Regulation of growth factor receptor downregulation by a deubiquitinating enzyme UBPY

1.通过去泛素化酶UBPY调节表皮生长因子（EGF）受体下调配体激活的EGF受体的泛素化作为靶向蛋白质进行溶酶体运输的信号。我们发现UBPY去泛素化早期内体上激活的EGF受体并延迟其下调。这些结果表明，通过从受体中去除“溶酶体靶向信号”并抑制其向溶酶体的运输，UBPY负性调节EGF受体的下调。2.去泛素化酶UBPY对早期内体形态的调节通过表达一个显性的-阴性突变体或siRNA导致泛素化蛋白在早期内体上的积累和细胞器的形态畸变。电镜观察显示，UBPY抑制细胞早期内体异常聚集。这些结果表明，蛋白质泛素化水平的调节 ...更多信息 14-3-3蛋白在M期对UBPY去泛素化活性的调节我们发现UBPY通过一个共有的14 - 3-3-结合基序（Rb-SP）与14 -3-3蛋白结合，<680>这种结合需要S^的磷酸化<680>。我们还发现14-3-3的结合抑制了UBPY的催化活性。最后，UBPY被去磷酸化，与14-3-3蛋白质解离，并在M期被催化活化，4.去泛素化酶AMSH定位于早期内体的机制我们发现AMSH及其同源物AMSH-like protein（AMSH-LP）通过一种新的~（18）-结合方式与网格蛋白重链的末端结构域结合。AMSH和AMSH-LP之间保守的30个氨基酸网格蛋白结合基序。从这些去泛素化酶中删除网格蛋白结合基序，以及siRNA介导的细胞网格蛋白敲除，导致它们错误定位于细胞质。这些结果表明AMSH和AMSH-LP通过与内体膜上的网格蛋白涂层相互作用锚定到早期内体。少

1.Regulation of the epidermal growth factor (EGF) receptor downregulation by a deubiquitinating enzyme UBPYUbiquitination of ligand-activated EGF receptor serves as a signal that targets the protein for lysosomal trafficking. We found that UBPY deubiquitinates activated EGF receptor on early endosomes and delays its downregulation. These results indicated that by removing the "lysosome-targeting signal" from the receptor and inhibiting its trafficking to lysosomes, UBPY regulates the downregulation of EGF receptor negatively.2.Regulation of the morphology of early endosomes by a deubiquitinating enzyme UBPYInhibition of the UBPY function by expressing a dominant-negative mutant or siRNA resulted in the accumulation of ubiquitinated proteins on early endosomes and the morphological aberration of the organelle. Electromicroscopic study showed that early endosomes are aberrantly aggregated in UBPY-inhibited cells. These results suggested that the regulation of the level of protein ubiquit … More ination on early endosomes by UBPY is essential for maintaining the morphology of the organelle.3.Regulation of the deubiquitinating activity of UBPY by 14-3-3 proteins in the M phaseWe found that UBPY binds 14-3-3 proteins via a consensus 14-3-3-binding motif, RSYS^<680>SP. The binding required the phosphorylation of S^<680>. We also found that the binding of 14-3-3 inhibits the catalytic activity of UBPY. Finally, UBPY is dephosphorylated, dissociated from 14-3-3 proteins, and catalytically activated in the M phase, suggesting that elevated activity of UBPY regulates the functions of early endosomes in the M phase.4.Mechanism of the localization of a deubiquitinating enzyme AMSH to early endosomesWe found that AMSH and its homolog AMSH-like protein (AMSH-LP) bind to the terminal domain of clathrin heavy chain via a novel ~30-amino-acid clathrin-binding motif which is conserved between AMSH and AMSH-LP. Deletion of the clathrin-binding motif from these deubiquitinating enzymes, as well as siRNA-mediated knockdown of cellular clathrin, led to their mislocalization to the cytoplasm. These results suggested that AMSH and AMSH-LP are anchored to early endosomes through the interaction with the clathrin coat on the endosomal membrane. Less