Purification of a model substrate for transcription factor phosphorylation by ERK2.
ERK2 belongs to the mitogen-activated protein kinase subfamily, which plays a pivotal role in cell signal transduction, in which it mediates effects on proliferation and differentiation by growth factors and hormones. While its cellular function has been under intense scrutiny since its initial discovery nearly 15 years ago, little progress has been made in understanding its kinetic mechanism. Such an understanding is important for the development of potent and specific inhibitors. A contributory factor has been the lack of a protein substrate suitable for rigorous mechanistic studies. Here we report the expression, purification, and characterization of the N-terminus (residues 1 through 138) of the transcription factor Ets-1, an excellent model substrate for ERK2 mechanistic studies. (His(6)-tagged)Ets-1(1-138) was expressed in Escherichia coli and rapidly purified in two steps by nickel-agarose-affinity chromatography, followed by high-resolution Mono-Q anion-exchange chromatography. A yield of 60 mg of the purified protein per liter of culture was obtained and could be stored conveniently at -80 degrees C in water. Rigorous characterization demonstrated that under the assay conditions, (His(6)-tagged)Ets-1(1-138) is exclusively phosphorylated on residue Thr-38 by ERK2 with the following Michaelis parameters: k(cat) = 17 s(-1), K(ATP)(m) = 140 microM, K(ATP)(i) = 68 microM, K(Ets-1)(m) = 19 microM, and K(Ets-1)(i) = 9.3 microM.
Waas
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WF
Division of Medicinal Chemistry, University of Texas at Austin 78712, USA.
Dalby
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KN
D000345:Affinity Labels; D004926:Escherichia coli / Q000235:genetics; D006639:Histidine; D007700:Kinetics; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism*; D008958:Models, Molecular; D010446:Peptide Fragments; D010766:Phosphorylation; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis* / Q000378:metabolism*; D050783:Proto-Oncogene Proteins c-ets; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D014157:Transcription Factors / Q000096:biosynthesis* / Q000378:metabolism*; D014170:Transformation, Genetic
D000345:Affinity Labels; D010446:Peptide Fragments; D050784:Proto-Oncogene Protein c-ets-1; D011518:Proto-Oncogene Proteins; D050783:Proto-Oncogene Proteins c-ets; D011994:Recombinant Proteins; D014157:Transcription Factors; D006639:Histidine; D019950:Mitogen-Activated Protein Kinase 1