Weis Center for Research, Geisinger Clinic, Danville, Pennsylvania 17822, USA.
Mol Cell Biol. 2010 Mar;30(6):1541-54. doi: 10.1128/MCB.00013-10. Epub 2010 Jan 19.
ACK (activated Cdc42-associated tyrosine kinase) (also Tnk2) is an ubiquitin-binding protein and plays an important role in ligand-induced and ubiquitination-mediated degradation of epidermal growth factor receptor (EGFR). Here we report that ACK is ubiquitinated by HECT E3 ubiquitin ligase Nedd4-1 and degraded along with EGFR in response to EGF stimulation. ACK interacts with Nedd4-1 through a conserved PPXY WW-binding motif. The WW3 domain in Nedd4-1 is critical for binding to ACK. Although ACK binds to both Nedd4-1 and Nedd4-2 (also Nedd4L), Nedd4-1 is the E3 ubiquitin ligase for ubiquitination of ACK in cells. Interestingly, deletion of the sterile alpha motif (SAM) domain at the N terminus dramatically reduced the ubiquitination of ACK by Nedd4-1, while deletion of the Uba domain dramatically enhanced the ubiquitination. Use of proteasomal and lysosomal inhibitors demonstrated that EGF-induced ACK degradation is processed by lysosomes, not proteasomes. RNA interference (RNAi) knockdown of Nedd4-1, not Nedd4-2, inhibited degradation of both EGFR and ACK, and overexpression of ACK mutants that are deficient in either binding to or ubiquitination by Nedd4-1 blocked EGF-induced degradation of EGFR. Our findings suggest an essential role of Nedd4-1 in regulation of EGFR degradation through interaction with and ubiquitination of ACK.
ACK（激活的 Cdc42 相关酪氨酸激酶）（也称为 Tnk2）是一种泛素结合蛋白，在配体诱导和泛素化介导的表皮生长因子受体（EGFR）降解中发挥重要作用。在这里，我们报告 ACK 通过 HECT E3 泛素连接酶 Nedd4-1 泛素化，并与 EGFR 一起在 EGF 刺激下降解。ACK 通过保守的 PPXY WW 结合基序与 Nedd4-1 相互作用。Nedd4-1 中的 WW3 结构域对于与 ACK 的结合至关重要。尽管 ACK 与 Nedd4-1 和 Nedd4-2（也称为 Nedd4L）都结合，但 Nedd4-1 是细胞中 ACK 泛素化的 E3 泛素连接酶。有趣的是，N 端的 sterile alpha motif (SAM) 结构域缺失会显著降低 Nedd4-1 对 ACK 的泛素化，而 Uba 结构域缺失会显著增强泛素化。使用蛋白酶体和溶酶体抑制剂表明，EGF 诱导的 ACK 降解是通过溶酶体而不是蛋白酶体进行的。RNA 干扰 (RNAi) 敲低 Nedd4-1，而不是 Nedd4-2，抑制了 EGFR 和 ACK 的降解，并且表达缺乏与 Nedd4-1 结合或泛素化的 ACK 突变体可阻止 EGF 诱导的 EGFR 降解。我们的研究结果表明，Nedd4-1 通过与 ACK 相互作用和泛素化在调节 EGFR 降解中起着重要作用。
