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. 2006 Sep 6;25(17):4008-19.
doi: 10.1038/sj.emboj.7601268. Epub 2006 Aug 24.

p97/DAP5 is a ribosome-associated factor that facilitates protein synthesis and cell proliferation by modulating the synthesis of cell cycle proteins

Sang Hyun Lee  1 , Frank McCormick
Affiliations

p97/DAP5 is a ribosome-associated factor that facilitates protein synthesis and cell proliferation by modulating the synthesis of cell cycle proteins

Sang Hyun Lee et al. EMBO J. .

Abstract

p97 (also referred to as DAP5, NAT1 or eIF4G2) has been proposed to act as a repressor of protein synthesis. However, we found that p97 is abundantly expressed in proliferating cells and p97 is recruited to ribosomes following growth factor stimulation. We also report that p97 binds eIF2beta through its C-terminal domain and localizes to ribosome through its N-terminal MIF4G domain. When overexpressed, p97 increases reporter luciferase activity. In contrast, overexpression of the C-terminal two-thirds of eukaryotic initiation factor 4GI (eIF4GI), a region that shares significant homology with p97, or the N-terminal MIF4G domain of p97 markedly inhibits reporter activity, the rate of global translation and cell proliferation. Conversely, downregulation of p97 levels by RNA interference also decreases the rate of global translation and inhibits cell proliferation. This coincides with an increase in p27/Kip1 protein levels and a marked decrease in CDK2 kinase activity. Taken together, our results demonstrate that p97 is functionally different from the closely related C-terminal two-thirds of eIF4GI and it can positively promote protein synthesis and cell proliferation.

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Figures

Figure 1
Figure 1
The C-terminal domain of p97 binds to eIF2β in vitro and in vivo. (A) (Top) The domains of p97 are shown schematically. The amino acids at the boundaries of these regions are indicated by numbers. (Bottom left) Coomassie staining of purified recombinant GST-tagged p97 proteins (1 μg each) used for in vitro GST-pulldown assay. (Bottom right) 293T total cell lysates were incubated with the indicated purified recombinant GST-tagged p97 proteins and glutathione-agarose beads. The beads were extensively washed and the bound eIF2β and eIF2α proteins were determined by immunoblot analysis. (B) In vitro GST-pulldown assay to identify the minimal region in p97 for eIF2β binding. (C) (Top) Amino-acid sequences of the minimal eIF2β binding region in p97 were aligned with corresponding amino-acid sequences in eIF5, eIF2Bɛ and eIF4GI. The positions of alanine scanning mutations in the minimal eIF2β binding region in p97 used for co-immunoprecipitation assay are shown. (Bottom left) Myc-tagged full-length or mutants of p97 were co-expressed in 293T cells with HA-tagged eIF2β. The cells were lysed and a 1/10 of total lysates used for co-immunoprecipitation was visualized with immunoblot analysis. (Bottom right) anti-Myc rabbit polyclonal antibody and protein A-agarose beads were added to an indicated total cell lysates. The beads were washed and the bound proteins were visualized with immunoblot analysis. (D, E) Myc-tagged full-length or mutants of p97 were expressed in 293T cells. Total cell lysates (D) or S100 supernatants (E) were prepared and subjected to immunoprecipitation analysis using anti-Myc antibody. Bound proteins were visualized with immunoblot analysis. (F) p97 binds eIF2β in vivo. (Top) 293T total cell lysates were incubated with goat polyclonal antibody raised against the N-terminal end of p97 and protein A-agarose beads. To determine the relative affinity of p97 to eIF2β, increasing amounts of purified GST-tagged N-terminal truncated p97 (GST-p97349−907) that cannot be recognized by the anti-p97 antibody were added during incubation. The beads were washed and bound eIF2β was visualized with immunoblot analysis. (Bottom) eIF2β bound to p97 was quantified with a densitometer. The amount of bound eIF2β proteins was plotted against the concentration of GST-p97349−907 protein.
Figure 2
Figure 2
p97 is a ribosome-associated protein which co-sediments with eIF2β and eIF2α in 40S ribosomal fractions in a growth factor dependent manner and the N-terminal MIF4G domain of p97 is required for ribosomal localization of p97. (A) Exponentially growing T98G cells were lysed in hypotonic lysis buffer. Fraction containing ribosomes was pelleted with centrifugation. The resulting pellets were washed with RSW buffer. An equal volume of lysates from each fraction was separated and analyzed by immunoblot. MEK1 was used as a marker for soluble cytoplasmic fraction and eIF4GI was used as a marker for pellet fractions contains ribosome-associated proteins. (B) T98G whole-cell extracts prepared using a lysis buffer containing ∼1.2% Triton X-100 and ∼1.2% deoxycholate were resolved on sucrose density gradient by centrifugation. Gradients were separated into 12 fractions. (Top) A half of each fraction was precipitated with TCA. p97 protein was identified with immunoblot analysis. (Bottom) The remaining half of each fraction was used to exact total RNA and subjected to gel analysis to determine the presences of 18S and 28S rRNAs. rRNAs were detected by ethidium bromide staining. (C) p97 co-sediments with eIF2β and eIF2α in ribosomal fraction in a growth factor dependent manner. Whole-cell extracts were prepared from either U87 cells grown exponentially in the presence of serum or U87 cells serum starved for 48 h. Cell extracts were resolved on sucrose density gradient. Top one-third of the gradient was separated into 26 fractions. Each fraction was precipitated with TCA and the presence of p97, eIF2β and eIF2α was determined by immunoblot analysis. (D) Myc-tagged MIF4G domain (Myc-p971−380) or Myc-tagged MIF4G deletion mutant of p97 (Myc-p97495−907) was expressed in HEK293 cells. Whole-cell extracts were prepared and resolved on sucrose density gradient. Gradients were separated into 12 fractions. A half of each fraction was precipitated with TCA and Myc-tagged proteins were identified with immunoblot analysis using anti-Myc polyclonal antibody.
Figure 3
Figure 3
Ectopic expression of p97 induces luciferase activity through its C-terminal domain and the MIF4G domain of p97 inhibits general protein synthesis and induces cell cycle arrest. (A, D) CMV-derived mammalian expression vectors encoding Myc-tagged full-length or the indicated mutants of p97 were co-transfected with pCDNA3 vector encoding firefly luciferase into 293T cells. At 20 h after transfection, cells were lysed and analyzed for luciferase activity. Average of luciferase activity from five independent experiments is shown with standard deviation. (B, E) The same amount of each lysates were separated and subjected to immunoblot analysis. (C) Northern blot analysis of luciferase mRNA in 293T cells. Total cellular RNA was isolated and 20 μg of total cellular RNA was subjected to Northern blot analysis for luciferase mRNA (Top). To ensure equal loading of RNAs, 28S and 18S rRNAs were visualized by staining with Ethidium Bromide (Bottom). Control refers to the cells without transfection (lane 6). (F) Myc-tagged wild type p97 or Myc-tagged MIF4G domain of p97 (Myc-p971−380) proteins were expressed in HEK293 cells. Cells were pulse-labeled with [35S]methionine and [35S]cysteine mix for 1 h, and total cell extracts were prepared and separated through SDS polyacrylamide gel. (Left) The loading of total protein was visualized with Coomassie staining. (Right) The same gel was dried and subjected to auto-radiography. (G) An aliquot of transfected HEK293 cells with an indicated DNA plasmid construct was stained with PI (PI) and subjected to FACS analysis.
Figure 4
Figure 4
p97 is functionally different from the closely related C-terminal two-thirds of eIF4GI. (A) The domains of eIF4GI and p97 are shown schematically. The amino acids at the boundaries of these regions are indicated by numbers. (B) CMV-derived mammalian expression vectors encoding eIF4GI or p97 were co-transfected with pCDNA3 encoding firefly luciferase into 293T cells. At 20 h after transfection, cells were lysed and 10 μl of each lysate was analyzed for luciferase activity. The average of luciferase activity from five independent experiments is shown with standard deviations. (C) The same amount of each lysates were separated and subjected to immunoblot analysis. (D) (Top) Coomassie staining of purified recombinant GST-eIF4GI and GST-p97 proteins (1 μg each) used for in vitro GST-pulldown assay. (Bottom) 293T total cell lysates were incubated with the indicated purified recombinant GST-tagged eIF4GI and GST-tagged p97 proteins and glutathione-agarose beads. The beads were extensively washed and the bound eIF2β was determined by immunoblot analysis. (E) Myc-tagged eIF4GI621−1560 proteins were expressed in 293T cells and subjected to immunoprecipitation and immunoblot analysis (lanes 1–4). 293T total cell lysates were incubated with GST-tagged eIF4GI621−1560 proteins and the bound proteins were determined with immunoblot analysis (lanes 5 and 6).
Figure 5
Figure 5
Downregulation of p97 protein levels by RNA interference inhibit cell proliferation and decreases a rate of general protein synthesis. (A) Control non-silencing or p97 siRNA was transfected into T98G cells. At 48 h after transfections, total lysates were prepared. Total lysates (20 μg) were separated and subjected to immunoblot analysis for p97 (Top) and β-actin for loading control (Bottom). (B) Control non-silencing (Left) or p97 siRNA (Right) were transfected into T98G cells and photographed 48 h after transfection. (C) T98G cells were labeled with Br-dU and subjected to FACS analysis. The percentages of Br-dU positive T98G cells from three independent experiments are shown. (D) Control non-silencing or p97 siRNA was transfected into T98G cells. At 48 h after transfection, cell extracts were prepared and resolved on sucrose density gradient. Gradients were separated into 36 fractions. RNA was isolated from each fraction quantified by absorbance at 260 nm. The amount of RNA in each fraction was measured at OD260 and plotted against fraction numbers. (E) Control non-silencing or p97 siRNA was transfected into T98G cells. At 48 h after transfection, cells were pulse-labeled with [35S]methionine and [35S]cysteine mix for 1 h and subjected to filter binding assay. The amount of [35S]methionine and [35S]cysteine incorporation into total protein was determined by liquid scintillation counting.
Figure 6
Figure 6
Downregulation of p97 proteins levels induces a reduction of luciferase mRNA in polysomes and luciferase activity. Control non-silencing or p97 siRNA was transfected into T98G cells. At 48 h after siRNA transfections, pCDNA3 vector encoding firefly luciferase was transfected. At 20 h after transfection, Cell extracts were prepared and resolved on sucrose density gradient. (A) Cell extracts (20 μg) was separated and subjected to immunoblot analysis for p97 and β-actin (Left). Cell extracts (20 μg) were analyzed for luciferase activity (Right). (B, C) Gradients were separated into 12 fractions and each fraction was used to exact total RNA and subjected to Northern blot analysis for luciferase mRNA and gel analysis to determine the presences of 18S and 28S rRNAs by ethidium bromide staining. (D) Intensity of bands corresponding to luciferase mRNA in each fraction was quantified by Storm-imager analysis using ImageQuant software and relative changes of luciferase mRNA were plotted against an indicated fraction.
Figure 7
Figure 7
Downregulation of p97 protein levels increases in p27/Kip1 protein levels and inhibits CDK2 kinase activity. (A) Control non-silencing siRNA or p97 siRNA (sequences are shown in experimental procedures) was transfected into T98G cells. Cells were lysed at indicated time points and subjected to immunoblot analysis. (B) p97 siRNA transfected T98G cells were collected at indicated time point, stained with PI, and subjected to FACS analysis. (C) Immunoprecipitation and kinase assay. Total proteins were extracted from T98G cells 48 h after transfections with indicated siRNAs and immunoprecipitated with antibodies for CDK2 and were subjected to kinase assay with Histone H1 protein as a substrate and immunoprecipitated CDK2 protein levels were determined with immunoblot analysis. (D) Control non-silencing or p97 siRNA was transfected into T98G cells. Cells were pulse-labeled with [35S]methionine and [35S]cysteine mix for 1 h, cell extracts were prepared. [35S]-labeled cell extracts were immunoprecipitated with anti-p27/Kip1 or anti-Cyclin E antibodies together with protein A-agarose beads. Bound proteins were separated and subjected to autoradiography. Intensity of bands corresponding to an indicated protein was quantified by Storm-imager analysis using ImageQuant software and relative fold changes of indicated protein bands were shown. (E) Control non-silencing siRNA or p97 siRNA was transfected into T98G cells. Cells were pulse-labeled with [35S]methionine and [35S]cysteine mix for 1 h and chased for an indicated time. [35S]-labeled cell extracts were immunoprecipitated with anti-p27/Kip1 antibody together with protein A-agarose beads. Bound proteins were separated and subjected to autoradiography (Top). Intensity of bands corresponding to p27/Kip1 protein was quantified by Storm-imager analysis using ImageQuant software and relative changes of indicated protein bands were plotted against an indicated chase time (Bottom).
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