RESUMO
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.
Assuntos
Epitopos/química , Epitopos/metabolismo , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Haptenos/química , Haptenos/metabolismo , Trifosfato de Adenosina/análogos & derivados , Sequência de Aminoácidos , Animais , Epitopos/imunologia , MAP Quinases Reguladas por Sinal Extracelular/genética , Duplicação Gênica , Haptenos/imunologia , Imunoglobulina G/química , Imunoglobulina G/metabolismo , Marcação por Isótopo/métodos , Camundongos , Camundongos Knockout , Organotiofosfatos/química , Organotiofosfatos/metabolismo , Especificidade por SubstratoRESUMO
Chemical genetic analysis of protein kinases involves engineering kinases to be uniquely sensitive to inhibitors and ATP analogs that are not recognized by wild-type kinases. Despite the successful application of this approach to over two dozen kinases, several kinases do not tolerate the necessary modification to the ATP binding pocket, as they lose catalytic activity or cellular function upon mutation of the 'gatekeeper' residue that governs inhibitor and nucleotide substrate specificity. Here we describe the identification of second-site suppressor mutations to rescue the activity of 'intolerant' kinases. A bacterial genetic selection for second-site suppressors using an aminoglycoside kinase APH(3')-IIIa revealed several suppressor hotspots in the kinase domain. Informed by results from this selection, we focused on the beta sheet in the N-terminal subdomain and generated a structure-based sequence alignment of protein kinases in this region. From this alignment, we identified second-site suppressors for several divergent kinases including Cdc5, MEKK1, GRK2 and Pto. The ability to identify second-site suppressors to rescue the activity of intolerant kinases should facilitate chemical genetic analysis of the majority of protein kinases in the genome.