Protein Science Department, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, USA.
Biochemistry. 2011 Sep 20;50(37):7964-76. doi: 10.1021/bi200542r. Epub 2011 Aug 26.
Kinases catalyze the transfer of γ-phosphate from ATP to substrate protein residues triggering signaling pathways responsible for a plethora of cellular events. Isolation and production of homogeneous preparations of kinases in their fully active forms is important for accurate in vitro measurements of activity, stability, and ligand binding properties of these proteins. Previous studies have shown that MEK1 can be produced in its active phosphorylated form by coexpression with RAF1 in insect cells. In this study, using activated MEK1 produced by in vitro activation by RAF1 (pMEK1(in vitro)), we demonstrate that the simultaneous expression of RAF1 for production of activated MEK1 does not result in stoichiometric phosphorylation of MEK1. The pMEK1(in vitro) showed higher specific activity toward ERK2 protein substrate compared to the pMEK1 that was activated via coexpression with RAF1 (pMEK1(in situ)). The two pMEK1 preparations showed quantitative differences in the phosphorylation of T-loop residue serine 222 by Western blotting and mass spectrometry. Finally, pMEK1(in vitro) showed marked differences in the ligand binding properties compared to pMEK1(in situ). Contrary to previous findings, pMEK1(in vitro) bound allosteric inhibitors U0126 and PD0325901 with a significantly lower affinity than pMEK1(in situ) as well as its unphosphorylated counterpart (npMEK1) as demonstrated by thermal-shift, AS-MS, and calorimetric studies. The differences in inhibitor binding affinity provide direct evidence that unphosphorylated and RAF1-phosphorylated MEK1 form distinct inhibitor sites.
激酶催化 ATP 的 γ-磷酸基团向底物蛋白残基的转移，触发负责多种细胞事件的信号通路。分离和生产激酶的均相制剂，使其处于完全活性状态，对于准确测量这些蛋白质的体外活性、稳定性和配体结合特性非常重要。先前的研究表明，MEK1 可以与 RAF1 在昆虫细胞中共表达，以其活性磷酸化形式产生。在本研究中，使用通过 RAF1（pMEK1（in vitro））体外激活产生的激活 MEK1（pMEK1（in vitro）），我们证明同时表达 RAF1 生产激活的 MEK1 不会导致 MEK1 的化学计量磷酸化。与通过与 RAF1 共表达激活的 pMEK1（pMEK1（in situ））相比，pMEK1（in vitro）对 ERK2 蛋白底物表现出更高的比活性。两种 pMEK1 制剂在 T 环残基丝氨酸 222 的磷酸化通过 Western blot 和质谱分析显示出定量差异。最后，pMEK1（in vitro）与 pMEK1（in situ）相比，在配体结合特性方面表现出明显差异。与先前的发现相反，pMEK1（in vitro）与别构抑制剂 U0126 和 PD0325901 的结合亲和力明显低于 pMEK1（in situ）以及其未磷酸化的对应物（npMEK1），这通过热移、AS-MS 和量热研究得到证实。抑制剂结合亲和力的差异提供了直接证据，表明未磷酸化和 RAF1 磷酸化的 MEK1 形成不同的抑制剂结合位点。
