RESUMO
Large-scale comparative phosphoproteomics studies have frequently been done on whole cells or organs by conventional bottom-up mass spectrometry approaches, that is, at the phosphopeptide level. Using this approach, there is no way to know which protein isoforms the phosphopeptide signal originated from. Also, as a consequence of the scale of these studies, important information on the localization of phosphorylation sites in subcellular compartments is not surveyed. As a case study, we investigated whether the isoforms of dynamin I (dynI), at the whole brain and subcellular level, had differential phosphorylation. We first established that the dynI isoforms xa, xb, and xd were expressed in nerve terminals. Our investigation revealed that dynI xa was constitutively phosphorylated to a higher extent than the other isoforms despite identical sequences in the phosphorylated subdomains. DynI xa had a 10-fold higher stoichiometry of diphosphorylation at Ser-774 and Ser-778 than dynI xb and xd combined. Diphosphorylation was 2-fold enriched in nerve terminals relative to whole brain and was preferentially targeted for stimulus-dependent dephosphorylation. Phospho-Ser-851 and Ser-857 were depleted from nerve terminals. Our data reveals major differential phosphorylation of dynI phosphosites in different variants and in different neuronal compartments that would be completely imperceptible to a large-scale phosphoproteomics approach.
Assuntos
Encéfalo/metabolismo , Dinamina I/metabolismo , Espaço Intracelular/metabolismo , Proteômica/métodos , Vesículas Sinápticas/metabolismo , Sequência de Aminoácidos , Animais , Cromatografia Líquida , Biologia Computacional , Dinamina I/genética , Eletroforese em Gel de Poliacrilamida/métodos , Componentes do Gene , Isoenzimas/metabolismo , Espectrometria de Massas , Dados de Sequência Molecular , Fosforilação , RatosRESUMO
Synaptic vesicle endocytosis (SVE) is triggered by calcineurin-mediated dephosphorylation of the dephosphin proteins. SVE is maintained by the subsequent rephosphorylation of the dephosphins by unidentified protein kinases. Here, we show that cyclin-dependent kinase 5 (Cdk5) phosphorylates dynamin I on Ser 774 and Ser 778 in vitro, which are identical to its endogenous phosphorylation sites in vivo. Cdk5 antagonists and expression of dominant-negative Cdk5 block phosphorylation of dynamin I, but not of amphiphysin or AP180, in nerve terminals and inhibit SVE. Thus Cdk5 has an essential role in SVE and is the first dephosphin kinase identified in nerve terminals.