RESUMO
AFAP-110 has an intrinsic ability to alter actin filament integrity as an actin filament crosslinking protein. This capability is regulated by a carboxy terminal leucine zipper (Lzip) motif. The Lzip motif facilitates self-association stabilizing the AFAP-110 multimers. Deletion of the Lzip motif (AFAP-110(Deltalzip)) reduces the stability of the AFAP-110 multimer and concomitantly increases its ability to crosslink actin filaments, in vitro, and to activate cSrc and alter actin filament integrity, in vivo. We sought to determine how the Lzip motif regulates AFAP-110 function. Substitution of the c-Fos Lzip motif in place of the AFAP-110 Lzip motif (AFAP-110(fos)) was predicted to preserve the alpha-helical structure while changing the sequence. To alter the structure of the alpha-helix, a leucine to proline mutation was generated in the AFAP-110 alpha-helical Lzip motif (AFAP-110(581P)), which largely preserved the sequence. The helix mutants, AFAP-110(Deltalzip), AFAP-110(fos), and AFAP-110(581P), demonstrated reduced multimer stability with an increased capacity to crosslink actin filaments, in vitro, relative to AFAP-110. An analysis of opposing binding sites indicated that the carboxy terminus/Lzip motif can contact sequences within the amino terminal pleckstrin homology (PH1) domain indicating an auto-inhibitory mechanism for regulating multimer stability and actin filament crosslinking. In vivo, only AFAP-110(Deltalzip) and AFAP-110(581P) were to activate cSrc and to alter cellular actin filament integrity. These data indicate that the intrinsic ability of AFAP-110 to crosslink actin filaments is dependent upon both the sequence and structure of the Lzip motif, while the ability of the Lzip motif to regulate AFAP-110-directed activation of cSrc and changes in actin filament integrity in vivo is dependent upon the structure or presence of the Lzip motif. We hypothesize that the intrinsic ability of AFAP-110 to crosslink actin filaments or activate cSrc are distinct functions.
Assuntos
Citoesqueleto de Actina/fisiologia , Zíper de Leucina/fisiologia , Proteínas dos Microfilamentos/fisiologia , Fosfoproteínas/fisiologia , Animais , Western Blotting , Células COS , Chlorocebus aethiops , Cromatografia Líquida , Clonagem Molecular , Componentes do Gene/genética , Componentes do Gene/fisiologia , Genes fos/genética , Glutationa Transferase/genética , Glutationa Transferase/metabolismo , Proteínas de Fluorescência Verde , Zíper de Leucina/genética , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Proteínas dos Microfilamentos/química , Proteínas dos Microfilamentos/genética , Microscopia Eletrônica , Microscopia de Fluorescência , Modelos Biológicos , Mutagênese Sítio-Dirigida , Fosfoproteínas/química , Fosfoproteínas/genética , Ligação Proteica/fisiologia , Estrutura Quaternária de Proteína , Estrutura Secundária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Quinases da Família src/metabolismoRESUMO
c-Src and c-Yes are highly homologous members of the Src family of non-receptor tyrosine kinases. The overall sequence similarity between c-Src and c-Yes allows them to perform many overlapping functions. However, the phenotypes of the c-src and c-yes knockout mice, and cells derived from them, are quite different, indicating functional specificity between the two proteins. Specifically, c-src-/- cells are deficient in several processes that require dynamic regulation of the actin cytoskeleton. In order to begin to understand why c-Yes is unable to compensate for c-Src signaling, we used a series of Src/Yes chimeras in which the non-catalytic functional domains of Src527F were replaced by those of c-Yes. Using chicken embryo fibroblasts as a model system, our results indicate that the c-Yes N-terminal SH4-Unique domains are sufficient to inhibit the ability of Src527F to alter cell morphology, induce actin filament rearrangements or stimulate motility or invasive potential. The data also indicate that the SH4-Unique-SH3-SH2 domains of c-Yes work cooperatively and prevent activation of signaling proteins associated with Src527F transformation, including activation of phosphatidylinositol 3-kinase, phosphorylation of c-Raf and Akt and downregulation of RhoA-GTP. These data indicate that c-Yes may not modulate signals associated with c-Src-induced changes in actin filament integrity and may explain why c-Yes fails to compensate for c-Src signaling in src-/- cells.
Assuntos
Citoesqueleto de Actina/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Tirosina Quinases/deficiência , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais/genética , Domínios de Homologia de src/fisiologia , Quinases da Família src , Animais , Proteína Tirosina Quinase CSK , Movimento Celular/genética , Tamanho Celular/genética , Células Cultivadas , Embrião de Galinha , Fibroblastos/citologia , Fibroblastos/enzimologia , Regulação Enzimológica da Expressão Gênica/genética , Sistema de Sinalização das MAP Quinases/genética , Invasividade Neoplásica/genética , Fosfatidilinositol 3-Quinases/metabolismo , Estrutura Terciária de Proteína/genética , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-akt , Proteínas Proto-Oncogênicas c-raf/metabolismo , Proteínas Proto-Oncogênicas c-yes , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Proteína rhoA de Ligação ao GTP/metabolismoRESUMO
The Src family of nonreceptor tyrosine kinases plays an important role in modulating signals that affect growth cone extension, neuronal differentiation, and brain development. Recent reports indicate that the Src SH2/SH3 binding partner AFAP-110 has the capacity to modulate actin filament integrity as a cSrc activating protein and as an actin filament bundling protein. Both AFAP-110 and a brain specific isoform called AFAP-120 (collectively referred to as AFAP) exist at high levels in chick embryo brain. We sought to identify the localization of AFAP in mouse brain in order to identify its expression pattern and potential role as a cellular modulator of Src family kinase activity and actin filament integrity in the brain. In E16 mouse embryos, AFAP expression levels were very high and concentrated in the olfactory bulb, cortex, forebrain, cerebellum, and various peripheral sensory structures. In P3 mouse pups, overall expression was reduced compared to E16 embryos, and AFAP was found primarily in olfactory bulb, cortex, and cerebellum. AFAP expression levels were significantly reduced in adult mice, with high expression levels only detected in the olfactory bulb. Western blot analysis indicated that concentrated expression of AFAP correlates well with the AFAP-120 isoform, which appears to be a splice variant of AFAP-110. As the expression pattern of AFAP overlaps with the reported expression patterns of cSrc and Fyn, we hypothesize that AFAP is positioned to modulate signal transduction cascades that direct activation of these nonreceptor tyrosine kinases and concomitant cellular changes that occur in actin filaments during brain development.
Assuntos
Encéfalo , Encéfalo/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas dos Microfilamentos/metabolismo , Fosfoproteínas/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Processamento Alternativo , Animais , Animais Recém-Nascidos , Anticorpos , Western Blotting , Encéfalo/anatomia & histologia , Encéfalo/embriologia , Células COS , Bases de Dados de Ácidos Nucleicos , Embrião de Mamíferos , Imuno-Histoquímica , Camundongos , Bulbo Olfatório/metabolismo , Isoformas de Proteínas , Alinhamento de Sequência , Quinases da Família src/metabolismoRESUMO
The actin filament-associated protein and Src-binding partner, AFAP-110, is an adaptor protein that links signaling molecules to actin filaments. AFAP-110 binds actin filaments directly and multimerizes through a leucine zipper motif. Cellular signals downstream of Src(527F) can regulate multimerization. Here, we determined recombinant AFAP-110 (rAFAP-110)-bound actin filaments cooperatively, through a lateral association. We demonstrate rAFAP-110 has the capability to cross-link actin filaments, and this ability is dependent on the integrity of the carboxy terminal actin binding domain. Deletion of the leucine zipper motif or PKC phosphorylation affected AFAP-110's conformation, which correlated with changes in multimerization and increased the capability of rAFAP-110 to cross-link actin filaments. AFAP-110 is both a substrate and binding partner of PKC. On PKC activation, stress filament organization is lost, motility structures form, and AFAP-110 colocalizes strongly with motility structures. Expression of a deletion mutant of AFAP-110 that is unable to bind PKC blocked the effect of PMA on actin filaments. We hypothesize that upon PKC activation, AFAP-110 can be cooperatively recruited to newly forming actin filaments, like those that exist in cell motility structures, and that PKC phosphorylation effects a conformational change that may enable AFAP-110 to promote actin filament cross-linking at the cell membrane.
