MARK2/EMMK1/Par-1Bα phosphorylation of Rab11-family interacting protein 2 is necessary for the timely establishment of polarity in Madin-Darby canine kidney cells
MARK2/EMMK1/Par-1Bα phosphorylation of Rab11-family interacting protein 2 is necessary for the timely establishment of polarity in Madin-Darby canine kidney cells
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DOI：
10.1091/mbc.e05-08-0736
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发表时间：
2006-08-01
影响因子：
3.3
通讯作者：
Goldenring, James R.
中科院分区：
文献类型：
--
作者：
Ducharme, Nicole A.;Hales, Chadwick M.;Goldenring, James R.
Rab11a、肌球蛋白Vb和Rab11家族相互作用蛋白2(FIP2)调控上皮细胞质膜循环。本研究试图通过鉴定修饰Rab11-FIP2的激酶活性来更全面地表征Rab11-FIP2的功能。我们发现胃微粒体膜提取液可使丝氨酸227上的Rab11-FIP2磷酸化。我们鉴定了使Rab11-FIP2磷酸化的蛋白为Mark2/EMK1/PAR-1Bα(Mark2)，而重组Mark2仅在丝氨酸227上使Rab11-FIP2磷酸化。我们建立了稳定表达增强型绿色荧光蛋白-Rab11-FIP2野生型或非磷酸化突变体[Rab11-FIP2(S227A)]的Madin-Darby犬肾(MDCK)细胞系。对这些细胞系的分析表明，Rab11-FIP2除了在质膜循环系统中发挥作用外，还具有新的作用。在钙开关实验中，表达Rab11-FIP2(S227A)的细胞在及时重建含有p120的连接复合体方面存在缺陷。然而，Rab11-FIP2(S227A)不影响循环系统成分的定位，也不影响顶端再循环和跨细胞途径的正常功能。这些结果表明，Mark2对丝氨酸227上Rab11-FIP2的磷酸化调节了另一条调节上皮极性建立的途径。
Rab11a, myosin Vb, and the Rab11-family interacting protein 2 (FIP2) regulate plasma membrane recycling in epithelial cells. This study sought to characterize more fully Rab11-FIP2 function by identifying kinase activities modifying Rab11-FIP2. We have found that gastric microsomal membrane extracts phosphorylate Rab11-FIP2 on serine 227. We identified the kinase that phosphorylated Rab11-FIP2 as MARK2/EMK1/Par-1B alpha (MARK2), and recombinant MARK2 phosphorylated Rab11-FIP2 only on serine 227. We created stable Madin-Darby canine kidney (MDCK) cell lines expressing enhanced green fluorescent protein-Rab11-FIP2 wild type or a nonphosphorylatable mutant [Rab11-FIP2(S227A)]. Analysis of these cell lines demonstrates a new role for Rab11-FIP2 in addition to that in the plasma membrane recycling system. In calcium switch assays, cells expressing Rab11-FIP2(S227A) showed a defect in the timely reestablishment of p120-containing junctional complexes. However, Rab11-FIP2(S227A) did not affect localization with recycling system components or the normal function of apical recycling and transcytosis pathways. These results indicate that phosphorylation of Rab11-FIP2 on serine 227 by MARK2 regulates an alternative pathway modulating the establishment of epithelial polarity.