RESUMO
The cadherin-catenin adhesion complex is a key contributor to epithelial tissue stability and dynamic cell movements during development and tissue renewal. How this complex is regulated to accomplish these functions is not fully understood. We identified several phosphorylation sites in mammalian αE-catenin (also known as catenin α-1) and Drosophila α-Catenin within a flexible linker located between the middle (M)-region and the carboxy-terminal actin-binding domain. We show that this phospho-linker (P-linker) is the main phosphorylated region of α-catenin in cells and is sequentially modified at casein kinase 2 and 1 consensus sites. In Drosophila, the P-linker is required for normal α-catenin function during development and collective cell migration, although no obvious defects were found in cadherin-catenin complex assembly or adherens junction formation. In mammalian cells, non-phosphorylatable forms of α-catenin showed defects in intercellular adhesion using a mechanical dispersion assay. Epithelial sheets expressing phosphomimetic forms of α-catenin showed faster and more coordinated migrations after scratch wounding. These findings suggest that phosphorylation and dephosphorylation of the α-catenin P-linker are required for normal cadherin-catenin complex function in Drosophila and mammalian cells.
Assuntos
Caderinas/metabolismo , Caseína Quinase II/metabolismo , Caseína Quinase I/metabolismo , Adesão Celular , Drosophila melanogaster/metabolismo , alfa Catenina/metabolismo , Actinas/metabolismo , Sequência de Aminoácidos , Animais , Apoptose , Western Blotting , Caderinas/genética , Caseína Quinase I/genética , Caseína Quinase II/genética , Membrana Celular/metabolismo , Movimento Celular , Proliferação de Células , Células Cultivadas , Neoplasias do Colo/metabolismo , Neoplasias do Colo/patologia , Cães , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Feminino , Imunofluorescência , Humanos , Imunoprecipitação , Células Madin Darby de Rim Canino , Dados de Sequência Molecular , Ovário/citologia , Ovário/metabolismo , Fosforilação , Estrutura Terciária de Proteína , Homologia de Sequência de Aminoácidos , alfa Catenina/química , alfa Catenina/genéticaRESUMO
Assembly factors (AFs) prevent premature translation initiation on small (40S) ribosomal subunit assembly intermediates by blocking ligand binding. However, it is unclear how AFs are displaced from maturing 40S ribosomes, if or how maturing subunits are assessed for fidelity, and what prevents premature translation initiation once AFs dissociate. Here we show that maturation involves a translation-like cycle whereby the translation factor eIF5B, a GTPase, promotes joining of large (60S) subunits with pre-40S subunits to give 80S-like complexes, which are subsequently disassembled by the termination factor Rli1, an ATPase. The AFs Tsr1 and Rio2 block the mRNA channel and initiator tRNA binding site, and therefore 80S-like ribosomes lack mRNA or initiator tRNA. After Tsr1 and Rio2 dissociate from 80S-like complexes Rli1-directed displacement of 60S subunits allows for translation initiation. This cycle thus provides a functional test of 60S subunit binding and the GTPase site before ribosomes enter the translating pool.
