Phosphorylation of claudin-3 at threonine 192 by cAMP-dependent protein kinase regulates tight junction barrier function in ovarian cancer cells
Phosphorylation of claudin-3 at threonine 192 by cAMP-dependent protein kinase regulates tight junction barrier function in ovarian cancer cells
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DOI：
10.1074/jbc.m502003200
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发表时间：
2005-07-15
影响因子：
4.8
通讯作者：
Morin, PJ
中科院分区：
文献类型：
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作者：
D'Souza, T;Agarwal, R;Morin, PJ
紧密连接蛋白（Claudin）是紧密连接（TJs）的形成和功能所必需的膜蛋白。在细胞渗透性和极性中具有重要作用的TJ的破坏被认为有助于上皮肿瘤发生。Claudin-3和Claudin-4在卵巢癌中经常过表达，但参与这些蛋白质调控的分子途径尚不清楚。有趣的是，一些研究已经证明了磷酸化在调节TJ复合物中的作用，尽管claudin磷酸化的证据很少。在这里，我们发现，claudin-3和-4可以在卵巢癌细胞中磷酸化。谷胱甘肽S-转移酶融合构建体的体外磷酸化试验表明，claudin-3的C末端是cAMP依赖性蛋白激酶（PKA）的优良底物。使用定点突变，我们在claudin-3 C末端第192位氨基酸处鉴定了PKA磷酸化位点。过表达含有T192 D突变的蛋白质，模拟磷酸化状态，导致卵巢癌细胞系OVCA 433中TJ强度降低。我们的研究结果表明，密蛋白-3磷酸化PKA，激酶经常在卵巢癌中激活，可能提供了一种机制，在这种癌症中的TJ中断。此外，我们的研究结果可能对正常上皮细胞中TJ的调节具有普遍意义。
Claudins are integral membrane proteins essential in the formation and function of tight junctions (TJs). Disruption of TJs, which have essential roles in cell permeability and polarity, is thought to contribute to epithelial tumorigenesis. Claudin-3 and -4 are frequently overexpressed in ovarian cancer, but the molecular pathways involved in the regulation of these proteins are unclear. Interestingly, several studies have demonstrated a role for phosphorylation in the regulation of TJ complexes, although evidence for claudin phosphorylation is scarce. Here, we showed that claudin-3 and -4 can be phosphorylated in ovarian cancer cells. In vitro phosphorylation assays using glutathione S-transferase fusion constructs demonstrated that the C terminus of claudin-3 is an excellent substrate for cAMP-dependent protein kinase (PKA). Using site-directed mutagenesis, we identified a PKA phosphorylation site at amino acid 192 in the C terminus of claudin-3. Overexpression of the protein containing a T192D mutation, mimicking the phosphorylated state, resulted in a decrease in TJ strength in ovarian cancer cell line OVCA433. Our results suggest that claudin-3 phosphorylation by PKA, a kinase frequently activated in ovarian cancer, may provide a mechanism for the disruption of TJs in this cancer. In addition, our findings may have general implications for the regulation of TJs in normal epithelial cells.