Saito Satoko, Matsui Hiroyuki, Kawano Miyuki, Kumagai Keigo, Tomishige Nario, Hanada Kentaro, Echigo Seishi, Tamura Shinri, Kobayashi Takayasu
Department of Biochemistry, Institute of Development, Aging and Cancer, Tohoku University, 4-1 Seiryo-machi, Aoba-ku, Sendai 980-8575, Japan.
J Biol Chem. 2008 Mar 7;283(10):6584-93. doi: 10.1074/jbc.M707691200. Epub 2007 Dec 28.
Protein phosphatase 2Cepsilon (PP2Cepsilon), a mammalian PP2C family member, is expressed in various tissues and is implicated in the negative regulation of stress-activated protein kinase pathways. We show that PP2Cepsilon is an endoplasmic reticulum (ER) transmembrane protein with a transmembrane domain at the amino terminus and the catalytic domain facing the cytoplasm. Yeast two-hybrid screening of a human brain library using PP2Cepsilon as bait resulted in the isolation of a cDNA that encoded vesicle-associated membrane protein-associated protein A (VAPA). VAPA is an ER resident integral membrane protein involved in recruiting lipid-binding proteins such as the ceramide transport protein CERT to the ER membrane. Expression of PP2Cepsilon resulted in dephosphorylation of CERT in a VAPA expression-dependent manner, which was accompanied by redistribution of CERT from the cytoplasm to the Golgi apparatus. The expression of PP2Cepsilon also enhanced the association between CERT and VAPA. In addition, knockdown of PP2Cepsilon expression by short interference RNA attenuated the interaction between CERT and VAPA and the sphingomyelin synthesis. These results suggest that CERT is a physiological substrate of PP2Cepsilon and that dephosphorylation of CERT by PP2Cepsilon may play an important role in the regulation of ceramide trafficking from the ER to the Golgi apparatus.
蛋白磷酸酶2Cε（PP2Cε）是哺乳动物PP2C家族的成员，在多种组织中表达，并参与应激激活蛋白激酶途径的负调控。我们发现PP2Cε是一种内质网（ER）跨膜蛋白，其氨基末端有一个跨膜结构域，催化结构域面向细胞质。以PP2Cε为诱饵对人脑文库进行酵母双杂交筛选，分离出一个编码囊泡相关膜蛋白相关蛋白A（VAPA）的cDNA。VAPA是一种内质网驻留整合膜蛋白，参与将脂质结合蛋白如神经酰胺转运蛋白CERT招募到内质网膜上。PP2Cε的表达导致CERT以VAPA表达依赖的方式去磷酸化，同时伴随着CERT从细胞质重新分布到高尔基体。PP2Cε的表达还增强了CERT与VAPA之间的结合。此外，通过短干扰RNA敲低PP2Cε的表达减弱了CERT与VAPA之间的相互作用以及鞘磷脂的合成。这些结果表明CERT是PP2Cε的生理底物，并且PP2Cε对CERT的去磷酸化可能在神经酰胺从内质网到高尔基体的转运调节中起重要作用。
