Gur Ibrahim, Fujiwara Kazushiro, Hasegawa Koichi, Yoshikawa Kazuaki
Laboratory of Regulation of Neuronal Development, Institute for Protein Research, Osaka University, Suita, Osaka, Japan.
PLoS One. 2014 Jun 9;9(6):e99503. doi: 10.1371/journal.pone.0099503. eCollection 2014.
Necdin, a pleiotropic protein that promotes differentiation and survival of mammalian neurons, is a member of MAGE (melanoma antigen) family proteins that share a highly conserved MAGE homology domain. Several MAGE proteins interact with ubiquitin E3 ligases and modulate their activities. However, it remains unknown whether MAGE family proteins interact with SUMO (small ubiquitin-like modifier) E3 ligases such as PIAS (protein inhibitor of activated STAT) family, Nsmce2/Mms21 and Cbx4/Pc2. In the present study, we examined whether necdin interacts with these SUMO E3 ligases. Co-immunoprecipitation analysis revealed that necdin, MAGED1, MAGEF1 and MAGEL2 bound to PIAS1 but not to Nsmce2 or Cbx4. These SUMO E3 ligases bound to MAGEA1 but failed to interact with necdin-like 2/MAGEG1. Necdin bound to PIAS1 central domains that are highly conserved among PIAS family proteins and suppressed PIAS1-dependent sumoylation of the substrates STAT1 and PML (promyelocytic leukemia protein). Remarkably, necdin promoted degradation of PIAS1 via the ubiquitin-proteasome pathway. In transfected HEK293A cells, amino- and carboxyl-terminally truncated mutants of PIAS1 bound to necdin but failed to undergo necdin-dependent ubiquitination. Both PIAS1 and necdin were associated with the nuclear matrix, where the PIAS1 terminal deletion mutants failed to localize, implying that the nuclear matrix is indispensable for necdin-dependent ubiquitination of PIAS1. Our data suggest that necdin suppresses PIAS1 both by inhibiting SUMO E3 ligase activity and by promoting ubiquitin-dependent degradation.
Necdin是一种促进哺乳动物神经元分化和存活的多效性蛋白，是MAGE（黑色素瘤抗原）家族蛋白的成员，该家族蛋白共享一个高度保守的MAGE同源结构域。几种MAGE蛋白与泛素E3连接酶相互作用并调节其活性。然而，MAGE家族蛋白是否与SUMO（小泛素样修饰物）E3连接酶相互作用，如PIAS（活化STAT的蛋白抑制剂）家族、Nsmce2/Mms21和Cbx4/Pc2，仍不清楚。在本研究中，我们检测了Necdin是否与这些SUMO E3连接酶相互作用。免疫共沉淀分析显示，Necdin、MAGED1、MAGEF1和MAGEL2与PIAS1结合，但不与Nsmce2或Cbx4结合。这些SUMO E3连接酶与MAGEA1结合，但未能与necdin样2/MAGEG1相互作用。Necdin与PIAS家族蛋白中高度保守的PIAS1中央结构域结合，并抑制PIAS1依赖的底物STAT1和PML（早幼粒细胞白血病蛋白）的SUMO化。值得注意的是，Necdin通过泛素-蛋白酶体途径促进PIAS1的降解。在转染的HEK293A细胞中，PIAS1的氨基和羧基末端截短突变体与Necdin结合，但未能发生Necdin依赖的泛素化。PIAS1和Necdin都与核基质相关，而PIAS1末端缺失突变体未能定位到核基质，这意味着核基质对于Necdin依赖的PIAS1泛素化是必不可少的。我们的数据表明，Necdin通过抑制SUMO E3连接酶活性和促进泛素依赖的降解来抑制PIAS1。
