Plakophilin 1 stimulates translation by promoting eIF4A1 activity - PubMed (original) (raw)
Plakophilin 1 stimulates translation by promoting eIF4A1 activity
Annika Wolf et al. J Cell Biol. 2010.
Abstract
Plakophilins 1-3 (PKP1-3) are desmosomal proteins of the p120(ctn) family of armadillo-related proteins that are essential for organizing the desmosomal plaque. Recent findings identified PKPs in stress granules, suggesting an association with the translational machinery. However, a role of PKPs in controlling translation remained elusive so far. In this study, we show a direct association of PKP1 with the eukaryotic translation initiation factor 4A1 (eIF4A1). PKP1 stimulated eIF4A1-dependent translation via messenger RNA cap and encephalomyocarditis virus internal ribosomal entry site (IRES) structures, whereas eIF4A1-independent translation via hepatitis C virus IRES was not affected. PKP1 copurified with eIF4A1 in the cap complex, and its overexpression stimulated eIF4A1 recruitment into cap-binding complexes. At the molecular level, PKP1 directly promoted eIF4A1 adenosine triphosphatase activity. The stimulation of translation upon PKP1 overexpression correlated with the up-regulation of proliferation and cell size. In conclusion, these findings identify PKP1 as a regulator of translation and proliferation via modulation of eIF4A1 activity and suggest that PKP1 controls cell growth in physiological and pathological conditions.
Figures
Figure 1.
PKP1 colocalizes with eIF4A1. (A and B) HaCaT cells were exposed to 1 mM arsenate or 3 mM H2O2 for 1-h treatment or were left untreated, fixed, and stained for PKP1 and the SG marker TIAR (A) or for PKP1 and subunits of the initiation complex (eIF4A1, -4E, and -4G; B). (C and D) HaCaT cells were transfected with PKP1-DsRed and GFP-eIF4A1 (C) or myc-PKP1 and Flag-eIF4A1 (D). After 24 h, cells were treated with H2O2 (C) or arsenate (D) or left untreated, fixed, and stained with myc and Flag antibodies (D). Nuclei were labeled with DAPI. (A–D) Dashed boxes indicate the enlarged areas. Bars: (A–D, left) 10 µm; (A–D, right) 5 µm.
Figure 2.
PKP1 interacts with eIF4A1. (A) Yeast two-hybrid analysis. YRG2 cells were transformed with PKP1, -2, or -3 constructs and eIF4A1. Transformants were plated on selection plates lacking tryptophan and leucine (−WL) and reporter plates lacking tryptophan, leucine, and histidine (−WLH). (B) GST pull-down assays. GST-tagged eIF4A1 and GST (control) were immobilized on glutathione beads and probed for an interaction with PKP1 in the absence or presence of RNase A. Binding of His-tagged PKP1 to GST-tagged eIF4A1 was determined by Western blotting. (C) BiFC analysis. HeLa cells were cotransfected with the indicated constructs. At 24 h, transfected cells were identified by staining for the Flag and HA epitopes. YFP indicates the BiFC signal. (D) YFP fluorescence intensity was quantified by FACS. Mean values of three independent experiments counting >20,000 cells each are shown. PKP1 repeats were used as reference. Error bars indicate the SD. ***, P ≤ 0.0005. wt, wild type. Bars, 20 µm.
Figure 3.
Depletion of PKP1 or eIF4A1 does not prevent SG formation. (A and B) HaCaT cells were transfected with siRNAs for PKP1 or eIF4A1 or a control siRNA (c). Knockdown efficiencies were determined at 48 h on the mRNA level by qRT-PCR (mean values of five independent experiments; **, P ≤ 0.005; ***, P ≤ 0.0005; A) and on the protein level by Western blot (B). Error bars indicate the SD. (C and D) Cells were treated with 1 mM arsenate for 1 h and stained for eIF4A1 and PKP1 (C) or TIAR and PKP1 or TIAR and eIF4A1 (D). Bars, 20 µm.
Figure 4.
PKP1 stimulates eIF4A1-dependent translation. (A–C) The FFL reporter construct was cotransfected with the indicated GFP plasmids and siRNAs into HEK293 cells. Luciferase activity was determined and normalized to the luciferase mRNA level at 48 h. (D and E) In vitro–transcribed reporter mRNAs as indicated were translated in rabbit reticulocyte lysates in the absence or presence of recombinant PKP1. The products were resolved by SDS-PAGE and subjected to autoradiography (E). (D) Newly translated protein was quantified relative to buffer controls (no PKP1). The 35S intensity in the absence of PKP1 was set to 100%. (A–D) Data represent the mean of three independent experiments. Error bars indicate the SD. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005. wt, wild type.
Figure 5.
PKP1 stimulates eIF4A1 activity and its recruitment to the cap-binding complex. (A) ATPase assays were performed in the presence of 500 µM ATP, 1 µg poly(A) RNA, and PKP1 or PKP3, eIF4A1, or eIF4A1 + PKP as indicated. (B) Activity in the presence of PKP1 and eIF4A1 is presented relative to the sum of the individual activities. eIF4A1 activity was set to 100%. (C–G) Lysates from untransfected HaCaT cells showing endogenous proteins (C) and extracts from HEK293 cells (D–G) transfected with GFP-PKP1 or GFP alone (D and E) or in combination with control or eIF4A1 siRNA (F and G) were incubated with m7GTP-Sepharose. Bound protein was eluted in SDS buffer and characterized by Western blotting (WB) with the indicated antibodies. Proteins in the eluates were quantified relative to eIF4E (E) or relative to eluates from control siRNA–transfected cells (G). pg, plakoglobin. (A–G) Data represent the mean of three (A–E) and two (F and G) independent experiments. (A, B, E, and G) Error bars indicate the SD. *, P ≤ 0.05; **, P ≤ 0.005; ***, P ≤ 0.0005.
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