Claudin-16 is directly phosphorylated by protein kinase A independently of a vasodilator-stimulated phosphoprotein-mediated pathway
Claudin-16 is directly phosphorylated by protein kinase A independently of a vasodilator-stimulated phosphoprotein-mediated pathway
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DOI：
10.1002/jcp.21178
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发表时间：
2008-01-01
影响因子：
5.6
通讯作者：
Miwa, Masao
中科院分区：
文献类型：
--
作者：
Ikari, Akira;Ito, Midori;Miwa, Masao
Claudin-16 （CLDN-16）参与Henle粗升肢细胞旁Mg2+的重吸收。CLDN-16的紧密连接定位和Mg2+转运受cAMP/ pka依赖性磷酸化调控。在这里，我们研究了PKA是否以直接或间接的方式磷酸化CLDN-16。利用Tet-OFF系统，CLDN-16在Madin-Darby犬肾（MDCK）细胞中稳定表达。胎牛血清（FCS）可上调CLDN-16的磷酸化水平。这种磷酸化被PKA抑制剂N-[2-（对溴肉桂胺）乙基]-5-异喹啉磺酰胺二盐酸盐完全抑制。在没有FCS的情况下，二丁基cAMP （DBcAMP）以浓度依赖的方式增加了CLDN-16的磷酸丝氨酸水平。磷酸化的CLDN-16引起了Mg2+的经上皮电阻（TER）和经上皮转运的增加。血管扩张剂刺激磷酸化蛋白（VASP）在FCS或DBcAMP的存在下也被磷酸化。在谷胱甘肽- s -转移酶（GST）下拉实验中，CLDN-16的胞质羧基结构域与PKA相关，但与VASP无关。此外，在MDCK细胞中，PKA被CLDN-16免疫沉淀，而VASP则没有。在表达VASP去磷酸化突变体（Ser 160Ala）的细胞中，CLDN-16被DBcAMP磷酸化，并与紧密连接支架蛋白ZO-1相关，没有完整的细胞-细胞连接。我们认为PKA直接磷酸化CLDN-16，导致定位到紧密连接（TJs）和维持Mg2+重吸收。
Claudin-16 (CLDN-16) is involved in the paracellular reabsorption of Mg2+ in the thick ascending limb of Henle. The tight junctional localization and Mg2+ transport of CLDN-16 are regulated by cAMP/PKA-dependent phosphorylation. Here, we examined whether PKA phosphorylates CLDN-16 in a direct or indirect manner. CLDN-16 was stably expressed in Madin-Darby canine kidney (MDCK) cells using a Tet-OFF system. The phosphorylation of CLDN-16 is upregulated by fetal calf serum (FCS). This phosphorylation was completely inhibited by a PKA inhibitor, N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide dihydrochloride. Without FCS, dibutyryl cAMP (DBcAMP) increased the phosphoserine level of CLDN-16 in a concentration-dependent manner. The phosphorylated CLDN-16 elicited increases of transepithelial electrical resistance (TER) and transepithelial transport of Mg2+. Vasodilator-stimulated phosphoprotein (VASP) was also phosphorylated in the presence of FCS or DBcAMP. In the glutathione-S-transferase (GST) pull down assay, a cytosolic carboxyl domain of CLDN-16 was associated with PKA, but not with VASP. Furthermore, PKA was immunoprecipitated with CLDN-16 in MDCK cells, but VASP was not. In cells expressing a dephosphorylated mutant (Ser 160Ala) of VASP, CLDN-16 was phosphorylated by DBcAMP and was associated with ZO-1, a tight junctional-scaffolding protein, without integral cell-cell junctions. We suggest that PKA directly phosphorylates CLDN-16, resulting in the localization to tight junctions (TJs) and the maintenance of Mg2+ reabsorption.