Scott Gregory K, Gu Feng, Crump Colin M, Thomas Laurel, Wan Lei, Xiang Yang, Thomas Gary
Vollum Institute, L-474, 3181 SW Sam Jackson Park Road, Portland, OR 97239, USA.
EMBO J. 2003 Dec 1;22(23):6234-44. doi: 10.1093/emboj/cdg596.
PACS-1 is a cytosolic sorting protein that directs the localization of membrane proteins in the trans-Golgi network (TGN)/endosomal system. PACS-1 connects the clathrin adaptor AP-1 to acidic cluster sorting motifs contained in the cytoplasmic domain of cargo proteins such as furin, the cation-independent mannose-6-phosphate receptor and in viral proteins such as human immunodeficiency virus type 1 Nef. Here we show that an acidic cluster on PACS-1, which is highly similar to acidic cluster sorting motifs on cargo molecules, acts as an autoregulatory domain that controls PACS-1-directed sorting. Biochemical studies show that Ser278 adjacent to the acidic cluster is phosphorylated by CK2 and dephosphorylated by PP2A. Phosphorylation of Ser278 by CK2 or a Ser278-->Asp mutation increased the interaction between PACS-1 and cargo, whereas a Ser278-->Ala substitution decreased this interaction. Moreover, the Ser278-->Ala mutation yields a dominant-negative PACS-1 molecule that selectively blocks retrieval of PACS-1-regulated cargo molecules to the TGN. These results suggest that coordinated signaling events regulate transport within the TGN/endosomal system through the phosphorylation state of both cargo and the sorting machinery.
PACS-1是一种胞质分选蛋白，可指导膜蛋白在反式高尔基体网络（TGN）/内体系统中的定位。PACS-1将网格蛋白衔接蛋白AP-1与货物蛋白（如弗林蛋白酶、不依赖阳离子的甘露糖-6-磷酸受体）细胞质结构域中包含的酸性簇分选基序相连，也与病毒蛋白（如人类免疫缺陷病毒1型Nef）中的此类基序相连。在此我们表明，PACS-1上一个与货物分子上的酸性簇分选基序高度相似的酸性簇，作为一个自我调节结构域，控制着PACS-1介导的分选过程。生化研究表明，酸性簇附近的Ser278被CK2磷酸化，并被PP2A去磷酸化。CK2使Ser278磷酸化或Ser278突变为天冬氨酸会增加PACS-1与货物之间的相互作用，而Ser278突变为丙氨酸则会降低这种相互作用。此外，Ser278突变为丙氨酸会产生一个显性负性PACS-1分子，该分子可选择性地阻断PACS-1调节的货物分子向TGN的回收。这些结果表明，协调的信号事件通过货物和分选机制的磷酸化状态来调节TGN/内体系统内的运输。
