Department of Chemistry, University of Washington, Box 351700, Seattle, WA 98195-1700, USA.
Chembiochem. 2013 Jan 21;14(2):209-16. doi: 10.1002/cbic.201200673. Epub 2013 Jan 14.
Protein kinases are essential enzymes for cellular signaling, and are often regulated by participation in protein complexes. The mitogen-activated protein kinase (MAPK) p38 is involved in multiple pathways, and its regulation depends on its interactions with other signaling proteins. However, the identification of p38-interacting proteins is challenging. For this reason, we have developed label transfer reagents (LTRs) that allow labeling of p38 signaling complexes. These LTRs leverage the potency and selectivity of known p38 inhibitors to place a photo-crosslinker and tag in the vicinity of p38 and its binding partners. Upon UV irradiation, proteins that are in close proximity to p38 are covalently crosslinked, and labeled proteins are detected and/or purified with an orthogonal chemical handle. Here we demonstrate that p38-selective LTRs selectively label a diversity of p38 binding partners, including substrates, activators, and inactivators. Furthermore, these LTRs can be used in immunoprecipitations to provide low-resolution structural information on p38-containing complexes.
蛋白激酶是细胞信号转导的必需酶，通常通过参与蛋白质复合物来调节。丝裂原活化蛋白激酶（MAPK）p38 参与多种途径，其调节取决于与其他信号蛋白的相互作用。然而，p38 相互作用蛋白的鉴定具有挑战性。为此，我们开发了标签转移试剂（LTR），可用于标记 p38 信号复合物。这些 LTR 利用已知的 p38 抑制剂的效力和选择性，在 p38 及其结合伙伴的附近放置一个光交联剂和标签。经紫外线照射后，与 p38 近距离接触的蛋白质会发生共价交联，然后用正交化学接头检测和/或纯化标记的蛋白质。在这里，我们证明 p38 选择性 LTR 可选择性标记多种 p38 结合伙伴，包括底物、激活剂和抑制剂。此外，这些 LTR 可用于免疫沉淀，以提供包含 p38 的复合物的低分辨率结构信息。
