RESUMO
The synthesis and biochemical characterization of AX4697, a fluorescent, bisindolylmaleimide-derived probe for PKCalpha and beta, is described. AX4697 was able to quantify changes in PKC expression in drug-treated Jurkat cells and was shown to covalently label PKCalpha on C619, a residue that sits just outside the active site.
Assuntos
Corantes Fluorescentes/síntese química , Corantes Fluorescentes/farmacologia , Indóis/síntese química , Indóis/farmacologia , Maleimidas/síntese química , Maleimidas/farmacologia , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C/metabolismo , Domínio Catalítico , Desenho de Fármacos , Corantes Fluorescentes/química , Humanos , Indóis/química , Células Jurkat , Maleimidas/química , Estrutura Molecular , Proteína Quinase C beta , Relação Estrutura-AtividadeRESUMO
Characterization and functional annotation of the large number of proteins predicted from genome sequencing projects poses a major scientific challenge. Whereas several proteomics techniques have been developed to quantify the abundance of proteins, these methods provide little information regarding protein function. Here, we present a gel-free platform that permits ultrasensitive, quantitative, and high-resolution analyses of protein activities in proteomes, including highly problematic samples such as undiluted plasma. We demonstrate the value of this platform for the discovery of both disease-related enzyme activities and specific inhibitors that target these proteins.
Assuntos
Peptídeos/análise , Proteômica/métodos , Animais , Sítios de Ligação , Eletroforese Capilar , Camundongos , Mapeamento de Peptídeos , Peptídeos/química , Serina Endopeptidases/análise , Serina Endopeptidases/químicaRESUMO
Endothelial cells approaching confluence exhibit marked decreases in tyrosine phosphorylation of receptor tyrosine kinases and adherens junctions proteins, required for cell cycle arrest and adherens junctions stability. Recently, we demonstrated a close correlation in endothelial cells between membrane cholesterol and tyrosine phosphorylation of adherens junctions proteins. Here, we probe the mechanistic basis for this correlation. We find that as endothelial cells reach confluence, the tyrosine phosphatase SHP-2 is recruited to a low-density membrane fraction in a cholesterol-dependent manner. Binding of SHP-2 to this fraction was not abolished by phenyl phosphate, strongly suggesting that this binding was mediated by other regions of SHP-2 beside its SH2 domains. Annexin II, previously implicated in cholesterol trafficking, was associated in a complex with SHP-2, and both proteins localized to adhesion bands in confluent endothelial monolayers. These studies reveal a novel, cholesterol-dependent mechanism for the recruitment of signaling proteins to specific plasma membrane domains via their interactions with annexin II.