A semisynthetic epitope for kinase substrates.
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.
Allen
Jasmina J
JJ
Graduate Program in Chemistry and Chemical Biology, University of California, San Francisco, San Francisco, California 94143, USA.
Li
Manqing
M
Brinkworth
Craig S
CS
Paulson
Jennifer L
JL
Wang
Dan
D
Hübner
Anette
A
Chou
Wen-Hai
WH
Davis
Roger J
RJ
Burlingame
Alma L
AL
Messing
Robert O
RO
Katayama
Carol D
CD
Hedrick
Stephen M
SM
Shokat
Kevan M
KM
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives; D000595:Amino Acid Sequence; D000818:Animals; D000939:Epitopes / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D048049:Extracellular Signal-Regulated MAP Kinases / Q000235:genetics / Q000378:metabolism*; D020440:Gene Duplication; D006241:Haptens / Q000737:chemistry* / Q000276:immunology / Q000378:metabolism*; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism; D007553:Isotope Labeling / Q000379:methods; D051379:Mice; D018345:Mice, Knockout; D063086:Organothiophosphates / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity
D000939:Epitopes; D006241:Haptens; D007074:Immunoglobulin G; D063086:Organothiophosphates; C022571:adenosine 5'-O-(3-thiotriphosphate); D000255:Adenosine Triphosphate; D048049:Extracellular Signal-Regulated MAP Kinases