A semisynthetic epitope for kinase substrates.
Nat Methods
; 4(6): 511-6, 2007 Jun.
Article
em En
| MEDLINE
| ID: mdl-17486086
ABSTRACT
The ubiquitous nature of protein phosphorylation makes it challenging to map kinase-substrate relationships, which is a necessary step toward defining signaling network architecture. To trace the activity of individual kinases, we developed a semisynthetic reaction scheme, which results in the affinity tagging of substrates of the kinase in question. First, a kinase, engineered to use a bio-orthogonal ATPgammaS analog, catalyzes thiophosphorylation of its direct substrates. Second, alkylation of thiophosphorylated serine, threonine or tyrosine residues creates an epitope for thiophosphate ester-specific antibodies. We demonstrated the generality of semisynthetic epitope construction with 13 diverse kinases JNK1, p38alpha MAPK, Erk1, Erk2, Akt1, PKCdelta, PKCepsilon, Cdk1/cyclinB, CK1, Cdc5, GSK3beta, Src and Abl. Application of this approach, in cells isolated from a mouse that expressed endogenous levels of an analog-specific (AS) kinase (Erk2), allowed purification of a direct Erk2 substrate.
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
MAP Quinases Reguladas por Sinal Extracelular
/
Haptenos
/
Epitopos
Limite:
Animals
Idioma:
En
Revista:
Nat Methods
Assunto da revista:
TECNICAS E PROCEDIMENTOS DE LABORATORIO
Ano de publicação:
2007
Tipo de documento:
Article
País de afiliação:
Estados Unidos