Regulation of growth factor receptor downregulation by a deubiquitinating enzyme UBPY

1.通过去泛素化酶UBPY调节EGF受体的下调配体激活的EGF受体的泛素化是以溶酶体转运为靶的蛋白的信号。我们发现UBPY去泛素化激活了早期内小体上的EGF受体，并延缓了其下调。这些结果表明，UBPY通过去除受体上的“溶酶体靶向信号”并抑制其向溶酶体的转运，负向地调节EGF受体的下调。2.脱泛素酶UBPY通过表达显性负性突变体或siRNA抑制UBPY的功能，导致泛素化蛋白在早期内体上的积累和细胞器的形态异常。电子显微镜研究表明，在UbPY抑制的细胞中，早期的内吞体会异常聚集。这些结果表明，对…泛素蛋白水平的调节3.14-3-3蛋白对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结合基序与14-3-3蛋白结合。这种结合需要S^&lt；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，从14-3-3蛋白解离，并在M期被催化激活，这表明UBPY活性的提高调节了早期内吞体内的功能。4.脱泛素酶AMSH定位于早期内体的机制我们发现AMSH及其同系物AMSH-LP通过一个新的~30个氨基酸的clathrin结合基序与cathrin重链结合，该基序保守于AMSH和AMSH-LP之间。这些脱泛素化酶中分子筛蛋白结合基序的缺失，以及siRNA介导的细胞内分子筛蛋白的敲除，导致它们错误地定位在细胞质中。这些结果表明，AMSH和AMSH-LP是通过与内体膜上的网状蛋白涂层相互作用而锚定在早期的内体上的。较少

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