title: Tuning a three-component reaction for trapping kinase substrate complexes.
abstract: 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.
authors.affiliation: Howard Hughes Medical Institute, Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94107, USA.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Statsuk
authors.forename: Dustin J
authors.initials: DJ
authors.lastname: Maly
authors.forename: Markus A
authors.initials: MA
authors.lastname: Seeliger
authors.forename: Miles A
authors.initials: MA
authors.lastname: Fabian
authors.forename: William H
authors.initials: WH
authors.lastname: Biggs
authors.forename: David J
authors.initials: DJ
authors.lastname: Lockhart
authors.forename: Patrick P
authors.initials: PP
authors.lastname: Zarrinkar
authors.forename: John
authors.initials: J
authors.lastname: Kuriyan
authors.forename: Kevan M
authors.initials: KM
authors.lastname: Shokat
mesh_terms: 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
chemical_list: D003432:Cross-Linking Reagents; D005456:Fluorescent Dyes; D010455:Peptides; D011506:Proteins; D013876:Thiophenes; D010770:Phosphotransferases; D008239:Lysine; D000241:Adenosine; D003545:Cysteine