Purification of a model substrate for transcription factor phosphorylationby ERK2

ERK2 belongs to the mitogen-activated protein kinase subfamily, which plays a pivotal role in cell signal transduction, in which it mediates effects o n proliferation and differentiation by growth factors and hormones. While i ts cellular function has been under intense scrutiny since its initial disc overy nearly 15 years ago, little progress has been made in understanding i ts kinetic mechanism. Such an understanding is important for the developmen t of potent and specific inhibitors. A contributory factor has been the lac k of a protein substrate suitable for rigorous mechanistic studies. Here we report the expression, purification, and characterization of the N-terminu s (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) w as expressed in Escherichia coli and rapidly purified in two steps by nicke l-agarose-affinity chromatography, followed by high-resolution Mono-Q anion -exchange chromatography. A yield of 60 mg of the purified protein per lite r of culture was obtained and could be stored conveniently at -80 degreesC in water. Rigorous characterization demonstrated that under the assay condi tions, (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-m(ATP) = 140 muM, K-i(ATP) = 68 muM, K-m(Ets-1) = 19 muM, and K-i(Ets-1) = 9.3 muM. (C) 2001 Academic Press.