kinase activity. These findings provide a model for understanding how ß1-containing integrins interact with and activate Abl family kinases.
Assuntos
Integrina beta1/metabolismo , Proteínas Tirosina Quinases/metabolismo , Sequência de Aminoácidos , Domínio Catalítico , Ativação Enzimática , Células HEK293 , Humanos , Integrina beta1/química , Dados de Sequência Molecular , Fosforilação , Domínios e Motivos de Interação entre Proteínas , Mapeamento de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/químicaRESUMO
Both Arp2/3 complex and the Abl2/Arg nonreceptor tyrosine kinase are essential to form and maintain diverse actin-based structures in cells, including cell edge protrusions in fibroblasts and cancer cells and dendritic spines in neurons. The ability of Arg to promote cell edge protrusions in fibroblasts does not absolutely require kinase activity, raising the question of how Arg might modulate actin assembly and turnover in the absence of kinase function. Arg has two distinct actin-binding domains and interacts physically and functionally with cortactin, an activator of the Arp2/3 complex. However, it was not known whether and how Arg influences actin filament stability, actin branch formation, or cofilin-mediated actin severing or how cortactin influences these reactions of Arg with actin. Arg or cortactin bound to actin filaments stabilizes them from depolymerization. Low concentrations of Arg and cortactin cooperate to stabilize filaments by slowing depolymerization. Arg stimulates formation of actin filament branches by Arp2/3 complex and cortactin. An Arg mutant lacking the C-terminal calponin homology actin-binding domain stimulates actin branch formation by the Arp2/3 complex, indicative of autoinhibition. ArgΔCH can stimulate the Arp2/3 complex even in the absence of cortactin. Arg greatly potentiates cofilin severing of actin filaments, and cortactin attenuates this enhanced severing. The ability of Arg to stabilize filaments, promote branching, and increase severing requires the internal (I/L)WEQ actin-binding domain. These activities likely underlie important roles that Arg plays in the formation, dynamics, and stability of actin-based cellular structures.
Assuntos
Citoesqueleto de Actina/metabolismo , Complexo 2-3 de Proteínas Relacionadas à Actina/metabolismo , Actinas/metabolismo , Cortactina/metabolismo , Proteínas Tirosina Quinases/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Proteína 2 Relacionada a Actina , Actinas/química , Animais , Arginina/farmacologia , Cofilina 1/metabolismo , Citoesqueleto/metabolismo , Humanos , Camundongos , Microscopia de Fluorescência , Fosforilação/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacosRESUMO
The closely related Abl family kinases, Arg and Abl, play important non-redundant roles in the regulation of cell morphogenesis and motility. Despite similar N-terminal sequences, Arg and Abl interact with different substrates and binding partners with varying affinities. This selectivity may be due to slight differences in amino acid sequence leading to differential interactions with target proteins. We report that the Arg Src homology (SH) 2 domain binds two specific phosphotyrosines on cortactin, a known Abl/Arg substrate, with over 10-fold higher affinity than the Abl SH2 domain. We show that this significant affinity difference is due to the substitution of arginine 161 and serine 187 in Abl to leucine 207 and threonine 233 in Arg, respectively. We constructed Abl SH2 domains with R161L and S187T mutations alone and in combination and find that these substitutions are sufficient to convert the low affinity Abl SH2 domain to a higher affinity "Arg-like" SH2 domain in binding to a phospho-cortactin peptide. We crystallized the Arg SH2 domain for structural comparison to existing crystal structures of the Abl SH2 domain. We show that these two residues are important determinants of Arg and Abl SH2 domain binding specificity. Finally, we expressed Arg containing an "Abl-like" low affinity mutant Arg SH2 domain (L207R/T233S) and find that this mutant, although properly localized to the cell periphery, does not support wild type levels of cell edge protrusion. Together, these observations indicate that these two amino acid positions confer different binding affinities and cellular functions on the distinct Abl family kinases.
Assuntos
Cortactina/química , Proteínas Proto-Oncogênicas c-abl/química , Substituição de Aminoácidos , Animais , Células Cultivadas , Cortactina/genética , Cortactina/metabolismo , Cristalografia por Raios X , Fibroblastos , Humanos , Camundongos , Camundongos Knockout , Mutação de Sentido Incorreto , Ligação Proteica , Proteínas Proto-Oncogênicas c-abl/genética , Proteínas Proto-Oncogênicas c-abl/metabolismo , Relação Estrutura-Atividade , Domínios de Homologia de srcRESUMO
Dendritic spine loss is observed in many psychiatric disorders, including schizophrenia, and likely contributes to the altered sense of reality, disruption of working memory, and attention deficits that characterize these disorders. ErbB4, a member of the EGF family of receptor tyrosine kinases, is genetically associated with schizophrenia, suggesting that alterations in ErbB4 function contribute to the disease pathology. Additionally, ErbB4 functions in synaptic plasticity, leading us to hypothesize that disruption of ErbB4 signaling may affect dendritic spine development. We show that dendritic spine density is reduced in the dorsomedial prefrontal cortex of ErbB4 conditional whole-brain knockout mice. We find that ErbB4 localizes to dendritic spines of excitatory neurons in cortical neuronal cultures and is present in synaptic plasma membrane preparations. Finally, we demonstrate that selective ablation of ErbB4 from excitatory neurons leads to a decrease in the proportion of mature spines and an overall reduction in dendritic spine density in the prefrontal cortex of weanling (P21) mice that persists at 2 months of age. These results suggest that ErbB4 signaling in excitatory pyramidal cells is critical for the proper formation and maintenance of dendritic spines in excitatory pyramidal cells.
Assuntos
Espinhas Dendríticas/fisiologia , Regulação da Expressão Gênica/genética , Neurônios/ultraestrutura , Córtex Pré-Frontal/citologia , Receptor ErbB-4/deficiência , Fatores Etários , Animais , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/metabolismo , Fracionamento Celular , Células Cultivadas , Espinhas Dendríticas/metabolismo , Proteína 4 Homóloga a Disks-Large , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Guanilato Quinases/metabolismo , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Transgênicos , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Nestina/genética , Nestina/metabolismo , Neurônios/metabolismo , Córtex Pré-Frontal/embriologia , Córtex Pré-Frontal/crescimento & desenvolvimento , Receptor ErbB-4/genética , Sinapses/metabolismo , TransfecçãoRESUMO
The Abelson (Abl) non-receptor tyrosine kinase regulates the cytoskeleton during multiple stages of neural development, from neurulation, to the articulation of axons and dendrites, to synapse formation and maintenance. We previously showed that Abl is genetically linked to the microtubule (MT) plus end tracking protein (+TIP) CLASP in Drosophila. Here we show in vertebrate cells that Abl binds to CLASP and phosphorylates it in response to serum or PDGF stimulation. In vitro, Abl phosphorylates CLASP with a Km of 1.89 µM, indicating that CLASP is a bona fide substrate. Abl-phosphorylated tyrosine residues that we detect in CLASP by mass spectrometry lie within previously mapped F-actin and MT plus end interaction domains. Using purified proteins, we find that Abl phosphorylation modulates direct binding between purified CLASP2 with both MTs and actin. Consistent with these observations, Abl-induced phosphorylation of CLASP2 modulates its localization as well as the distribution of F-actin structures in spinal cord growth cones. Our data suggest that the functional relationship between Abl and CLASP2 is conserved and provides a means to control the CLASP2 association with the cytoskeleton.
Assuntos
Actinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Microtúbulos/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Citoesqueleto de Actina/metabolismo , Sequência de Aminoácidos , Animais , Células COS , Adesão Celular/efeitos dos fármacos , Chlorocebus aethiops , Cones de Crescimento/efeitos dos fármacos , Cones de Crescimento/metabolismo , Células HEK293 , Humanos , Proteínas Associadas aos Microtúbulos/química , Microtúbulos/efeitos dos fármacos , Dados de Sequência Molecular , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Fator de Crescimento Derivado de Plaquetas/farmacologia , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Frações Subcelulares/efeitos dos fármacos , Frações Subcelulares/metabolismo , Especificidade por Substrato/efeitos dos fármacos , XenopusRESUMO
Soluble amyloid-ß oligomers (Aßo) trigger Alzheimer's disease (AD) pathophysiology and bind with high affinity to cellular prion protein (PrP(C)). At the postsynaptic density (PSD), extracellular Aßo bound to lipid-anchored PrP(C) activates intracellular Fyn kinase to disrupt synapses. Here, we screened transmembrane PSD proteins heterologously for the ability to couple Aßo-PrP(C) with Fyn. Only coexpression of the metabotropic glutamate receptor, mGluR5, allowed PrP(C)-bound Aßo to activate Fyn. PrP(C) and mGluR5 interact physically, and cytoplasmic Fyn forms a complex with mGluR5. Aßo-PrP(C) generates mGluR5-mediated increases of intracellular calcium in Xenopus oocytes and in neurons, and the latter is also driven by human AD brain extracts. In addition, signaling by Aßo-PrP(C)-mGluR5 complexes mediates eEF2 phosphorylation and dendritic spine loss. For mice expressing familial AD transgenes, mGluR5 antagonism reverses deficits in learning, memory, and synapse density. Thus, Aßo-PrP(C) complexes at the neuronal surface activate mGluR5 to disrupt neuronal function.
Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Neurônios/metabolismo , Proteínas PrPC/metabolismo , Proteínas Proto-Oncogênicas c-fyn/metabolismo , Receptores de Glutamato Metabotrópico/fisiologia , Transdução de Sinais/fisiologia , Doença de Alzheimer/fisiopatologia , Animais , Cálcio/metabolismo , Células Cultivadas , Quinase do Fator 2 de Elongação/metabolismo , Células HEK293 , Humanos , Camundongos , Oócitos , Fosforilação , Densidade Pós-Sináptica/metabolismo , Receptor de Glutamato Metabotrópico 5 , XenopusRESUMO
BACKGROUND: Epithelial morphogenesis is a dynamic process that involves coordination of signaling and actin cytoskeletal rearrangements. PRINCIPAL FINDINGS: We analyzed the contribution of the branched actin regulator WAVE2 in the development of 3-dimensional (3D) epithelial structures. WAVE2-knockdown (WAVE2-KD) cells formed large multi-lobular acini that continued to proliferate at an abnormally late stage compared to control acini. Immunostaining of the cell-cell junctions of WAVE2-KD acini revealed weak and heterogeneous E-cadherin staining despite little change in actin filament localization to the same junctions. Analysis of cadherin expression demonstrated a decrease in E-cadherin and an increase in N-cadherin protein and mRNA abundance in total cell lysates. In addition, WAVE2-KD cells exhibited an increase in the mRNA levels of the epithelial-mesenchymal transition (EMT)-associated transcription factor Twist1. KD of Twist1 expression in WAVE2-KD cells reversed the cadherin switching and completely rescued the aberrant 3D morphological phenotype. Activity of the WAVE2 complex binding partner Abl kinase was also increased in WAVE2-KD cells, as assessed by tyrosine phosphorylation of the Abl substrate CrkL. Inhibition of Abl with STI571 rescued the multi-lobular WAVE2-KD 3D phenotype whereas overexpression of Abl kinase phenocopied the WAVE2-KD phenotype. CONCLUSIONS: The WAVE2 complex regulates breast epithelial morphology by a complex mechanism involving repression of Twist1 expression and Abl kinase activity. These data reveal a critical role for WAVE2 complex in regulation of cellular signaling and epithelial morphogenesis.