Division of Pharmaceutical Cell Biology, Graduate School of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan.
Biol Pharm Bull. 2010;33(6):951-7. doi: 10.1248/bpb.33.951.
Nedd4-interacting protein 2 (NDFIP2) has three transmembrane domains and interacts with multiple Nedd4 family ubiquitin ligases through polyprolinetyrosine (PY) motifs located in its N-terminal cytoplasmic domain. It has been postulated that NDFIP2 acts as an adaptor for the ubiquitylation of substrates with Nedd4 ubiquitin ligase. However, whether NDFIP2 promotes or inhibits the ubiquitylation of Nedd4 substrates is still under debate. We show here that although NDFIP2 is detected in the Golgi/trans-Golgi network (TGN) area, it is rapidly delivered to and degraded in lysosomes with its half-life ca. 1.5 h. Intriguingly, knockdown (KD) of NDFIP2 with small interfering RNA (siRNA) impaired both the formation and function of gap junctions. Indeed, KD of NDFIP2 destabilized the gap junction protein connexin43 that contains PY motif. In support of this, overexpression of NDFIP2 stabilized connexin43 and enhanced the formation of gap junctions. Furthermore, the PY motifs of NDFIP2, which are required for its interaction with Nedd4, Atrophin-1 interacting protein (AIP) 4 (AIP4)/Itch, and AIP2/WWP2, were necessary for the targeting of NDFIP2 to lysosomes and/or the stability of connexin43 and gap junctions. Collectively these findings suggest that NDFIP2 may inhibit the Nedd4-dependent ubiquitylation of membrane proteins containing PY motifs, such as connexin43, in a competitive manner.
Nedd4 相互作用蛋白 2（NDFIP2）具有三个跨膜结构域，通过其细胞质 N 端结构域中的多脯氨酸-酪氨酸（PY）基序与多种 Nedd4 家族泛素连接酶相互作用。据推测，NDFIP2 作为带有 Nedd4 泛素连接酶的底物泛素化的衔接蛋白发挥作用。然而，NDFIP2 是否促进或抑制 Nedd4 底物的泛素化仍存在争议。我们在这里表明，尽管 NDFIP2 检测到在高尔基/反高尔基网络（TGN）区域，但它在溶酶体中迅速被递送到并降解，半衰期约为 1.5 小时。有趣的是，用小干扰 RNA（siRNA）敲低（KD）NDFIP2 会损害间隙连接的形成和功能。事实上，NDFIP2 的 KD 使含有 PY 基序的间隙连接蛋白连接蛋白 43（connexin43）不稳定。确实，NDFIP2 的过表达稳定了 connexin43 并增强了间隙连接的形成。此外，NDFIP2 与 Nedd4、Atrophin-1 相互作用蛋白（AIP）4（AIP4）/Itch 和 AIP2/WWP2 相互作用所必需的 PY 基序对于 NDFIP2 靶向溶酶体和/或 connexin43 和间隙连接的稳定性是必需的。总之，这些发现表明，NDFIP2 可能以竞争性方式抑制含有 PY 基序的膜蛋白（如 connexin43）的 Nedd4 依赖性泛素化。
