title: Isolation and Characterization of Intrinsically Active (MEK-Independent) Mutants of Mpk1/Erk.
abstract: The extracellular-regulated kinase (Erk) pathway is a major determinant in the control of diverse cellular processes, such as proliferation, differentiation, survival, and motility. The pathway executes its effects through kinases of the Erk family. Erks are not only critical for a variety of physiological processes, but are also associated with neurodegenerative diseases, cardiovascular diseases, diabetes and a large number of human cancers. However, the exact role of each Erk molecule in these biological and pathological processes is not fully determined. An efficient strategy for revealing these roles is to activate each Erk isoform individually, in a signal independent manner, and to monitor the molecular, physiological, and pathological effects. This could be achieved by developing intrinsically active variants for each Erk isoform and splicing variant and expressing these molecules individually in biological systems. A screening method that selects for relevant and useful active mutants of Erks is described in this chapter. The main principle of the method is to screen for mutants of Erk that function in the total absence of their relevant MEKs. Another principle is that the screen should be unbiased toward particular domains or mechanisms of action. We describe how these principles are combined into a screen that takes advantage of the yeast Mpk1/Erk pathway. Following the description of how intrinsically active Mpk1 molecules are isolated, we provide comprehensive and detailed descriptions of the methods used to characterize their catalytic activity, autophosphorylation capabilities, and phosphorylation status, as well as the methods used to determine the precise phosphorylated sites. The principles of the screen and the methods described here could be easily adapted for any Erk molecule in any organism.
authors.affiliation: Department of Biological Chemistry, The Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel.
authors.forename: Tal
authors.initials: T
authors.lastname: Goshen-Lago
authors.affiliation: Faculty of Biology, Technion - Israel Institute of Technology, Haifa, 32000, Israel.
authors.forename: Dganit
authors.initials: D
authors.lastname: Melamed
authors.affiliation: Faculty of Biology, Technion - Israel Institute of Technology, Haifa, 32000, Israel.
authors.forename: Arie
authors.initials: A
authors.lastname: Admon
authors.affiliation: Department of Biological Chemistry, The Institute of Life Science, The Hebrew University of Jerusalem, Jerusalem, 91904, Israel. engelber@mail.huji.ac.il.|CREATE-NUS-HUJ Cellular & Molecular Mechanisms of Inflammation Programme, National University of Singapore, 1 CREATE WAY, Innovation Wing, #03-09, Singapore, 138602, Singapore. engelber@mail.huji.ac.il.|Department of Microbiology, Yong Loo Lin School of Medicine, National University of Singapore, Singapore, 117456, Singapore. engelber@mail.huji.ac.il.
authors.forename: David
authors.initials: D
authors.lastname: Engelberg
mesh_terms: D002853:Chromatography, Liquid; D048049:Extracellular Signal-Regulated MAP Kinases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism*; D015870:Gene Expression; D006801:Humans; D020929:Mitogen-Activated Protein Kinase Kinases / Q000378:metabolism*; D050505:Mutant Proteins; D010766:Phosphorylation; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D053719:Tandem Mass Spectrometry; D015003:Yeasts / Q000378:metabolism
chemical_list: D050505:Mutant Proteins; D011993:Recombinant Fusion Proteins; D048049:Extracellular Signal-Regulated MAP Kinases; D020929:Mitogen-Activated Protein Kinase Kinases
keywords: Active variants; Autophosphorylation; Erk; LC/MS/MS; MAP kinase; MKK1/2; Mek1; Mpk1; Yeast