Okumura Fumihiko, Hatakeyama Shigetsugu, Matsumoto Masaki, Kamura Takumi, Nakayama Keiichi I
Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan.
J Biol Chem. 2004 Dec 17;279(51):53533-43. doi: 10.1074/jbc.M402916200. Epub 2004 Oct 5.
E4B (also known as UFD2a) is a mammalian homolog of Saccharomyces cerevisiae Ufd2, which was originally described as a ubiquitin chain assembly factor (E4). E4B is a U-box-type ubiquitin-protein isopeptide ligase (E3) and likely functions as either an E3 or an E4. With a yeast two-hybrid screen, we have now identified FEZ1 (fasciculation and elongation protein zeta 1) as a protein that interacts with E4B. FEZ1 is implicated in neuritogenesis when phosphorylated by protein kinase Czeta (PKCzeta). Interaction between E4B and FEZ1 in mammalian cells was enhanced by coexpression of constitutively active PKCzeta. E4B mediated the polyubiquitylation of FEZ1 but did not affect its intracellular stability, suggesting that such modification of FEZ1 is not a signal for its proteolysis. Polyubiquitylation of FEZ1 by E4B required Lys(27) of ubiquitin. Expression of a dominant-negative mutant of E4B in rat pheochromocytoma PC12 cells resulted in inhibition of neurite extension induced either by nerve growth factor or by coexpression of FEZ1 and constitutively active PKCzeta. These findings indicate that E4B serves as a ubiquitin ligase for FEZ1 and thereby regulates its function but not its degradation.
E4B（也称为UFD2a）是酿酒酵母Ufd2的哺乳动物同源物，最初被描述为一种泛素链组装因子（E4）。E4B是一种U-box型泛素-蛋白质异肽连接酶（E3），可能作为E3或E4发挥作用。通过酵母双杂交筛选，我们现已鉴定出FEZ1（成束和延伸蛋白ζ1）为一种与E4B相互作用的蛋白质。当被蛋白激酶Cζ（PKCζ）磷酸化时，FEZ1与神经突形成有关。在哺乳动物细胞中，组成型活性PKCζ的共表达增强了E4B与FEZ1之间的相互作用。E4B介导了FEZ1的多聚泛素化，但不影响其细胞内稳定性，这表明FEZ1的这种修饰不是其蛋白水解的信号。E4B对FEZ1的多聚泛素化需要泛素的赖氨酸（Lys）27。在大鼠嗜铬细胞瘤PC12细胞中表达E4B的显性负性突变体导致神经生长因子或FEZ1与组成型活性PKCζ共表达诱导的神经突延伸受到抑制。这些发现表明，E4B作为FEZ1的泛素连接酶，从而调节其功能而非降解。
