C3M, Inserm, Equipe labellisée Ligue contre le Cancer, University of Côte d'Azur, Nice, F-06204, France.
FPP at IGF, CNRS, Inserm, University of Montpellier, Montpellier, F-34094, France.
Sci Rep. 2018 Jan 23;8(1):1410. doi: 10.1038/s41598-018-19471-2.
The regulation of Rac1 by HACE1-mediated ubiquitination and proteasomal degradation is emerging as an essential element in the maintenance of cell homeostasis. However, how the E3 ubiquitin ligase activity of HACE1 is regulated remains undetermined. Using a proteomic approach, we identified serine 385 as a target of group-I PAK kinases downstream Rac1 activation by CNF1 toxin from pathogenic E. coli. Moreover, cell treatment with VEGF also promotes Ser-385 phosphorylation of HACE1. We have established in vitro that HACE1 is a direct target of PAK1 kinase activity. Mechanistically, we found that the phospho-mimetic mutant HACE1(S385E), as opposed to HACE1(S385A), displays a lower capacity to ubiquitinate Rac1 in cells. Concomitantly, phosphorylation of Ser-385 plays a pivotal role in controlling the oligomerization state of HACE1. Finally, Ser-385 phosphorylated form of HACE1 localizes in the cytosol away from its target Rac1. Together, our data point to a feedback inhibition of HACE1 ubiquitination activity on Rac1 by group-I PAK kinases.
HACE1 介导的 Rac1 泛素化和蛋白酶体降解的调节作用正成为维持细胞内稳态的重要因素。然而，HACE1 的 E3 泛素连接酶活性如何被调节仍不清楚。我们采用蛋白质组学方法，发现组 I PAK 激酶是 Rac1 激活后 CNF1 毒素（来自致病性大肠杆菌）的下游靶点，可将 HACE1 丝氨酸 385 磷酸化。此外，细胞用 VEGF 处理也会促进 HACE1 的 Ser-385 磷酸化。我们已经在体外证实 HACE1 是 PAK1 激酶活性的直接靶标。从机制上讲，我们发现与 HACE1(S385A)相比，磷酸化模拟突变体 HACE1(S385E)在细胞中对 Rac1 的泛素化能力较低。同时，Ser-385 的磷酸化在控制 HACE1 的寡聚状态方面起着关键作用。最后，HACE1 的 Ser-385 磷酸化形式定位于细胞质中，远离其靶标 Rac1。总之，我们的数据表明组 I PAK 激酶对 Rac1 的 HACE1 泛素化活性具有反馈抑制作用。
