ABSTRACT
ß-Arrestin is a scaffold protein that regulates signal transduction by seven transmembrane-spanning receptors. Among other functions it is also critically required for Wnt/ß-catenin signal transduction. In the present study we provide for the first time a mechanistic basis for the ß-arrestin function in Wnt/ß-catenin signaling. We demonstrate that ß-arrestin is required for efficient Wnt3a-induced Lrp6 phosphorylation, a key event in downstream signaling. ß-Arrestin regulates Lrp6 phosphorylation via a novel interaction with phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2)-binding protein Amer1/WTX/Fam123b. Amer1 has been shown very recently to bridge Wnt-induced and Dishevelled-associated PtdIns(4,5)P2 production to the phosphorylation of Lrp6. Using fluorescence recovery after photobleaching we show here that ß-arrestin is required for the Wnt3a-induced Amer1 membrane dynamics and downstream signaling. Finally, we show that ß-arrestin interacts with PtdIns kinases PI4KIIα and PIP5KIß. Importantly, cells lacking ß-arrestin showed higher steady-state levels of the relevant PtdInsP and were unable to increase levels of these PtdInsP in response to Wnt3a. In summary, our data show that ß-arrestins regulate Wnt3a-induced Lrp6 phosphorylation by the regulation of the membrane dynamics of Amer1. We propose that ß-arrestins via their scaffolding function facilitate Amer1 interaction with PtdIns(4,5)P2, which is produced locally upon Wnt3a stimulation by ß-arrestin- and Dishevelled-associated kinases.
Subject(s)
Adaptor Proteins, Signal Transducing/metabolism , Arrestins/metabolism , Cell Membrane/metabolism , Low Density Lipoprotein Receptor-Related Protein-6/metabolism , Tumor Suppressor Proteins/metabolism , Wnt3A Protein/metabolism , Adaptor Proteins, Signal Transducing/genetics , Animals , Arrestins/genetics , Blotting, Western , Cells, Cultured , Dishevelled Proteins , Embryo, Mammalian/cytology , Fibroblasts/cytology , Fibroblasts/metabolism , HEK293 Cells , Humans , Low Density Lipoprotein Receptor-Related Protein-6/genetics , Mice , Mice, Knockout , Microscopy, Confocal , Minor Histocompatibility Antigens , Phosphatidylinositol 4,5-Diphosphate/metabolism , Phosphoproteins/genetics , Phosphoproteins/metabolism , Phosphorylation , Phosphotransferases (Alcohol Group Acceptor)/genetics , Phosphotransferases (Alcohol Group Acceptor)/metabolism , Protein Binding , RNA Interference , Tumor Suppressor Proteins/genetics , Wnt3A Protein/genetics , beta-ArrestinsABSTRACT
Hypoxia leads to the upregulation of a variety of genes mediated largely via the hypoxia inducible transcription factor (HIF). Prominent HIF-regulated target genes such as the vascular endothelial growth factor (VEGF), the glucose transporter 1 (Glut-1), or erythropoietin (EPO) help to assure survival of cells and organisms in a low oxygenated environment. Here, we are the first to report the hypoxic regulation of the sperm associated antigen 4 (SPAG4). SPAG4 is a member of the cancer testis (CT) gene family and to date little is known about its physiological function or its involvement in tumor biology. A number of CT family candidate genes are therefore currently being investigated as potential cancer markers, due to their predominant testicular expression pattern. We analyzed RNA and protein expression by RNAse protection assay, immunofluorescent as well as immunohistological stainings. To evaluate the influence of SPAG4 on migration and invasion capabilities, siRNA knockdown as well as transient overexpression was performed prior to scratch or invasion assay analysis. The hypoxic regulation of SPAG4 is clearly mediated in a HIF-1 and VHL dependent manner. We furthermore show upregulation of SPAG4 expression in human renal clear cell carcinoma (RCC) and co-localization within the nucleolus in physiological human testis tissue. SPAG4 knockdown reduces the invasion capability of RCC cells in vitro and overexpression leads to enhancement of tumor cell migration. Together, SPAG4 could possibly play a role in the invasion capability and growth of renal tumors and could represent an interesting target for clinical intervention.
