A mechanism-based cross-linker for the identification of kinase-substrate pairs.
The reversible phosphorylation of proteins is one of the most important mechanisms for the regulation of signal transduction cascades. Recently, there has been substantial progress made in the identification of new phosphoproteins and phosphorylation sites. Unfortunately, there are very few methods available that allow this information to be used to identify the upstream kinase responsible for the phosphorylation event. Herein, we describe a new method that allows the cross-linking of a substrate of interest to its upstream kinase. This method relies upon a novel, mechanism-based cross-linker and the replacement of the phosphorylated residue with a cysteine residue. The application of this method to a number of kinase-peptide substrate pairs is described.
Maly
Dustin J
DJ
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, California 94107, USA.
Allen
Jasmina A
JA
Shokat
Kevan M
KM
D000241:Adenosine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry* / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism; D010766:Phosphorylation; D011494:Protein Kinases / Q000737:chemistry* / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000737:chemistry / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D012694:Serine / Q000737:chemistry / Q000378:metabolism; D013379:Substrate Specificity; D013912:Threonine / Q000737:chemistry / Q000378:metabolism; D009764:o-Phthalaldehyde / Q000737:chemistry / Q000378:metabolism
D003432:Cross-Linking Reagents; D011518:Proto-Oncogene Proteins; D013912:Threonine; D012694:Serine; D009764:o-Phthalaldehyde; C010959:5'-(4-fluorosulfonylbenzoyl)adenosine; D000255:Adenosine Triphosphate; D011494:Protein Kinases; D017346:Protein Serine-Threonine Kinases; D051057:Proto-Oncogene Proteins c-akt; D000241:Adenosine; D003545:Cysteine