RESUMO
The regulation and function of peptidylarginine deiminase isoform VI (PAD6), which is a highly abundant protein associated with the cytoplasmic lattices in mammalian oocytes, is poorly understood so far. It has been shown previously, that 14-3-3 proteins, a class of regulatory adapter proteins ubiquitous in eukaryotes, bind to PAD6 in vivo in a phosphorylation dependent manner. Here we identify possible 14-3-3 binding sites in human PAD6 by in silico methods, looking for conserved, surface exposed serine residues. Two of these sites were confirmed as 14-3-3 binding sites by fluorescence polarization competition and X-ray crystallography. We furthermore suggest a role of RSK-type kinases in the phosphorylation of one of these two binding sites and provide evidence in the form of in vitro kinase assays with p70S6 kinase and RSK1.
Assuntos
Proteínas 14-3-3/química , Biomarcadores Tumorais/química , Exonucleases/química , Hidrolases/química , Sequência de Aminoácidos , Sítios de Ligação , Cristalografia por Raios X , Exorribonucleases , Humanos , Interações Hidrofóbicas e Hidrofílicas , Modelos Moleculares , Dados de Sequência Molecular , Fragmentos de Peptídeos/química , Fosfoproteínas/química , Fosforilação , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Processamento de Proteína Pós-Traducional , Estrutura Secundária de Proteína , Proteína-Arginina Desiminase do Tipo 6 , Desiminases de Arginina em Proteínas , Proteínas Quinases S6 Ribossômicas 90-kDa/químicaRESUMO
The Ras-RAF-mitogen-activated protein kinase (Ras-RAF-MAPK) pathway is overactive in many cancers and in some developmental disorders. In one of those disorders, namely, Noonan syndrome, nine activating C-RAF mutations cluster around Ser(259), a regulatory site for inhibition by 14-3-3 proteins. We show that these mutations impair binding of 14-3-3 proteins to C-RAF and alter its subcellular localization by promoting Ras-mediated plasma membrane recruitment of C-RAF. By presenting biophysical binding data, the 14-3-3/C-RAFpS(259) crystal structure, and cellular analyses, we indicate a mechanistic link between a well-described human developmental disorder and the impairment of a 14-3-3/target protein interaction. As a broader implication of these findings, modulating the C-RAFSer(259)/14-3-3 protein-protein interaction with a stabilizing small molecule may yield a novel potential approach for treatment of diseases resulting from an overactive Ras-RAF-MAPK pathway.
