Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India.
Centre for Interdisciplinary Research in Basic Sciences, Jamia Millia Islamia, Jamia Nagar, New Delhi 110025, India.
Int J Biol Macromol. 2016 Dec;93(Pt A):1147-1154. doi: 10.1016/j.ijbiomac.2016.09.087. Epub 2016 Sep 24.
Microtubule affinity regulating Kinase 4 (MARK4) belongs to the family of AMP-activated protein kinase. It phosphorylates microtubule associated proteins at specific sites (Serine in KXGS motifs) in the microtubule-binding repeats. In our previous studies, two constructs, namely, kinase domain with 59 N-terminal residues (residues 1-310) and only kinase domain (residues 59-310) of MARK4 show aggregation at physiological pH. However, these two constructs were stable at extremes of pH conditions. Now the question arises: how is MARK4 stable at physiological pH in-vivo? To answer this question, we have successfully cloned, expressed, and purified UBA-domain along with the kinase domain of MARK4 and performed spectroscopic measurements and activity assays. We observed a pronounced secondary and tertiary structure and ATPase activity in the MARK4 at physiological pH. In conclusion, UBA domain may be important to maintain the structure, stability and activity of MARK4 under physiological conditions.
微管亲和调节激酶 4（MARK4）属于 AMP 激活蛋白激酶家族。它在微管结合重复序列中的特定位点（KXGS 模体中的丝氨酸）磷酸化微管相关蛋白。在我们之前的研究中，两种构建体，即具有 59 个 N 端残基（残基 1-310）的激酶结构域和仅激酶结构域（残基 59-310）的 MARK4，在生理 pH 下显示聚集。然而，这两种构建体在 pH 极端条件下是稳定的。现在的问题是：MARK4 在体内生理 pH 下是如何稳定的？为了回答这个问题，我们成功地克隆、表达和纯化了 MARK4 的 UBA 结构域和激酶结构域，并进行了光谱测量和活性测定。我们在生理 pH 下观察到 MARK4 具有明显的二级和三级结构和 ATP 酶活性。总之，UBA 结构域可能对 MARK4 在生理条件下的结构、稳定性和活性很重要。
