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. 2002 May 1;30(9):1919-28.
doi: 10.1093/nar/30.9.1919.

S6 phosphorylation-independent pathways regulate translation of 5'-terminal oligopyrimidine tract-containing mRNAs in differentiating hematopoietic cells

Diane Barth-Baus  1 , Carl A Stratton, Lou Parrott, Howard Myerson, Oded Meyuhas, Dennis J Templeton, Gary E Landreth, Jack O Hensold
Affiliations

S6 phosphorylation-independent pathways regulate translation of 5'-terminal oligopyrimidine tract-containing mRNAs in differentiating hematopoietic cells

Diane Barth-Baus et al. Nucleic Acids Res. .

Abstract

Synthesis of new ribosomes is an energy costly and thus highly regulated process. Ribosomal protein synthesis is controlled by regulating translation of the corresponding ribosomal protein (rp)mRNAs. In mammalian cells a 5'-terminal oligopyrimidine tract (TOP) is a conserved feature of these mRNAs that has been demonstrated to be essential for their translational regulation. Translation of TOP mRNAs has been proposed to be regulated by phosphorylation of ribosomal protein S6, which is a common effect of mitogenic stimulation of cells. However, as demonstrated here, S6 phosphorylation is not detectable in murine erythroleukemia (MEL) or other hematopoietic cells. The absence of S6 phosphorylation appears to be due to the action of a phosphatase that acts downstream of S6 kinase, presumably on S6 itself. Despite the absence of changes in S6 phosphorylation, translation of TOP mRNAs is repressed during differentiation of MEL cells. These data demonstrate the existence of a mechanism for regulating S6 phosphorylation that is distinct from kinase activation, as well as the existence of mechanisms for regulating translation of TOP mRNAs that are independent of S6 phosphorylation.

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Figures

Figure 1
Figure 1
Ribosomal protein S6 is not phosphorylated in MEL cells. Da3/EpoR and MEL cells were incubated overnight in medium containing 0.5% serum, then washed and resuspended in phosphate-free medium for 4 h with [32P]orthophosphate added for the final 2 h of incubation. The cultures were then split into two equal aliquots and erythropoietin added to one of the Da3/EpoR aliquots and A23187 to one of the MEL aliquots. Following an additional 30 min incubation extracts were prepared from the cells. Ribosomes were isolated by sedimentation through sucrose and the recovered ribosomal proteins separated by electrophoresis in 15% polyacrylamide Laemmli gels and visualized by autoradiography. The labels above the lanes indicate the cell type and treatment relevant for the isolated proteins. The positions of migration of the molecular size markers are shown at the left of the autoradiograph. Ribosomal protein S6 has previously been demonstrated to migrate at 32 kDa. The doublet near the 17.9 kDa molecular size marker is the acidic ribosomal phosphoproteins, P1 and P2.
Figure 2
Figure 2
PHAS I is constitutively phosphorylated in MEL cells. (A) Quiescent Da3/EpoR cells were exposed to erythropoietin for the times indicated. Cell extracts were prepared and the proteins separated by electrophoresis in 12.5% polyacrylamide gels under denaturing conditions. The separated proteins were transferred to PVDF membranes and PHAS I proteins detected by immunoblotting. Immunoreactive proteins were visualized by chemiluminscence. The most rapidly migrating α-isoform is unphosphorylated while the β- and γ-isoforms represent singly and doubly phosphorylated forms of PHAS I. (B) MEL cells were maintained overnight in 0.5% serum, then switched to serum-free medium and incubated for an additional 60 min. For the final 30 min incubation inducers of differentiation, either DMSO or A231287, were added as indicated. For reference, an extract of Da3/EpoR cells that had been exposed to erythropoietin for 5 min was included in the left-most lane of the gel that included the MEL cell extracts. (C) Quiescent Da3/EpoR cells were exposed to the indicated inhibitors for 30 min prior to the addition of erythropoietin (Epo). Following an additional 30 min incubation in the presence of both erythropoietin and the indicated inhibitor, cell extracts were prepared and analyzed by western blotting, as previously described. The inhibitors are indicated above the relevant lanes: LY, LY294002; Rap, rapamycin; PD, PD98059; BIM, bis-indolylmaleimide. (D) MEL cells were deprived of growth factors, as described previously, and exposed to signal transduction inhibitors. Extracts were prepared and PHAS I phosphorylation assessed as described above.
Figure 3
Figure 3
S6K is constitutively active in MEL cells. Quiescent Da3/EpoR cells were stimulated with erythropoietin for 30 min. MEL cells were maintained under similar conditions, without added erythropoietin. (A) The 70 kDa ribosomal protein S6 kinase (p70 S6k) was immunoprecipitated from cell extracts as described in the text. Kinase activity was assessed using purified 40S ribosomal subunits as substrate (left). Phosphorylation of S6K was assessed by immunoblotting the precipitated protein and is shown on the right. The phosphorylated isoforms of the kinase are detected by their slower mobility during electrophoresis. (B) Cells were grown in the presence of the indicated inhibitor for 60 min prior to extract preparation and p70s6k activity assessed by in vitro kinase assay. The extent of S6 phosphorylation is indicated graphically, relative to the corresponding extract prepared from cells that had not been exposed to inhibitors. Kinase activity in MEL cells was standardized to that in the erythropoietin-stimulated Da3/EpoR cells in the absence of inhibitors, which was arbitrarily set at 100.
Figure 4
Figure 4
Calyculin A exposure of MEL cells results in S6 phosphorylation. (A) MEL cells were incubated for 4 h in phosphate-free medium with [32P]orthophosphate added for the final 2 h of incubation. The culture was then split into three equal aliquots and anisomycin (Anis) or calyculin A (Cal) added to two of the aliquots. The remaining aliquot served as the untreated control (Con). Following an additional 30 min of incubation, extracts were prepared from the cells and ribosomes were obtained by sedimentation through sucrose. The recovered ribosomal proteins were separated by electrophoresis in 15% polyacrylamide gels under denaturing conditions and visualized by autoradiography. Due to the increase in background phosphorylation in the calyculin A-treated cells, the amount of material loaded in this lane was reduced by 50%, to allow for a more balanced exposure of the autoradiograph. The mobility of the indicated molecular size markers are shown to the left of the autoradiograph. The two phosphoproteins near the 17.9 kDa molecular size marker are the acidic ribosomal phosphoproteins, P1 and P2. (B) MEL cell phosphoproteins were metabolically labeled with and without the addition of calyculin A (Cal). Acid-soluble cytosolic proteins were extracted, separated by electrophoresis in 12% polyacrylamide gels and phosphorylated proteins identified by autoradiography. To identify the position of migration of ribosomal protein S6, a partially purified ribosomal preparation from cells that had been metabolically labeled with [32P]orthophosphate in the the presence of calyculin A was run in an adjacent gel lane. (C) BaF3 proteins were labeled with [32P]orthophosphate. Calyculin A and erythropoietin were added for the final 30 min incubation, as indicated. The r-proteins were recovered as described above, except that the concentration of KCl in the sucrose buffer was increased to 500 mM to reduce background due to loosely adherent proteins. Under these conditions the acidic ribosomal proteins are lost in the supernatants. The recovered r-proteins were analyzed by electrophoresis and autoradiography.
Figure 5
Figure 5
S6 phosphorylation is suppressed in primary myeloid but not erythroid cells. Normal human CD34+ cells were expanded by incubation in medium containing either erythropoietin (EPO) or G-CSF, as described in the text. (A) Cell phenotype was assessed by FACS analysis using the following myeloid cell surface markers: CD13, CD14 and CD33. CD71 was used as an erythroid marker. (B) S6 phosphorylation was assessed by metabolic labeling as previously described. Erythropoietin (EPO) was added to an aliquot of the expanded erythroid cells and G-CSF added to the expanded myeloid cells for the final 30 min incubation, as indicated above each lane. Ribosomal proteins were recovered by sedimentation through sucrose containing 500 mM KCl, separated by electrophoresis and visualized by autoradiography. Ribosomal protein S6 is indicated by the arrow. As previously described, the acidic ribosomal phosphoproteins are not recovered during sedimentation at this concentration of KCl. (C) The recovered ribosomal proteins were visualized by silver staining.
Figure 6
Figure 6
Inducers of MEL cell differentiation repress translation of TOP mRNAs. A HGH cDNA that had been ligated 3′ to the rpS16 promoter and 5′-UTR was stably expressed in MEL cells. As a control a similar construct in which the TOP had been mutated (C→A) was similarly expressed. Cytosolic RNAs were fractionated by sedimentation through sucrose gradients and HGH mRNA identified by northern blot hybridization of the gradient fractions as described in Materials and Methods. Translation of each transcript was assessed under normal growth conditions and following 8 h exposure to the inducer of differentiation A23187. Results were quantified with a phosphorimager. The northern blots are displayed in the bottom panels and distribution of the mRNAs in gradient fractions is shown graphically at the top (plotted as a percentage of total HGH hybridization signal). Ethidium stained gels shown below each northern blot demonstrate the effect of the inducer on the amount of polyribosomes as well as providing a reference point for the sedimentation of 40S and 60S subunits (as indicated by the peak accumulation of 18S and 28S rRNA). The fractions corresponding to the position of sedimentation of individual 40S and 60S ribosomal subunits, as well as the disomes, trisomes and higher order polysomes (as determined by UV absorbance tracings of the fractionated material) are indicated above the northern blots. (Note that under these salt conditions the monosome peak is too small to be well visualized.) Arrows placed above the graphs similarly indicate positions of sedimentation of fractions containing 40S and 60S subunits as well as disomes, trisomes and higher order polysomes.
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References

    1. Schmidt E. (1999) The role of c-myc in cellular growth control. Oncogene, 18, 2988–2996. - PubMed
    1. Warner J. (1999) The economics of ribosomal biosynthesis in yeast. Trends Biochem. Sci., 24, 437–440. - PubMed
    1. Meyuhas O. and Hornstein,E. (2000) Translational control of TOP mRNAs. In Sonenberg,N., Hershey,J. and Mathews,M. (eds), Translational Control of Gene Expression. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, pp. 671–693.
    1. Jefferies H., Reinhard,C., Kozma,S. and Thomas,G. (1994) Rapamycin selectively represses translation of the “polypyrimidine tract” mRNA family. Proc. Natl Acad. Sci. USA, 91, 4441–4445. - PMC - PubMed
    1. Terada N., Patel,H., Takase,K., Kohno,K., Nairn,A. and Gelfand,E. (1994) Rapamycin selectively inhibits translation of mRNAs encoding elongation factors and ribosomal proteins. Proc. Natl Acad. Sci. USA, 91, 11477–11481. - PMC - PubMed

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