title: Identification of extracellular signal-regulated kinase 1 (ERK1) direct substrates using stable isotope labeled kinase assay-linked phosphoproteomics.
abstract: Kinase mediated phosphorylation signaling is extensively involved in cellular functions and human diseases, and unraveling phosphorylation networks requires the identification of substrates targeted by kinases, which has remained challenging. We report here a novel proteomic strategy to identify the specificity and direct substrates of kinases by coupling phosphoproteomics with a sensitive stable isotope labeled kinase reaction. A whole cell extract was moderately dephosphorylated and subjected to in vitro kinase reaction under the condition in which (18)O-ATP is the phosphate donor. The phosphorylated proteins are then isolated and identified by mass spectrometry, in which the heavy phosphate (+85.979 Da) labeled phosphopeptides reveal the kinase specificity. The in vitro phosphorylated proteins with heavy phosphates are further overlapped with in vivo kinase-dependent phosphoproteins for the identification of direct substrates with high confidence. The strategy allowed us to identify 46 phosphorylation sites on 38 direct substrates of extracellular signal-regulated kinase 1, including multiple known substrates and novel substrates, highlighting the ability of this high throughput method for direct kinase substrate screening.
authors.affiliation: From the ‡Departments of Biochemistry.
authors.forename: Liang
authors.initials: L
authors.lastname: Xue
authors.affiliation: §Horticulture and Landscape Architecture.
authors.forename: Pengcheng
authors.initials: P
authors.lastname: Wang
authors.affiliation: ¶Mathematics.
authors.forename: Pianpian
authors.initials: P
authors.lastname: Cao
authors.affiliation: From the ‡Departments of Biochemistry, §Horticulture and Landscape Architecture.
authors.forename: Jian-Kang
authors.initials: JK
authors.lastname: Zhu
authors.affiliation: From the ‡Departments of Biochemistry, ‖Medicinal Chemistry and Molecular Pharmacology, **Chemistry, and ‡‡Purdue University Center for Cancer Research, Purdue University, West Lafayette, Indiana 47907 watao@purdue.edu.
authors.forename: W Andy
authors.initials: WA
authors.lastname: Tao
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism; D033942:Adaptor Proteins, Vesicular Transport / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000737:chemistry; D051017:Apoptosis Regulatory Proteins / Q000378:metabolism; D017360:Arabidopsis; D002460:Cell Line; D003598:Cytoskeletal Proteins; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism*; D020555:GTP-Binding Protein Regulators / Q000378:metabolism; D057809:HEK293 Cells; D006801:Humans; D007553:Isotope Labeling; D020935:MAP Kinase Signaling System / Q000502:physiology*; D010766:Phosphorylation; D040901:Proteomics / Q000379:methods*; D016601:RNA-Binding Proteins; D036226:Seedlings; D020741:rho GTP-Binding Proteins
chemical_list: D048868:Adaptor Proteins, Signal Transducing; D033942:Adaptor Proteins, Vesicular Transport; D051017:Apoptosis Regulatory Proteins; C414431:BAG3 protein, human; C422985:CDC42EP4 protein, human; D003598:Cytoskeletal Proteins; C486204:EPN2 protein, human; D020555:GTP-Binding Protein Regulators; D016601:RNA-Binding Proteins; C004282:periodate-oxidized adenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D048049:Extracellular Signal-Regulated MAP Kinases; D020741:rho GTP-Binding Proteins