Inhibition of gap junction activity through the release of the C1B domain of protein kinase cγ (PKCγ) from 14-3-3 -: Identification of PKCγ-binding sites
Inhibition of gap junction activity through the release of the C1B domain of protein kinase cγ (PKCγ) from 14-3-3 -: Identification of PKCγ-binding sites
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DOI：
10.1074/jbc.m403040200
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发表时间：
2004-12-10
影响因子：
4.8
通讯作者：
Takemoto, DJ
中科院分区：
文献类型：
--
作者：
Nguyen, TA;Takemoto, LJ;Takemoto, DJ
我们之前的研究表明，胰岛素样生长因子- 1或晶状体上皮源性生长因子增加了蛋白激酶Cgamma （PKCgamma）向膜的易位和PKCgamma对Cx43的磷酸化，并导致随后的间隙连接活性降低(Nguyen， T. a ., Boyle， D. L., Wagner， L. M., Shinohara， T., and Takemoto， D. J. (2003) Exp. Eye res76, 565-572；林博士、博伊尔博士和竹本博士（2003）《调查》。角膜切削。科学44,1160-1168)。晶状体上皮细胞的间隙连接活性受pkγ介导的Cx43磷酸化调控。PKCgamma的活性受到生长因子调控的二酰基甘油合成增加的刺激，但受到细胞质对接蛋白（如14-3-3）的抑制。在这里，我们已经确定了pkgamma - c1b结构域中负责其与14-3-3 - epsilon相互作用的两个位点。两个位点C1B1（残基101-112）和C1B5（残基141-151）位于PKCgamma的C1结构域内。C1B1和/或C1B5合成肽可直接竞争14-3-3 -3epsilon的结合，导致体内或体外内源性细胞从14-3-3 -3epsilon释放PKCgamma，激活PKCgamma酶活性，磷酸化PKCgamma，随后PKCgamma转运到膜上，抑制间隙连接活性。与对照相比，用C1B1或C1B5肽处理的细胞的间隙连接活性降低了至少5倍。与对照细胞相比，100 muM的C1B1或C1B5肽也导致Cx43斑块形成减少10或4倍。通过高压液相色谱法和基质辅助激光解吸电离飞行时间质谱法验证了这些合成肽被细胞吸收。我们已经证明PKCgamma的活性和定位受其与PKCgamma的C1B结构域的14-3-3 - epsilon结合的调节。与这些PKCgamma区域相对应的合成肽成功地竞争了14-3-3 -3epsilon与内源性PKCepsilon的结合，从而抑制了间隙连接的活性。这表明合成肽可以用于外源性调节间隙连接。
We have shown previously that insulin-like growth factor-I or lens epithelium-derived growth factor increases the translocation of protein kinase Cgamma (PKCgamma) to the membrane and the phosphorylation of Cx43 by PKCgamma and causes a subsequent decrease of gap junction activity (Nguyen, T. A., Boyle, D. L., Wagner, L. M., Shinohara, T., and Takemoto, D. J. (2003) Exp. Eye Res. 76, 565-572; Lin, D., Boyle, D. L., and Takemoto, D. J. ( 2003) Investig. Ophthalmol. Vis. Sci. 44, 1160-1168). Gap junction activity in lens epithelial cells is regulated by PKCgamma-mediated phosphorylation of Cx43. PKCgamma activity is stimulated by growth factor-regulated increases in the synthesis of diacylglycerol but is inhibited by cytosolic docking proteins such as 14-3-3. Here we have identified two sites on the PKCgamma-C1B domain that are responsible for its interaction with 14-3-3epsilon. Two sites, C1B1 (residues 101-112) and C1B5 (residues 141-151), are located within the C1 domain of PKCgamma. C1B1 and/or C1B5 synthetic peptides can directly compete for the binding of 14-3-3epsilon, resulting in the release of endogenous cellular PKCgamma from 14-3-3epsilon, in vivo or in vitro, in activation of PKCgamma enzyme activity, phosphorylation of PKCgamma, in the subsequent translocation of PKCgamma to the membrane, and in inhibition of gap junction activity. Gap junction activity was decreased by at least 5-fold in cells treated with C1B1 or C1B5 peptides when compared with a control. 100 muM of C1B1 or C1B5 peptides also caused a 10- or 4-fold decrease of Cx43 plaque formation compared with control cells. The uptake of these synthetic peptides into cells was verified by using high pressure liquid chromatography and matrix-assisted laser desorption ionization time-of-flight-mass spectrometry. We have demonstrated that the activity and localization of PKCgamma are regulated by its binding to 14-3-3epsilon at the C1B domain of PKCgamma. Synthetic peptides corresponding to these regions of PKCgamma successfully competed for the binding of 14-3-3epsilon to endogenous PKCepsilon, resulting in inhibition of gap junction activity. This demonstrates that synthetic peptides can be used to exogenously regulate gap junctions.