Mass spectrometry approaches to study mammalian kinase and phosphatase associated proteins.
Reversible phosphorylation events regulate critical aspects of cellular biology by affecting protein conformation, cellular localization, enzymatic activity and associations with interaction partners. Kinases and phosphatases interact not only with their substrates but also with regulatory subunits and other proteins, including scaffolds. In recent years, affinity purification coupled to mass spectrometry (AP-MS) has proven to be a powerful tool to identify protein-protein interactions (PPIs) involving kinases and phosphatases. In this review we outline general considerations for successful AP-MS, and describe strategies that we have used to characterize the interactions of kinases and phosphatases in human cells.
Samuel Lunenfeld Research Institute at Mount Sinai Hospital, 600 University Ave., Rm 992, Toronto, ON, Canada M5G 1X5.
Michelle J
MJ
Kean
Amber L
AL
Couzens
Anne-Claude
AC
Gingras
D000818:Animals; D002846:Chromatography, Affinity; D006801:Humans; D010749:Phosphoprotein Phosphatases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping / Q000379:methods*; D011494:Protein Kinases / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D059748:Proteolysis; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D014357:Trypsin / Q000737:chemistry
D011993:Recombinant Fusion Proteins; D011494:Protein Kinases; D010749:Phosphoprotein Phosphatases; D014357:Trypsin