RESUMO
The spectrum of congenital anomalies affecting either the upper tract (kidneys and ureters) or lower tract (reproductive organs) of the genitourinary (GU) system are fundamentally linked by the developmental origin of multiple GU tissues, including the kidneys, gonads, and reproductive ductal systems: the intermediate mesoderm. Although â¼31% of DiGeorge/del22q11.2 syndrome patients exhibit GU defects, little focus has been placed on the molecular etiology of GU defects in this syndrome. Among del22q11.2 patients exhibiting GU anomalies, we have mapped the smallest relevant region to only five genes, including CRKLCRKL encodes a src-homology adaptor protein implicated in mediating tyrosine kinase signaling, and is expressed in the developing GU-tract in mice and humans. Here we show that Crkl mutant embryos exhibit gene dosage-dependent growth restriction, and homozygous mutants exhibit upper GU defects at a microdissection-detectable rate of 23%. RNA-sequencing revealed that 52 genes are differentially regulated in response to uncoupling Crkl from its signaling pathways in the developing kidney, including a fivefold up-regulation of Foxd1, a known regulator of nephron progenitor differentiation. Additionally, Crkl heterozygous adult males exhibit cryptorchidism, lower testis weight, lower sperm count, and subfertility. Together, these data indicate that CRKL is intimately involved in normal development of both the upper and lower GU tracts, and disruption of CRKL contributes to the high incidence of GU defects associated with deletion at 22q11.2.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Cromossomos Humanos Par 22/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genitália , Proteínas Nucleares/metabolismo , Sistema Urinário , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Cromossomos Humanos Par 22/genética , Feminino , Genitália/anormalidades , Genitália/embriologia , Humanos , Masculino , Camundongos , Camundongos Knockout , Proteínas Nucleares/genética , Sistema Urinário/anormalidades , Sistema Urinário/embriologiaRESUMO
22q11 deletion (del22q11) syndrome is characterized genetically by heterozygous deletions within chromosome 22q11 and clinically by a constellation of congenital malformations of the aortic arch, heart, thymus, and parathyroid glands described as DiGeorge syndrome (DGS). Here, we report that compound heterozygosity of mouse homologs of two 22q11 genes, CRKL and TBX1, results in a striking increase in the penetrance and expressivity of a DGS-like phenotype compared to heterozygosity at either locus. Furthermore, we show that these two genes have critical dose-dependent functions in pharyngeal segmentation, patterning of the pharyngeal apparatus along the anteroposterior axis, and local regulation of retinoic acid (RA) metabolism and signaling. We can partially rescue one salient feature of DGS in Crkl+/-;Tbx1+/- embryos by genetically reducing the amount of RA produced in the embryo. Thus, we suggest that del22q11 is a contiguous gene syndrome involving dose-sensitive interaction of CRKL and TBX1 and locally aberrant RA signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Síndrome de DiGeorge/metabolismo , Deleção de Genes , Proteínas Nucleares/metabolismo , Transdução de Sinais/fisiologia , Proteínas com Domínio T/metabolismo , Tretinoína/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/deficiência , Animais , Aorta/embriologia , Aorta/metabolismo , Aorta/patologia , Região Branquial/embriologia , Região Branquial/metabolismo , Região Branquial/patologia , Cromossomos Humanos Par 22 , Sistema Enzimático do Citocromo P-450/genética , Sistema Enzimático do Citocromo P-450/metabolismo , Modelos Animais de Doenças , Embrião de Mamíferos , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Genótipo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Humanos , Hibridização In Situ/métodos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/deficiência , Ácido Retinoico 4 Hidroxilase , Proteínas com Domínio T/deficiência , Timo/embriologia , Timo/metabolismo , Timo/patologiaRESUMO
Deletions on chromosome 22q11.21 disrupt pharyngeal and cardiac development and cause DiGeorge and related human syndromes. CRKL (CRK-Like) lies within 22q11.21, and Crkl-/- mice have phenotypic features of 22q11 deletion (del22q11) syndromes. While human FGF8 does not localize to 22q11, deficiency of Fgf8 also generates many features of del22q11 syndrome in mice. Since Fgf8 signals via receptor-type tyrosine kinases, and Crk family adaptor proteins transduce intracellular signals downstream of tyrosine kinases, we investigated whether Crkl mediates Fgf8 signaling. In addition to discovering genetic interactions between Crkl and Fgf8 during morphogenesis of structures affected in del22q11 syndrome, we found that Fgf8 induces tyrosine phosphorylation of FgfRs 1 and 2 and their binding to Crkl. Crkl is required for normal cellular responses to Fgf8, including survival and migration, Erk activation, and target gene expression. These findings provide mechanistic insight into disrupted intercellular interactions in the pathogenesis of malformations seen in del22q11 syndrome.
Assuntos
Cromossomos Humanos Par 22 , Síndrome de DiGeorge/metabolismo , Fator 8 de Crescimento de Fibroblasto/metabolismo , Deleção de Genes , Proteínas Proto-Oncogênicas c-crk/deficiência , Transdução de Sinais/fisiologia , Animais , Apoptose , Western Blotting/métodos , Osso e Ossos/embriologia , Osso e Ossos/metabolismo , Sistema Cardiovascular/embriologia , Sistema Cardiovascular/metabolismo , Contagem de Células/métodos , Células Cultivadas , Fatores Quimiotáticos/metabolismo , Síndrome de DiGeorge/genética , Modelos Animais de Doenças , Embrião de Mamíferos , Ativação Enzimática , Imunofluorescência/métodos , Regulação da Expressão Gênica no Desenvolvimento/genética , Genótipo , Humanos , Camundongos , Camundongos Knockout , Modelos Biológicos , Crista Neural/metabolismo , Faringe/embriologia , Faringe/metabolismo , Fenótipo , Receptores de Fatores de Crescimento de Fibroblastos/metabolismo , Fatores de TempoRESUMO
Adapter proteins CRK and CRKL participate in a variety of signaling pathways, including cell adhesion, and fate regulation of mammalian cells. However, the molecular functions of CRK/CRKL in epigenetic regulation remain largely unknown. Here, we developed a pipeline to evaluate cell morphology using high-content image analysis combined with chemical screening of kinase and epigenetic modulators. We found that CRK/CRKL modulates gene regulatory networks associated with cell morphology through epigenetic alteration in mouse embryonic fibroblasts. Integrated epigenome and transcriptome analyses revealed that CRK/CRKL is involved in super-enhancer activity and upregulation of Cdt1, Rin1, and Spp1 expression for the regulation of cell morphology. Screening of a library of 80 epigenetic inhibitors showed that histone H3 modifiers, euchromatic histone methyltransferase 2 and mitogen- and stress-activated kinase 1, may be important for CRK/CRKL-mediated morphological changes. Taken together, our results indicate that CRK/CRKL plays a critical role in gene regulatory networks through epigenetic modification. DATABASES: Chromatin immunoprecipitation sequencing and RNA sequencing data were deposited in the DNA Data Bank of Japan under DRA011080 and DRA011081 accession numbers, respectively.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/genética , Epigênese Genética/genética , Adesões Focais/genética , Antígenos de Histocompatibilidade/genética , Histona-Lisina N-Metiltransferase/genética , Proteínas Proto-Oncogênicas c-crk/genética , Animais , Proteínas de Ciclo Celular/genética , Forma Celular/genética , Proteínas de Ligação a DNA/genética , Fibroblastos/metabolismo , Fibroblastos/patologia , Histonas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Programas de Rastreamento , Camundongos , Osteopontina/genética , Fosfotransferases/genética , Fosfotransferases/isolamento & purificação , Transdução de Sinais/genéticaRESUMO
CRK and CRKL (CRK-like) encode adapter proteins with similar biochemical properties. Here, we show that a 50% reduction of the family-combined dosage generates developmental defects, including aspects of DiGeorge/del22q11 syndrome in mice. Like the mouse homologs of two 22q11.21 genes CRKL and TBX1, Crk and Tbx1 also genetically interact, thus suggesting that pathways shared by the three genes participate in organogenesis affected in the syndrome. We also show that Crk and Crkl are required during mesoderm development, and Crk/Crkl deficiency results in small cell size and abnormal mesenchyme behavior in primary embryonic fibroblasts. Our systems-wide analyses reveal impaired glycolysis, associated with low Hif1a protein levels as well as reduced histone H3K27 acetylation in several key glycolysis genes. Furthermore, Crk/Crkl deficiency sensitizes MEFs to 2-deoxy-D-glucose, a competitive inhibitor of glycolysis, to induce cell blebbing. Activated Rapgef1, a Crk/Crkl-downstream effector, rescues several aspects of the cell phenotype, including proliferation, cell size, focal adhesions, and phosphorylation of p70 S6k1 and ribosomal protein S6. Our investigations demonstrate that Crk/Crkl-shared pathways orchestrate metabolic homeostasis and cell behavior through widespread epigenetic controls.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Síndrome de DiGeorge/metabolismo , Homeostase/genética , Proteínas Proto-Oncogênicas c-crk/metabolismo , Transdução de Sinais/genética , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Proliferação de Células/genética , Tamanho Celular , Células Cultivadas , Modelos Animais de Doenças , Feminino , Fibroblastos/metabolismo , Adesões Focais/metabolismo , Glucose/metabolismo , Glicólise/genética , Masculino , Mesoderma/crescimento & desenvolvimento , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fosforilação/genética , Proteínas Proto-Oncogênicas c-crk/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , TransfecçãoRESUMO
The oncoprotein c-Jun is a component of the activator protein-1 transcription factor complex, which is involved in cellular proliferation, transformation, and death. The stabilization of c-Jun is critically important for its function. The phosphorylation of c-Jun by c-Jun NH(2)-terminal kinase 1 and extracellular signal-regulated protein kinases reduces c-Jun ubiquitination resulting in increased stabilization of c-Jun. In this report, we showed that COOH-terminal Src kinase (CSK) binds with and phosphorylates c-Jun at Y26 and Y170. Phosphorylation of c-Jun by CSK, in opposition to c-Jun NH(2)-terminal kinase 1 and extracellular signal-regulated protein kinases, promoted c-Jun degradation and reduced stability. By promoting c-Jun degradation, CSK helps to maintain a low steady-state level of c-Jun, thereby inhibiting activator protein-1 activity and cell transformation caused by c-Jun. These results indicated that this function of CSK controls cell proliferation under normal growth conditions and may have implications for CSK loss of function in carcinogenesis.
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Quinases da Família src/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular Tumoral , Células HeLa , Humanos , Camundongos , Proteína Quinase 8 Ativada por Mitógeno/metabolismo , Dados de Sequência Molecular , Células NIH 3T3 , Fosforilação , Ligação Proteica , Fator de Transcrição AP-1/metabolismo , Ubiquitina/metabolismoRESUMO
During brain development, many neurons migrate long distances before settling and differentiating. These migrations are coordinated to ensure normal development. The secreted protein Reelin controls the locations of many types of neurons, and its absence causes the classic "Reeler" phenotype. Reelin action requires tyrosine phosphorylation of the intracellular protein Dab1 by Src-family kinases. However, little is known about signaling pathways downstream of Dab1. Here, we identify several proteins in embryonic brain extract that bind to tyrosine-phosphorylated, but not non-phosphorylated, Dab1. Of these, the Crk-family proteins (CrkL, CrkI, and CrkII ), bind significant quantities of Dab1 when embryonic cortical neurons are exposed to Reelin. CrkL binding to Dab1 involves two tyrosine phosphorylation sites, Y220 and 232, that are critical for proper positioning of migrating cortical plate neurons. CrkL also binds C3G, an exchange factor (GEF) for the small GTPase Rap1 that is activated in other systems by tyrosine phosphorylation. We report that Reelin stimulates tyrosine phosphorylation of C3G and activates Rap1. C3G and Rap1 regulate adhesion of fibroblasts and other cell types. Regulation of Crk/CrkL, C3G, and Rap1 by Reelin may be involved in coordinating neuron migrations during brain development.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Moléculas de Adesão Celular Neuronais/metabolismo , Proteínas da Matriz Extracelular/metabolismo , Fator 2 de Liberação do Nucleotídeo Guanina/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Proteínas Nucleares/metabolismo , Proteínas rap1 de Ligação ao GTP/metabolismo , Animais , Baculoviridae , Western Blotting , Moléculas de Adesão Celular Neuronais/farmacologia , Células Cultivadas , Cromatografia de Afinidade , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/efeitos dos fármacos , Proteínas da Matriz Extracelular/farmacologia , Vetores Genéticos , Espectrometria de Massas , Camundongos , Proteínas Nucleares/isolamento & purificação , Fosforilação/efeitos dos fármacos , Testes de Precipitina , Proteína Reelina , Serina Endopeptidases , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , TransfecçãoRESUMO
Members of the Src family of tyrosine kinases function to phosphorylate focal adhesion (FA) proteins. To explore the overlapping functions of Src kinases, we have targeted Csk, a negative regulator of the Src family, to FA structures. Expression of FA-targeted Csk (FA-Csk) effectively reduced the active form (nonphosphorylated at the C-terminal regulatory tyrosine) of Src members in the cell. We found that fibroblasts expressing FA-Csk lost integrin-mediated adhesion. Activated Src (SrcY529F) as well as activation of putative Src signaling mediators (Fak, Cas, Crk/CrkL, C3G, and Rap1) blocked the effect of FA-Csk in a manner dependent on Rap1. SrcY529F also inhibited activated Ras-induced cell detachment but failed to rescue detachment caused by an activated mutant of Raf1 (Raf-BXB) that Rap1 cannot inhibit. Although normal spreading onto fibronectin was restored by the beta(1) integrin affinity-activating antibody TS2/16 in cells expressing FA-Csk or Raf-BXB, FAs were lost in these cells. On the other hand, Rap1 activation could restore FAs in cells expressing FA-Csk. Activation of the executioner caspase, caspase 3, is essential for many forms of apoptosis. While a caspase 3 inhibitor (Z-DEVD-FMK) inhibited cell detachment triggered by activation of caspase 8, this inhibitor had no effect on cell detachment caused by FA-Csk. Likewise, overexpression of an activated Akt made cells resistant to the effect of caspase 8 activation, but not to the effect of FA-Csk. It is therefore likely that the primary cause of cell rounding and detachment induced by FA-Csk involves dysfunction of FAs rather than caspase-mediated apoptosis that may result from possible loss of survival signals mediated by Src family kinases. We suggest that endogenous Src family kinases are essential for FAs through activation of Rap1 in fibroblasts.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Adesões Focais/fisiologia , Integrinas/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Quinases da Família src/metabolismo , Animais , Inibidores de Caspase , Caspases/metabolismo , Adesão Celular/fisiologia , Linhagem Celular , Ativação Enzimática , Inibidores Enzimáticos/metabolismo , Matriz Extracelular/química , Matriz Extracelular/metabolismo , Fibroblastos/citologia , Fibroblastos/metabolismo , Adesões Focais/enzimologia , Genes Reporter , Humanos , Marcação In Situ das Extremidades Cortadas , Proteínas de Membrana/genética , Camundongos , Microscopia Confocal , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Proteínas Quinases/metabolismo , Transporte Proteico/fisiologia , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas de Protozoários/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais/fisiologia , Transgenes , Proteínas rap1 de Ligação ao GTP/metabolismo , Proteínas Ativadoras de ras GTPase/metabolismo , Quinases da Família src/genéticaRESUMO
The adapter protein Crk-Like (CrkL) can associate with the Src substrate p130(Cas) (Cas). The biological role of CrkL downstream of Cas, however, has been largely obscure. Consistent with the ability of CrkL to biochemically associate with Cas, we found that Src triggers translocation of CrkL to focal adhesions (FAs) in a manner dependent on Cas. Forced localization of CRKL to FAs (FA-CRKL) by itself was sufficient to induce activation of Rac1 and Cdc42 and rescued haptotaxis defects of mouse embryonic fibroblasts (MEFs) lacking Src, Yes, and Fyn, three broadly expressed Src family members required for integrin-induced migration. Consistent with Rac1 activation, FA-CRKL induced cotranslocation of a Rac1 activator, Dock1, to focal adhesions. These results therefore indicate a role for CrkL in mediating Src signaling by activating small G proteins at focal adhesions. Furthermore, MEFs lacking CrkL show impaired integrin-induced migration despite expression of a closely related protein, Crk-II, in these cells. These results therefore provide formal evidence that CrkL plays a specific role in integrin-induced migration as a downstream mediator of Src.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Adesões Focais/fisiologia , Integrinas/fisiologia , Proteínas Nucleares/fisiologia , Quinases da Família src/fisiologia , Animais , Movimento Celular/fisiologia , Células Cultivadas , Citoesqueleto/fisiologia , Fibroblastos/fisiologia , Humanos , Técnicas In Vitro , Camundongos , Camundongos Knockout , Proteínas Nucleares/química , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Proteína cdc42 de Ligação ao GTP/metabolismo , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
The integrin family of cell adhesion receptors are important for a diverse set of biological responses during development. Although many integrins have been shown to engage a similar set of cytoplasmic effector proteins in vitro, the importance of these proteins in the biological events mediated by different integrin receptors and ligands is uncertain. We have examined the role of one of the best-characterized integrin effectors, the focal adhesion protein paxillin, by disruption of the paxillin gene in mice. Paxillin was found to be critically involved in regulating the development of mesodermally derived structures such as heart and somites. The phenotype of the paxillin(-/-) mice closely resembles that of fibronectin(-/-) mice, suggesting that paxillin is a critical transducer of signals from fibronectin receptors during early development. Paxillin was also found to play a critical role in fibronectin receptor biology ex vivo since cultured paxillin-null fibroblasts display abnormal focal adhesions, reduced cell migration, inefficient localization of focal adhesion kinase (FAK), and reduced fibronectin-induced phosphorylation of FAK, Cas, and mitogen-activated protein kinase. In addition, we found that paxillin-null fibroblasts show some defects in the cortical cytoskeleton and cell spreading on fibronectin, raising the possibility that paxillin could play a role in structures distinct from focal adhesions. Thus, paxillin and fibronectin regulate some common embryonic developmental events, possibly due to paxillin modulation of fibronectin-regulated focal adhesion dynamics and organization of the membrane cytoskeletal structures that regulate cell migration and spreading.
Assuntos
Proteínas do Citoesqueleto/fisiologia , Desenvolvimento Embrionário e Fetal/fisiologia , Fibronectinas/fisiologia , Fosfoproteínas/fisiologia , Animais , Movimento Celular/fisiologia , Células Cultivadas , Proteínas do Citoesqueleto/deficiência , Proteínas do Citoesqueleto/genética , Desenvolvimento Embrionário e Fetal/genética , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Adesões Focais/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Marcação de Genes , Mesoderma/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Paxilina , Fenótipo , Fosfoproteínas/deficiência , Fosfoproteínas/genética , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Receptores de Fibronectina/fisiologia , Transdução de Sinais , Tirosina/metabolismoRESUMO
CRK and CRKL adapter proteins play essential roles in development and cancer through their SRC homology 2 and 3 (SH2 and SH3) domains. To gain insight into the origin of their shared functions, we have investigated their evolutionary history. We propose a term, crk/crkl ancestral (crka), for orthologs in invertebrates before the divergence of CRK and CRKL in the vertebrate ancestor. We have isolated two orthologs expressed in the choanoflagellate Monosiga brevicollis, a unicellular relative to the metazoans. Consistent with its highly-conserved three-dimensional structure, the SH2 domain of M. brevicollis crka1 can bind to the mammalian CRK/CRKL SH2 binding consensus phospho-YxxP, and to the SRC substrate/focal adhesion protein BCAR1 (p130CAS) in the presence of activated SRC. These results demonstrate an ancient origin of the CRK/CRKL SH2-target recognition specificity. Although BCAR1 orthologs exist only in metazoans as identified by an N-terminal SH3 domain, YxxP motifs, and a C-terminal FAT-like domain, some pre-metazoan transmembrane proteins include several YxxP repeats in their cytosolic region, suggesting that they are remotely related to the BCAR1 substrate domain. Since the tyrosine kinase SRC also has a pre-metazoan origin, co-option of BCAR1-related sequences may have rewired the crka-dependent network to mediate adhesion signals in the metazoan ancestor.
RESUMO
MAP kinase (MAPK) signaling results from activation of Raf kinases in response to external or internal stimuli. Here, we demonstrate that Raf kinase inhibitory protein (RKIP) regulates the activation of MAPK when B-Raf signaling is defective. We used multiple models including mouse embryonic fibroblasts (MEFs) and primary keratinocytes from RKIP- or Raf-deficient mice as well as allografts in mice to investigate the mechanism. Loss of B-Raf protein or activity significantly reduces MAPK activation in these cells. We show that RKIP depletion can rescue the compromised ERK activation and promote proliferation, and this rescue occurs through a Raf-1 dependent mechanism. These results provide formal evidence that RKIP is a bona fide regulator of Raf-1. We propose a new model in which RKIP plays a key role in regulating the ability of cells to signal through Raf-1 to ERK in B-Raf compromised cells.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Proteínas Proto-Oncogênicas B-raf/metabolismo , Animais , Proliferação de Células , Células Cultivadas , Ativação Enzimática , Sistema de Sinalização das MAP Quinases , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteína de Ligação a Fosfatidiletanolamina/antagonistas & inibidores , Proteína de Ligação a Fosfatidiletanolamina/genética , Proteínas Proto-Oncogênicas B-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-raf/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/genética , Proteínas Proto-Oncogênicas c-raf/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Transdução de SinaisRESUMO
CRKL (CRK-like) is an adapter protein predominantly phosphorylated in cells that express the tyrosine kinase p210(BCR-ABL), the fusion product of a (9;22) chromosomal translocation causative for chronic myeloid leukemia. It has been unclear, however, whether CRKL plays a functional role in p210(BCR-ABL) transformation. Here, we show that CRKL is required for p210(BCR-ABL) to support interleukin-3-independent growth of myeloid progenitor cells and long-term outgrowth of B-lymphoid cells from fetal liver-derived hematopoietic progenitor cells. Furthermore, a synthetic phosphotyrosyl peptide that binds to the CRKL SH2 domain with high affinity blocks association of endogenous CRKL with the p210(BCR-ABL) complex and reduces c-MYC levels in K562 human leukemic cells as well as in mouse hematopoietic cells transformed by p210(BCR-ABL) or the imatinib-resistant mutant T315I. These results indicate that the function of CRKL as an adapter protein is essential for p210(BCR-ABL)-induced transformation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Transformação Celular Neoplásica/metabolismo , Proteínas de Fusão bcr-abl/metabolismo , Células-Tronco Hematopoéticas/metabolismo , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Processos de Crescimento Celular/fisiologia , Transformação Celular Neoplásica/patologia , Células-Tronco Hematopoéticas/patologia , Humanos , Interleucina-3/farmacologia , Células K562 , Camundongos , Camundongos Transgênicos , Dados de Sequência Molecular , Fosforilação , Proteínas Proto-Oncogênicas c-myc/genética , Proteínas Proto-Oncogênicas c-myc/metabolismo , Fator de Transcrição STAT5/metabolismo , Proteína bcl-X/metabolismoRESUMO
The adapter protein CRKL is required for the normal development of multiple tissues that rely on fibroblast growth factor 8 (FGF8). The precise role of CRKL in receptor signaling has been unclear, however. To address this issue, we first modeled the three-dimensional structure of CRKL by molecular dynamics. By taking advantage of structural simulations, we performed in silico analysis of the interactions of the autophosphorylation sites of FGR receptor 1 (FGFR1) with the SH2 domain of CRKL or a highly related protein, CRK. As predicted by simulations, we confirm the specific physical interaction of phosphorylated Y463 (pY463) in FGFR1 with the CRKL SH2 domain at an affinity approximately 30-fold stronger than that of CRK. We also provide evidence that interactions outside of the core YXXP motif have a significant impact on physical association, which is consistent with predictions from molecular-dynamics simulations. Furthermore, we identify CRKL as an essential component of an FGF8-induced feed-forward loop permissive for efficient activation of the mitogen-activated protein kinase Erk1/2, as well as FGF8-induced anchorage-independent cell growth, using Crkl-deficient cells or a pY463 synthetic peptide. Although many cells generally require cell-matrix adhesion, our results demonstrate that CRKL permits cells to bypass the strict need for adhesion in response to FGF8 through direct interaction with receptor.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Retroalimentação Fisiológica/efeitos dos fármacos , Fator 8 de Crescimento de Fibroblasto/farmacologia , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Sequência de Aminoácidos , Animais , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Biologia Computacional , Simulação por Computador , Humanos , MAP Quinase Quinase 1/metabolismo , Camundongos , Modelos Biológicos , Modelos Moleculares , Dados de Sequência Molecular , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Peptídeos/química , Fosforilação/efeitos dos fármacos , Fosfotirosina/metabolismo , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-crk/química , Proteínas Proto-Oncogênicas c-crk/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Relação Estrutura-Atividade , Quinases raf/metabolismo , Domínios de Homologia de srcRESUMO
BACKGROUND: Raf Kinase Inhibitory Protein (RKIP, also PEBP1), a member of the Phosphatidylethanolamine Binding Protein family, negatively regulates growth factor signaling by the Raf/MAP kinase pathway. Since an organic compound, locostatin, was reported to bind RKIP and inhibit cell migration by a Raf-dependent mechanism, we addressed the role of RKIP in locostatin function. METHODS/FINDINGS: We analyzed locostatin interaction with RKIP and examined the biological consequences of locostatin binding on RKIP function. NMR studies show that a locostatin precursor binds to the conserved phosphatidylethanolamine binding pocket of RKIP. However, drug binding to the pocket does not prevent RKIP association with its inhibitory target, Raf-1, nor affect RKIP phosphorylation by Protein Kinase C at a regulatory site. Similarly, exposure of wild type, RKIP-depleted HeLa cells or RKIP-deficient (RKIP(-/-)) mouse embryonic fibroblasts (MEFs) to locostatin has no effect on MAP kinase activation. Locostatin treatment of wild type MEFs causes inhibition of cell migration following wounding. RKIP deficiency impairs migration further, indicating that RKIP protects cells against locostatin-mediated inhibition of migration. Locostatin treatment of depleted or RKIP(-/-) MEFs reveals cytoskeletal disruption and microtubule abnormalities in the spindle. CONCLUSIONS/SIGNIFICANCE: These results suggest that locostatin's effects on cytoskeletal structure and migration are caused through mechanisms independent of its binding to RKIP and Raf/MAP kinase signaling. The protective effect of RKIP against drug inhibition of migration suggests a new role for RKIP in potentially sequestering toxic compounds that may have deleterious effects on cells.
Assuntos
Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Oxazolidinonas/farmacologia , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Animais , Linhagem Celular Tumoral , Movimento Celular , Citoesqueleto/metabolismo , Fibroblastos/metabolismo , Células HeLa , Humanos , Camundongos , Modelos Biológicos , Ligação Proteica , Proteínas Proto-Oncogênicas c-raf/metabolismo , Ratos , Transdução de SinaisRESUMO
BACKGROUND: Raf kinase inhibitory protein (RKIP) belongs to the phosphatidylethanolamine binding protein (PEBP) family that is expressed in both prokaryotic and euakaryotic organisms. OBJECTIVE: In this review, we discuss the role of RKIP as a modulator of signal transduction, the relationship of RKIP to other members of the PEBP family, and the role of RKIP in human health and disease. RESULTS/CONCLUSION: In mammals, RKIP regulates activation of MAPK, NF-kappaB and G protein coupled receptors (GPCRs). As a modulator of key signaling pathways, RKIP affects various cellular processes including cell differentiation, the cell cycle, apoptosis and cell migration. Emerging evidence suggests that RKIP is implicated in several human diseases or disorders, among them metastatic tumorigenesis and Alzheimer's disease.
Assuntos
Proteína de Ligação a Fosfatidiletanolamina/fisiologia , Apoptose/fisiologia , Divisão Celular/fisiologia , Transformação Celular Neoplásica , Instabilidade Genômica/fisiologia , Humanos , NF-kappa B/metabolismo , Metástase Neoplásica , Proteína de Ligação a Fosfatidiletanolamina/química , Proteína de Ligação a Fosfatidiletanolamina/efeitos dos fármacos , Conformação Proteica , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Quinases raf/metabolismoRESUMO
Matk/CHK knockout mice were reported to show no apparent phenotypic abnormalities. This was thought to be due to the homologous kinase Csk that compensates for Matk/CHK. Here, we present the first evidence that the nonreceptor tyrosine kinase, Matk/CHK, is an important modulator of immune cell signaling. We found that the frequency of primitive hematopoietic cells, the side population c-kit(+) Lin(-) Sca-1(+) (SPKLS) cells, in Matk/CHK(-/-) mice was increased 2.2-fold compared with the control mice. Moreover, Matk/CHK deficiency led to significantly higher pre-B cell colony formation following IL-7 stimulation. Interestingly, when mice received the in vivo antigen challenge of TNP-ovalbumin followed by restimulation, the Matk/CHK(-/-) lymph node and spleen cells produced significantly lower IFN-gamma levels compared with the respective wild-type cells. Our study indicates that Matk/CHK is not functionally redundant with Csk, and that this tyrosine kinase plays an important role as a regulator of immunologic responses.
Assuntos
Células-Tronco Hematopoéticas/citologia , Sistema Imunitário/citologia , Proteínas Tirosina Quinases/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Animais , Antígenos/farmacologia , Linfócitos B/citologia , Linhagem da Célula/efeitos dos fármacos , Interferon gama/biossíntese , Interleucina-7/farmacologia , Linfonodos/metabolismo , Camundongos , Camundongos Knockout , Proteínas Tirosina Quinases/deficiência , Proteínas Proto-Oncogênicas pp60(c-src)/deficiência , Transdução de Sinais , Baço/metabolismoRESUMO
Here we report that C-terminal Src kinase (Csk), a tyrosine kinase that negatively regulates the activity of Src and related kinases, is important for vascular development. In Csk(-/-) embryos, although vascular tubules were formed and organized into capillary-like networks during the initial genesis of blood vessels, the vessels failed to engage in normal sprout formation. In chimeric embryos containing both wild-type and Csk(-/-) cells, the presence of wild-type cells enabled Csk(-/-) endothelial cells to participate in branching morphogenesis. We suggest that wild-type cells may have supplied an angiogenic factor absent in Csk(-/-) cells. Despite the partial rescue of vascular development in chimeric embryos, the embryos failed to form vitelline vessels and died at E9.5. These results indicate that Csk is required both for angiogenic sprouting and vascular remodeling.
Assuntos
Capilares/embriologia , Neovascularização Fisiológica/fisiologia , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Animais , Proteína Tirosina Quinase CSK , Capilares/fisiologia , Quimera , Regulação da Expressão Gênica no Desenvolvimento , Camundongos , Camundongos Endogâmicos , Camundongos Mutantes , Quinases da Família src/metabolismoRESUMO
The adapter protein CrkL has been implicated in multiple signal transduction pathways in hematopoietic cells. In T lymphocytes, the recruitment of CrkL-C3G complexes has been correlated with hyporesponsiveness, implicating CrkL as a potential negative regulator. To test this hypothesis we examined T cell activation in CrkL-deficient mice. The CrkL(-/-) genotype was partially embryonic lethal. In viable CrkL(-/-) mice, peripheral blood counts were normal. The thymus from CrkL(-/-) mice had 40% fewer cells compared to littermates, but the proportion of thymocyte subsets was comparable. There was no discernable alteration in T cell function as reflected by T cell numbers, expression of memory markers, IL-2 production, proliferation, and differentiation into Th1/Th2 phenotypes. Immunization induced comparable levels of IgG2a and IgG1 antibodies. Chimeric mice, generated by transfer of CrkL(-/-) fetal liver cells into irradiated RAG2(-/-) recipients, also showed normal T cell function, arguing against selection via partial embryonic lethality. Our results indicate that CrkL is not absolutely required for T cell development or function, and argue against it being an essential component of a negative regulatory pathway in TCR signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Nucleares/fisiologia , Linfócitos T/fisiologia , Animais , Formação de Anticorpos , Diferenciação Celular , Proteínas de Ligação a DNA/fisiologia , Interferon gama/farmacologia , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/deficiência , Proteínas Nucleares/genética , Células Th1/fisiologia , Células Th2/fisiologiaRESUMO
We sought to determine the functional role of the CrkL adapter protein and downstream pathways in interferon signaling. In experiments using CrkL(--) mouse embryonic fibroblasts, we found that CrkL is required for IFN alpha-dependent gene transcription via GAS elements, apparently via the formation of DNA-binding complexes with Stat5. On the other hand, gene transcription via ISRE elements is intact in the absence of CrkL, indicating that the regulatory effects on gene transcription are mediated only via the formation of CrkL:Stat5 complexes. Our studies also indicate that activation of the small GTPase Rap1 by IFN alpha is defective in cells lacking CrkL, indicating that the protein plays a critical role in regulating activation of the growth inhibitory C3G/Rap1 pathway. The IFN alpha-inducible activation of the small GTPase Rap1 requires a functional N-terminus SH3 domain in the CrkL protein, while the C-terminus SH3 domain does not appear to play a role in such a CrkL-function. We also demonstrate that both the Tyk-2 and Jak-1 kinases are required for activation of the CrkL/Rap1 pathway, as the Type I IFN-dependent GTP-bound form of Rap1 is inhibited by overexpression of dominant-negative Tyk-2 or Jak-1 mutants and is defective in cells lacking Tyk-2 or Jak-1. Taken altogether, these findings indicate that CrkL provides an important link between Jak-kinases and downstream cascades that play critical roles in IFN-dependent transcriptional regulation and induction of growth inhibitory responses.