RESUMO
F-BAR proteins are multivalent adaptors that link plasma membrane and cytoskeleton and coordinate cellular processes such as membrane protrusion and migration. Yet, little is known about the function of F-BAR proteins in vivo. Here we report, that the F-BAR protein NOSTRIN is necessary for proper vascular development in zebrafish and postnatal retinal angiogenesis in mice. The loss of NOSTRIN impacts on the migration of endothelial tip cells and leads to a reduction of tip cell filopodia number and length. NOSTRIN forms a complex with the GTPase Rac1 and its exchange factor Sos1 and overexpression of NOSTRIN in cells induces Rac1 activation. Furthermore, NOSTRIN is required for fibroblast growth factor 2 dependent activation of Rac1 in primary endothelial cells and the angiogenic response to fibroblast growth factor 2 in the in vivo matrigel plug assay. We propose a novel regulatory circuit, in which NOSTRIN assembles a signalling complex containing FGFR1, Rac1 and Sos1 thereby facilitating the activation of Rac1 in endothelial cells during developmental angiogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Vasos Sanguíneos/embriologia , Proteínas de Ligação a DNA/fisiologia , Fatores de Crescimento de Fibroblastos/metabolismo , Neovascularização Fisiológica/genética , Proteínas Adaptadoras de Transdução de Sinal/química , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Animais Geneticamente Modificados , Animais Recém-Nascidos , Vasos Sanguíneos/crescimento & desenvolvimento , Vasos Sanguíneos/fisiologia , Células CHO , Células Cultivadas , Cricetinae , Cricetulus , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Embrião de Mamíferos , Embrião não Mamífero , Fatores de Crescimento de Fibroblastos/fisiologia , Camundongos , Camundongos Knockout , Modelos Biológicos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Peixe-Zebra/embriologia , Peixe-Zebra/genéticaRESUMO
BACKGROUND: The c-Cbl-associated protein (CAP), also known as ponsin, localizes to focal adhesions and stress fibers and is involved in signaling events. Phosphorylation has been described for the other two members of the sorbin homology family, vinexin and ArgBP2, but no data exist about the putative phosphorylation of CAP. According to previous findings, CAP binds to tyrosine kinase c-Abl. However, it is not known if CAP is a substrate of c-Abl or other tyrosine kinases or if phosphorylation regulates its localization. RESULTS: We here show that CAP is Tyr phosphorylated by and interacts with both c-Abl and c-Src. One major phosphorylation site, Tyr360, and two minor contributors Tyr326 and Tyr632 were identified as Abl phosphorylation sites, whereas Src preferentially phosphorylates Tyr326 and Tyr360. Phosphorylation of CAP was not necessary for its localization to focal adhesions and stress fibers, but Tyr326Phe substitution alters the function of CAP during cell spreading. CONCLUSION: This is the first demonstration of phosphorylation of CAP by any kinase. Our findings suggest that coordinated action of Src and Abl might regulate the function of CAP and reveal a functional role especially for the Src-mediated Tyr phosphorylation of CAP in cell spreading.
Assuntos
Proteínas dos Microfilamentos/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-abl/metabolismo , Tirosina/metabolismo , Substituição de Aminoácidos , Proteína Tirosina Quinase CSK , Linhagem Celular Tumoral , Adesões Focais/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Insulina/farmacologia , Proteínas dos Microfilamentos/análise , Mutagênese Sítio-Dirigida , Fosforilação , Fibras de Estresse/metabolismo , Vanadatos/farmacologia , Quinases da Família srcRESUMO
We recently observed that a novel, shortened variant of eNOS trafficking inducer (NOSTRIN) is expressed in cirrhotic liver. This shortened variant (NOSTRINbeta) lacks the first 78 amino acids of full-length NOSTRIN (NOSTRINalpha) and thus a substantial part of its F-BAR domain. In contrast to NOSTRINalpha, NOSTRINbeta mainly localizes to the cell nucleus. In this study, we show that nuclear import of NOSTRINbeta depends on two nuclear localization signals (aa 32-36: KKRK and aa 57-61: KAKKK). Each of the sequences is independently functional, but both are required to sustain nuclear localization of NOSTRINbeta. Export of NOSTRINbeta from the nucleus is facilitated by a CRM1-dependent mechanism relying on the nuclear export sequence LELEKERIQL (aa 135-145). Unlike NOSTRINbeta, the full-length variant NOSTRINalpha was conspicuously absent from the nucleus. This is most likely because of the fact that its N-terminal F-BAR domain, which is truncated in NOSTRINbeta, facilitates association with cellular membranes. NOSTRINbeta directly binds to the 5'-regulatory region of the NOSTRIN gene (bp -200 to -1), and overexpression of NOSTRINbeta strongly decreases transcription of a reporter gene under control of this DNA region. Taken together, our results suggest that nuclear NOSTRINbeta may negatively regulate transcription of the NOSTRIN gene.