Leucine zipper-mediated homo-oligomerization regulates the Rho-GEF activity of AKAP-Lbc
Leucine zipper-mediated homo-oligomerization regulates the Rho-GEF activity of AKAP-Lbc
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DOI：
10.1074/jbc.m414440200
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发表时间：
2005-04-15
影响因子：
4.8
通讯作者：
Diviani, D
中科院分区：
文献类型：
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作者：
Baisamy, L;Jurisch, N;Diviani, D
AKAP-Lbc 是 A 激酶锚定蛋白 (AKAP) 家族的新成员，其功能是 cAMP 依赖性蛋白激酶 (PKA) 靶向蛋白以及 RhoA 的鸟嘌呤核苷酸交换因子 (GEF)。我们最近证明，异源三聚体 G 蛋白 G(12) 的 α 亚基可刺激 AKAP-Lbc Rho-GEF 活性，而锚定的 PKA 对 AKAP-Lbc 的磷酸化会诱导 14-3-3 的募集，从而抑制其 GEF 功能。在本报告中，我们使用免疫共沉淀方法证明 AKAP-Lbc 可以在细胞内形成同源寡聚物。诱变研究表明，寡聚化是由位于锚定蛋白 C 末端区域的两个相邻的亮氨酸拉链基序介导的。最有趣的是，寡聚化的破坏导致 AKAP-Lbc 在基础条件下刺激细胞中 Rho-GTP 形成的能力急剧增加，这表明寡聚化使 AKAP-Lbc 维持在基础非活性状态。基于这些结果以及我们之前的研究结果表明 AKAP-Lbc 通过与 14-3-3 结合而失活，我们研究了 14-3-3 的调节作用可能需要 AKAP-Lbc 寡聚化的假设。最有趣的是，我们发现寡聚化能力受损的 AKAP-Lbc 突变体完全抵抗 PKA 和 14-3-3 的抑制作用。这表明仅当锚定蛋白处于寡聚状态时，14-3-3 才能负向调节 AKAP-Lbc 的 Rho-GEF 活性。总而言之，这些发现为寡聚化如何调节 Dbl 家族交换因子的活性提供了新的机制解释。
AKAP-Lbc is a novel member of the A-kinase anchoring protein (AKAPs) family, which functions as a cAMP-dependent protein kinase (PKA)-targeting protein as well as a guanine nucleotide exchange factor (GEF) for RhoA. We recently demonstrated that AKAP-Lbc Rho-GEF activity is stimulated by the alpha-subunit of the heterotrimeric G protein G(12), whereas phosphorylation of AKAP-Lbc by the anchored PKA induces the recruitment of 14-3-3, which inhibits its GEF function. In the present report, using co-immunoprecipitation approaches, we demonstrated that AKAP-Lbc can form homo-oligomers inside cells. Mutagenesis studies revealed that oligomerization is mediated by two adjacent leucine zipper motifs located in the C-terminal region of the anchoring protein. Most interestingly, disruption of oligomerization resulted in a drastic increase in the ability of AKAP-Lbc to stimulate the formation of Rho-GTP in cells under basal conditions, suggesting that oligomerization maintains AKAP-Lbc in a basal-inactive state. Based on these results and on our previous findings showing that AKAP-Lbc is inactivated through the association with 14-3-3, we investigated the hypothesis that AKAP-Lbc oligomerization might be required for the regulatory action of 14-3-3. Most interestingly, we found that mutants of AKAP-Lbc impaired in their ability to undergo oligomerization were completely resistant to the inhibitory effect of PKA and 14-3-3. This suggests that 14-3-3 can negatively regulate the Rho-GEF activity of AKAP-Lbc only when the anchoring protein is in an oligomeric state. Altogether, these findings provide a novel mechanistic explanation of how oligomerization can regulate the activity of exchange factors of the Dbl family.