Wang Ling, Fisher Laura A, Wahl James K, Peng Aimin
Department of Oral Biology, College of Dentistry, University of Nebraska Medical Center, Lincoln, Nebraska, USA.
Hybridoma (Larchmt). 2011 Oct;30(5):469-74. doi: 10.1089/hyb.2011.0051.
Mitosis is known to be regulated by protein kinases, including MPF, Plk1, Aurora kinases, and so on, which become active in M-phase and phosphorylate a wide range of substrates to control multiple aspects of mitotic entry, progression, and exit. Mechanistic investigations of these kinases not only provide key insights into cell cycle regulation, but also hold great promise for cancer therapy. Recent studies, largely in Xenopus, characterized a new mitotic kinase named Greatwall (Gwl) that plays essential roles in both mitotic entry and maintenance. In this study, we generated a panel of mouse monoclonal antibodies (MAbs) specific for Xenopus Gwl and characterized these antibodies for their utility in immunoblotting, immunoprecipitation, and immunodepletion in Xenopus egg extracts. Importantly, we generated an MAb that is capable of neutralizing endogenous Gwl. The addition of this antibody into M-phase extracts results in loss of mitotic phosphorylation of Gwl, Plk1, and Cdk1 substrates. These results illustrate a new tool to study loss-of-function of Gwl, and support its essential role in mitosis. Finally, we demonstrated the usefulness of the MAb against human Gwl/MASTL.
已知有丝分裂受蛋白激酶调控，包括成熟促进因子（MPF）、Polo样激酶1（Plk1）、极光激酶等，这些激酶在M期变得活跃，并使多种底物磷酸化，以控制有丝分裂进入、进程和退出的多个方面。对这些激酶的机制研究不仅为细胞周期调控提供了关键见解，也为癌症治疗带来了巨大希望。最近的研究主要在非洲爪蟾中进行，鉴定出一种名为长城激酶（Gwl）的新的有丝分裂激酶，它在有丝分裂进入和维持中都起着至关重要的作用。在本研究中，我们制备了一组针对非洲爪蟾Gwl的小鼠单克隆抗体（MAb），并对这些抗体在非洲爪蟾卵提取物中的免疫印迹、免疫沉淀和免疫去除方面的效用进行了表征。重要的是，我们制备了一种能够中和内源性Gwl的单克隆抗体。将这种抗体添加到M期提取物中会导致Gwl、Plk1和Cdk1底物的有丝分裂磷酸化丧失。这些结果说明了一种研究Gwl功能丧失的新工具，并支持其在有丝分裂中的重要作用。最后，我们证明了抗人Gwl/MASTL单克隆抗体的实用性。
