Bao Xiaoyong, Reuss Luis, Altenberg Guillermo A
Membrane Protein Laboratory, Sealy Center for Structural Biology, and Department of Physiology and Biophysics, The University of Texas Medical Branch, Galveston, TX 77555-0437, USA.
J Biol Chem. 2004 May 7;279(19):20058-66. doi: 10.1074/jbc.M311137200. Epub 2004 Feb 17.
Indirect evidence suggests that the permeability of connexin 43 (Cx43) gap-junctional channels (connexons) to small organic molecules (M(r) < 1,000) is decreased by protein kinase C (PKC)-mediated phosphorylation of Ser-368. However, it is currently unknown whether this effect is produced directly by phosphorylation of this residue or whether cytoplasmic regulatory factors are required for the decrease in Cx43 gap-junctional channel permeability. Here we studied the effects of PKC-mediated phosphorylation on purified recombinant wild-type Cx43 and a PKC-unresponsive mutant (S368A). Our studies show that (a) PKC phosphorylates Ser-368, (b) the phosphorylation by PKC of purified and reconstituted connexons abolishes sucrose and Lucifer Yellow permeability, (c) the regulation of Cx43 by PKC is the direct result of phosphorylation of Ser-368 and does not involve intermediary regulatory factors, and (d) phosphorylation of Ser-368 produces a conformational change in purified Cx43 as demonstrated by changes in intrinsic Trp fluorescence and proteolytic digestion pattern. We conclude that phosphorylation of Ser-368 by PKC induces a conformational change of Cx43 that results in a decrease in connexon permeability.
间接证据表明，蛋白激酶C（PKC）介导的Ser-368磷酸化可降低连接蛋白43（Cx43）间隙连接通道（连接子）对小有机分子（相对分子质量<1000）的通透性。然而，目前尚不清楚这种效应是由该残基的磷酸化直接产生的，还是Cx43间隙连接通道通透性降低需要细胞质调节因子。在这里，我们研究了PKC介导的磷酸化对纯化的重组野生型Cx43和PKC无反应突变体（S368A）的影响。我们的研究表明：（a）PKC使Ser-368磷酸化；（b）PKC对纯化和重组的连接子进行磷酸化会消除蔗糖和荧光素黄的通透性；（c）PKC对Cx43的调节是Ser-368磷酸化的直接结果，不涉及中间调节因子；（d）如内在色氨酸荧光和蛋白水解消化模式的变化所示，Ser-368的磷酸化会使纯化的Cx43发生构象变化。我们得出结论，PKC介导的Ser-368磷酸化诱导Cx43构象变化，导致连接子通透性降低。
