Department of Biochemistry and Molecular Biology, Eppley Institute for Research in Cancer and Allied Diseases, University of Nebraska Medical Center, Omaha, NE 68198, USA.
Mol Biol Cell. 2013 Mar;24(6):715-33. doi: 10.1091/mbc.E12-07-0537. Epub 2013 Jan 30.
The molecular mechanisms regulating the assembly of connexins (Cxs) into gap junctions are poorly understood. Using human pancreatic tumor cell lines BxPC3 and Capan-1, which express Cx26 and Cx43, we show that, upon arrival at the cell surface, the assembly of Cx43 is impaired. Connexin43 fails to assemble, because it is internalized by clathrin-mediated endocytosis. Assembly is restored upon expressing a sorting-motif mutant of Cx43, which does not interact with the AP2 complex, and by expressing mutants that cannot be phosphorylated on Ser-279 and Ser-282. The mutants restore assembly by preventing clathrin-mediated endocytosis of Cx43. Our results also document that the sorting-motif mutant is assembled into gap junctions in cells in which the expression of endogenous Cx43 has been knocked down. Remarkably, Cx43 mutants that cannot be phosphorylated on Ser-279 or Ser-282 are assembled into gap junctions only when connexons are composed of Cx43 forms that can be phosphorylated on these serines and forms in which phosphorylation on these serines is abolished. Based on the subcellular fate of Cx43 in single and contacting cells, our results document that the endocytic itinerary of Cx43 is altered upon cell-cell contact, which causes Cx43 to traffic by EEA1-negative endosomes en route to lysosomes. Our results further show that gap-junctional plaques formed of a sorting motif-deficient mutant of Cx43, which is unable to be internalized by the clathrin-mediated pathway, are predominantly endocytosed in the form of annular junctions. Thus the differential phosphorylation of Cx43 on Ser-279 and Ser-282 is fine-tuned to control Cx43's endocytosis and assembly into gap junctions.
连接子（Cx）组装成缝隙连接的分子机制尚未完全阐明。我们使用表达 Cx26 和 Cx43 的人胰腺肿瘤细胞系 BxPC3 和 Capan-1 表明，在到达细胞表面时，Cx43 的组装受到损害。连接蛋白 43 无法组装，因为它被网格蛋白介导的内吞作用内化。当表达不与 AP2 复合物相互作用的 Cx43 分拣基序突变体，以及表达不能在 Ser-279 和 Ser-282 上磷酸化的突变体时，组装会得到恢复。突变体通过阻止 Cx43 的网格蛋白介导的内吞作用来恢复组装。我们的结果还证明，在敲低内源性 Cx43 表达的细胞中，分拣基序突变体被组装成缝隙连接。值得注意的是，不能在 Ser-279 或 Ser-282 上磷酸化的 Cx43 突变体只有在连接子由可以在这些丝氨酸上磷酸化的 Cx43 形式和这些丝氨酸上磷酸化被废除的形式组成时，才能组装成缝隙连接。基于 Cx43 在单个和接触细胞中的亚细胞命运，我们的结果证明，在细胞接触时，Cx43 的内吞途径发生改变，导致 Cx43 通过 EEA1 阴性内体运输到溶酶体。我们的结果进一步表明，由不能通过网格蛋白介导的途径内化的 Cx43 分拣基序缺陷突变体形成的缝隙连接斑块主要以环形连接的形式被内吞。因此，Cx43 在 Ser-279 和 Ser-282 上的差异磷酸化被精细调节以控制 Cx43 的内吞作用和组装成缝隙连接。
