Department of Molecular Cytogenetics, Medical Research Institute and School of Biomedical Science, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo-ku, Tokyo 113-8510, Japan.
Department of Molecular Cytogenetics, Medical Research Institute and School of Biomedical Science, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo-ku, Tokyo 113-8510, Japan; Department of Genome Medicine, Hard Tissue Genome Research Center, Tokyo Medical and Dental University, 1-5-45 Yushima Bunkyo-ku, Tokyo 113-8510, Japan.
J Biol Chem. 2011 Dec 23;286(51):44086-44094. doi: 10.1074/jbc.M111.251694. Epub 2011 Oct 18.
LAPTM5 (lysosomal-associated protein multispanning transmembrane 5) is a membrane protein on the intracellular vesicles. We have previously demonstrated that the accumulation of LAPTM5-positive vesicles was closely associated with the programmed cell death occurring during the spontaneous regression of neuroblastomas. Although the accumulation of LAPTM5 protein might occur at the post-translational level, the molecular mechanism has been unclear. Here, we found that the level of LAPTM5 protein is regulated negatively by the degradation through ubiquitination by ITCH, an E3 ubiquitin ligase. ITCH directly binds to the PPxY motif of LAPTM5 via its WW domains and promotes ubiquitination through a HECT-type ligase domain. Overexpression of ITCH led to the degradation of LAPTM5 protein, and conversely, knockdown of ITCH by siRNA resulted in the stabilization of LAPTM5 protein. In addition, the inhibition of ITCH enhanced the cell death occurred by accumulation of LAPTM5 in neuroblastoma cells. These findings suggest that LAPTM5 is a novel substrate in terms of degradation by the ubiquitin ligase ITCH, and this system might act as a negative regulator in the spontaneous regression of neuroblastomas by preventing LAPTM5-mediated cell death.
LAPTM5（溶酶体相关蛋白多跨膜 5）是细胞内囊泡上的一种膜蛋白。我们之前已经证明，LAPTM5 阳性囊泡的积累与神经母细胞瘤自发消退过程中发生的程序性细胞死亡密切相关。虽然 LAPTM5 蛋白的积累可能发生在翻译后水平，但分子机制尚不清楚。在这里，我们发现 LAPTM5 蛋白的水平受到通过 ITCH（一种 E3 泛素连接酶）泛素化进行的降解的负调控。ITCH 通过其 WW 结构域直接与 LAPTM5 的 PPxY 基序结合，并通过 HECT 型连接酶结构域促进泛素化。ITCH 的过表达导致 LAPTM5 蛋白的降解，相反，通过 siRNA 敲低 ITCH 导致 LAPTM5 蛋白的稳定。此外，抑制 ITCH 增强了由于神经母细胞瘤细胞中 LAPTM5 积累而导致的细胞死亡。这些发现表明，LAPTM5 是泛素连接酶 ITCH 降解的一种新型底物，该系统可能通过防止 LAPTM5 介导的细胞死亡来作为神经母细胞瘤自发消退的负调节剂。
