Distinct Repression of Translation by Wortmannin and Rapamycin

Abstract

The role of phosphatidylinositol 3-kinase and FKSO6-binding protein . rapamycin-associated protein (FRAP) in translational control has been examined by treating RD-rhabdomyosarcoma cells with wortmannin and rapamycin and studying the effects on cell-growth, translation initiation, and protein synthesis. Whereas wortmannin and rapamycin exhibit subtle effects on global translation, examination of individual mRNAs in sucrose gradients and of individual proteins in two-dimensional polyacrylamide gels reveals that wortmannin and rapamycin exhibit distinct effects on the translation of individual mRNAs. Wortmannin represses the synthesis of a third of cellular proteins, whereas rapamycin affects a subset of these proteins. Since ribosomal protein S6 was rapidly dephosphorylated following wortmannin and rapamycin treatment, and the phosphorylation status of the eukaryotic initiation factor 4E was unchanged, our data imply that the p70 signalling pathway has at least one branch-point upstream of FRAP leading to an additional route of translational control.Keywords : translation ; wortmannin ; rapamycin ; ~7 0 "~~ signaling pathway.Protein synthesis is a key regulatory point in the control of gene expression and cell growth. Translation is mainly controlled at the level of initiation, and several critical components of this process are controlled by distinct signalling pathways. Translation initiation is generally considered to occur by a capdependent scanning mechanism where the 43s ribosomal preinitiation complex initially is brought into contact with the 5' terminus of the mRNA by binding to the eukaryotic initiation factor (eIF) 4G in the cap-binding complex eIF-4F. The eIF-4F complex also contains the cap-binding protein eIF-4E that recognises the 7-methyl-guanosine (m'G) structure and eIF-4A that, in conjunction with eIF-4B, exhibits an ATP-dependent RNA helicase activity. Besides the general translation apparatus, specific trunsacting factors may be required for translation of individual mRNAs (for reviews, see [1,21).The p70 ribosomal protein S6 kinase (p70'") plays an important role in the progression of cells from G I to S phase of the cell cycle [3] and in translational activation by insulin and growth factors (reviewed in [4] ; eEF-la, elongation factor la; 5'TOP, mRNA with a 5'-terminal cytosine followed by a short stretch of pyrimidines, IGF-11, insulin-like growth factor 11; 5'-UTR, 5'hntranslated region; GraP-DH, glyceraldehyde-3-phosphate dehydrogenase ; DMEM, Dulbecco's modified Eagles medium; 2D, two-dimensional. try, Rigshospitalet, DK-2100 Copenhagen, DenmarkEnzyme. Phosphatidylinositol 3-kinase (EC 2.7.1.137).stream of both phosphatidylinositol 3-kinase and the phosphatidylinositol-3-kinase-activated protein kinase B [7]. Moreover, Cdc42 and Racl have recently been reported to associate with ~7 0 '~~ [8]. Studies of the p7Whk pathway have been facilitated by the availability of wortmannin and rapamycin that inhibit phosphatidylinositol 3-kinase and FRAP, respectively. Wortmannin is a fungal metabol...

Mammalian Target of Rapamycin Positively Regulates Collagen Type I Production via a Phosphatidylinositol 3-Kinase-independent Pathway

“…For example, rapamycin and LY294002 inhibited nitric oxide production in Raw 264.7 cells, whereas wortmannin is ineffective (61). Likewise, studies in several different cell types have revealed that wortmannin and rapamycin exhibit distinct effects on the translation of individual mRNAs (62). Thus, it appears that there are multiple pathways leading to mTOR activation, which are utilized in different cellular processes.…”

p70 S6 Kinase-mediated Protein Synthesis Is a Critical Step for Vascular Endothelial Cell Proliferation

“…These results can be explained by the presence of an oligopyrimidine tract in the 5Ј-untranslated region of the cyclin D1 mRNA. However, other mRNA determinants could be involved, since about 30% of newly synthesized proteins are inhibited by rapamycin (19,44). Rapamycin's target is the immunophilin FK506-binding protein, which binds to the kinase FRAP/ mTOR.…”

Regulation of Ribosome Biogenesis by the Rapamycin-sensitive TOR-signaling Pathway inSaccharomyces cerevisiae

“…In serum-starved cells as well as in cells treated with the macrolide antibiotic rapamycin, S6 becomes rapidly dephosphorylated, and 5ЈTOP mRNAs are no longer translated. Analysis of rapamycin-treated cells reveals that the steady-state distribution of these mRNAs shifts from polyribosomes to smaller ribonucleoprotein particles of unknown composition (Jefferies et al, 1997;Pedersen et al, 1997). The existence of 5ЈTOP sequences in mice as well as Xenopus suggests that this mechanism is conserved among metazoans (Meyuhas et al, 1996).…”