Tandem fluorescence imaging of dynamic S-acylation and protein turnover.
Proc Natl Acad Sci U S A
; 107(19): 8627-32, 2010 May 11.
Article
em En
| MEDLINE
| ID: mdl-20421494
ABSTRACT
The functional significance and regulation of reversible S-acylation on diverse proteins remain unclear because of limited methods for efficient quantitative analysis of palmitate turnover. Here, we describe a tandem labeling and detection method to simultaneously monitor dynamic S-palmitoylation and protein turnover. By combining S-acylation and cotranslational fatty acid chemical reporters with orthogonal clickable fluorophores, dual pulse-chase analysis of Lck revealed accelerated palmitate cycling upon T-cell activation. Subsequent pharmacological perturbation of Lck palmitate turnover suggests yet uncharacterized serine hydrolases contribute to dynamic S-acylation in cells. In addition to dually fatty-acylated proteins, this tandem fluorescence imaging method can be generalized to other S-acylated proteins using azidohomoalanine as a methonine surrogate. The sensitivity and efficiency of this approach should facilitate the functional characterization of cellular factors and drugs that modulate protein S-acylation. Furthermore, diverse protein modifications could be analyzed with this tandem imaging method using other chemical reporters to investigate dynamic regulation of protein function.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Proteína Tirosina Quinase p56(lck) Linfócito-Específica
/
Imagem Molecular
Idioma:
En
Ano de publicação:
2010
Tipo de documento:
Article