Evidence that phosphorylation of human Upf1 protein varies with intracellular location and is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway

Human Upf1 protein (p), a group 1 RNA helicase, has recently been shown to function in nonsense-mediated mRNA decay (NMD) in mammalian cells. Here, we demonstrate that the estimated 3 x 10(6) copies of hUpf1p per exponentiall y growing HeLa cell are essentially equally distributed among polysomal, su bpolysomal, and ribosome-free fractions. We also demonstrate that hUpf1p bi nds RNA and is a phosphoprotein harboring phosphoserine and phosphothreonin e. hUpf1p is phosphorylated to the highest extent when polysome-associated and to the lowest extent when ribosome free. We find that serum-induced pho sphorylation of hUpf1p is inhibited by wortmannin at a concentration that s electively inhibits PI 3-kinase related kinases and, to a lesser extent, by rapamycin. These and other data suggest that phosphorylation is mediated b y a wortmannin-sensitve and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway. Comparisons are made of hUpf1 p to Upf1p and SMG-2, whic h are the orthologs to hUpf1p in Saccharomyces cerevisiae and Caenorhabditi s elegans, respectively.