Tuning a three-component reaction for trapping kinase substrate complexes.
The upstream protein kinases responsible for thousands of phosphorylation events in the phosphoproteome remain to be discovered. We developed a three-component chemical reaction which converts the transient noncovalent substrate-kinase complex into a covalently cross-linked product by utilizing a dialdehyde-based cross-linker, 1. Unfortunately, the reaction of 1 with a lysine in the kinase active site and an engineered cysteine on the substrate to form an isoindole cross-linked product could not be performed in the presence of competing cellular proteins due to nonspecific side reactions. In order to more selectively target the cross-linker to protein kinases in cell lysates, we replaced the weak, kinase-binding adenosine moiety of 1 with a potent protein kinase inhibitor scaffold. In addition, we replaced the o-phthaldialdehyde moiety in 1 with a less-reactive thiophene-2,3-dicarboxaldehyde moiety. The combination of these two structural modifications provides for cross-linking of a cysteine-containing substrate to its corresponding kinase in the presence of competing cellular proteins.
Statsuk
Alexander V
AV
Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94107, USA.
Maly
Dustin J
DJ
Seeliger
Markus A
MA
Fabian
Miles A
MA
Biggs
William H
WH
Lockhart
David J
DJ
Zarrinkar
Patrick P
PP
Kuriyan
John
J
Shokat
Kevan M
KM
D000241:Adenosine / Q000737:chemistry; D003432:Cross-Linking Reagents / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry; D006367:HeLa Cells; D006801:Humans; D008239:Lysine / Q000737:chemistry; D008956:Models, Chemical; D008968:Molecular Conformation; D010455:Peptides / Q000737:chemistry; D010766:Phosphorylation; D010770:Phosphotransferases / Q000737:chemistry* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry; D013876:Thiophenes / Q000737:chemistry
D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D010455:Peptides; D011506:Proteins; D013876:Thiophenes; D010770:Phosphotransferases; D008239:Lysine; D000241:Adenosine; D003545:Cysteine