Evidence that phosphorylation of human Upfl 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 hUpf1 p per exponentially growing HeLa cell are essentially equally distributed among polysomal, subpolysomal, and ribosome-free fractions. We also demonstrate that hUpf1p binds RNA and is a phosphoprotein harboring phosphoserine and phosphothreonine. hUpf1p is phosphorylated to the highest extent when polysome-associated and to the lowest extent when ribosome free. We find that serum-induced phosphorylation of hUpf1p is inhibited by wortmannin at a concentration that selectively inhibits PI 3-kinase related kinases and, to a lesser extent, by rapamycin. These and other data suggest that phosphorylation is mediated by a wortmannin-sensitive and rapamycin-sensitive PI 3-kinase-related kinase signaling pathway. Comparisons are made of hUpf1p to Upf1p and SMG-2, which are the orthologs to hUpf1p in Saccharomyces cerevisiae and Caenorhabditis elegans, respectively.
Pal
M
M
Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York 14263, USA.
Ishigaki
Y
Y
Nagy
E
E
Maquat
L E
LE
D000730:Androstadienes / Q000494:pharmacology*; D002460:Cell Line; D003513:Cycloheximide / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D007668:Kidney; D007700:Kinetics; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism*; D010766:Phosphorylation; D011132:Polyribosomes / Q000187:drug effects / Q000378:metabolism*; D020365:RNA Helicases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D029701:Saccharomyces cerevisiae Proteins; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D020123:Sirolimus / Q000494:pharmacology*; D013347:Subcellular Fractions / Q000378:metabolism; D015534:Trans-Activators; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D003513:Cycloheximide; D019869:Phosphatidylinositol 3-Kinases; C074151:NAM7 protein, S cerevisiae; D020365:RNA Helicases; C101828:UPF1 protein, human; D020123:Sirolimus; D000077191:Wortmannin