Skip to main page content
U.S. flag
See More

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2017 May 18;8(5):e2800.
doi: 10.1038/cddis.2017.204.

RACK1 depletion in the ribosome induces selective translation for non-canonical autophagy

Hag Dong Kim  1   2 , EunBin Kong  1 , YongJoong Kim  1   2 , Jin-Soo Chang  1 , Joon Kim  1   2
Affiliations

RACK1 depletion in the ribosome induces selective translation for non-canonical autophagy

Hag Dong Kim et al. Cell Death Dis. .

Abstract

RACK1, which was first demonstrated as a substrate of PKCβ II, functions as a scaffold protein and associates with the 40S small ribosomal subunit. According to previous reports, ribosomal RACK1 was also suggested to control translation depending on the status in translating ribosome. We here show that RACK1 knockdown induces autophagy independent of upstream canonical factors such as Beclin1, Atg7 and Atg5/12 conjugates. We further report that RACK1 knockdown induces the association of mRNAs of LC3 and Bcl-xL with polysomes, indicating increased translation of these proteins. Therefore, we propose that the RACK1 depletion-induced autophagy is distinct from canonical autophagy. Finally, we confirm that cells expressing mutant RACK1 (RACK1R36D/K38E) defective in ribosome binding showed the same result as RACK1-knockdown cells. Altogether, our data clearly show that the depletion of ribosomal RACK1 alters the capacity of the ribosome to translate specific mRNAs, resulting in selective translation of mRNAs of genes for non-canonical autophagy induction.

PubMed Disclaimer

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
(a) HT1080 cells were transfected with control or RACK1 siRNAs (50 pmol), and then incubated for 48 h. The cell lysates were subjected to immunoblot analysis using the indicated antibodies. LC3-II and p62/SQSTM1 are markers of autophagic flux (left panel). Intensities of LC3 and p62 proteins were normalized against that of tubulin proteins, and the relative expressions in RACK1 siRNA-treated cells compared with that in control cells was plotted (right panel). **P<0.01 (Student’s t-test). (b) Extracts of siRNA-transfected cells were pretreated with Bafilomycin A1 (1 μM) for 1 h, and subjected to immunoblot analysis using the indicated antibodies. (c) Each siRNA-treated HT1080 cells were lysed and extracts were subjected to immunoblot analysis using the indicated antibodies. (d) Immunoblot analysis using the indicated antibodies was performed after transient transfection of plasmids, pcDNA3-Flag vector or pcDNA3-Flag-RACK1 (WT) into HT1080 cells. (e and g) Indicated siRNAs (50 pmol) were transfected into EGFP-LC3 stable HT1080 cells. GFP-LC3 dots were detected by fluorescence microscopy (e), and the cell lysates were analyzed by immunoblotting using the indicated antibodies (g). (f) HT1080 cells were transfected with control or RACK1 siRNAs (50 pmol), CYTO-ID Green Detection Reagent is added, and then detected and analyzed. **P<0.01 (Student’s t-test). (h) After 48 h, GFP-LC3 stable HT1080 cells were treated with control or RACK1 siRNA (50 pmol), and then incubated with 50 nM LysoTracker for 30 min. After washing with PBS, images were obtained by florescence microscopy. The merged yellow signals of LysoTracker (red) and GFP-LC3 (green) indicated the autophagolysosome, which is formed by fusion of the lysosome and autophagosome
Figure 2
Figure 2
RACK1 depletion-induced autophagy is non-canonical. (a) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 48 h, followed by immunoblot analysis using the indicated antibodies (left panel). Intensities of Atg5/12 and Beclin1 proteins were normalized against that of tubulin proteins, and the relative expressions in RACK1 siRNA-treated cells compared with that in control cells were plotted (right panel). (b) HT1080 cells were transfected with siRNAs against control or RACK1 in combination with or without Beclin1 siRNA (100 pmol). After 48 h, these cells were analyzed by immunoblotting using the indicated antibodies. (c) HT1080 cells were transfected with control or Atg5 siRNA (100 pmol). After 24 h, the cells were re-transfected with control or RACK1 siRNA. After a further 48 h incubation, the cell extracts were subjected to immunoblot analysis using the indicated antibodies. (d) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 48 h, followed by immunoblot analysis using the indicated antibodies. (e) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 24 h. Extracts of siRNA-transfected cells were pretreated with rapamycin 1 μM for 24 h, followed by immunoblot analysis using the indicated antibodies. Intensities of LAMP1 and LAMP2 proteins were normalized against that of tubulin proteins, and the relative expressions in RACK1 siRNA-treated cells compared with that in control cells were plotted. *P<0.05, **P<0.01 (Student’s t-test)
Figure 3
Figure 3
Knockdown of RACK1 increases Bcl-xL protein level. (a) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 48 h, followed by immunoblot analysis using the indicated antibodies (left panel). Intensities of Bcl-xL protein was normalized against that of tubulin protein, and the relative expression in RACK1 siRNA-treated cells compared with that in control cells was plotted (right panel). **P<0.01 (Student’s t-test). (b) HT1080 cells were transfected with siRNAs against control or RACK1 in combination with or without Beclin1 siRNA (100 pmol). After 48 h, these cells were analyzed by immunoblotting using the indicated antibodies. (c) HT1080 cells transfected with control or RACK1 siRNAs (50 pmol) were incubated for 48 h. Extracts of siRNA-transfected cells were pretreated with rapamycin 1 μM for 24 h, followed by immunoblot analysis using the indicated antibodies. (d) Extracts obtained from cells transfected with each siRNA (20 pmol) were immunoprecipitated using Bcl-xL and Beclin1 antibodies. Total cell lysates and the immunoprecipitates were resolved and subjected to immunoblot analysis using the indicated antibodies
Figure 4
Figure 4
RACK1 depletion-induced autophagy is not a cell-line-specific phenomenon. (a–d and g–i) Control or RACK1 siRNAs were transfected (20 pmol) into HeLa (a), HDF (b), HepG2 and Hep3B (c, d), MCF7 and MDA-MB231 (g), MEF (h) and U2OS (i) cells. After 48 h, immunoblot analysis was performed using the indicated antibodies. (e, f and j) Indicated siRNAs (50 pmol) were transfected into EGFP-LC3 stably expressing HepG2, Hep3B and U2OS cells, followed by fluorescence microscopy (f and j) and immunoblot analyses using the indicated antibodies (e). (k) In each cell line, the intensities of LC3 proteins were normalized against that of tubulin protein, and the relative expression in RACK1 siRNA-treated cells compared with that in control cells was plotted. *P<0.05, **P<0.01 (Student’s t-test)
Figure 5
Figure 5
Increment of LC3 and Bcl-xL occurs at the translational level. (a) Control or RACK1 siRNA-transfected cells were incubated with 1 μM Bafilomycin A1 and 50 μg/ml cycloheximide for 1 h. The cell lysates were subjected to immunoblot analysis using the indicated antibodies. The numbers represent signal intensities normalized to the LC3-II level (first lane). (b) Relative LC3 and Bcl-xL mRNA levels in whole-cell lysates by real-time PCR. (c) Relative LC3 and Bcl-xL mRNA levels in polysomal fractions by real-time PCR. The mRNA levels were normalized to that of β-actin, and relative mRNA quantities divided by those in control cells are shown. *P<0.05, **P<0.01 (Student’s t-test). Data are representative of three independent experiments. (d) Control or RACK1 siRNAs were transfected (20 pmol) into ATG7 knockout MEF cells. Extracts of siRNA-transfected cells were pretreated with pepstatin A (2 ug/ml) for 16 h, and subjected to immunoblot analysis using the indicated antibodies
Figure 6
Figure 6
Overexpression of RACK1R36D/K38E also induces autophagy. HT1080 (a), HeLa (b), HepG2 and Hep3B (c) cells were transfected with pcDNA3-FLAG-RACK and RACK1R36D/K38E. After 2 days, the levels of various proteins were assessed by immunoblot analysis. (d) pcDNA-FLAG-RACK1 and pcDNA-FLAG-RACK1R36D/K38E were transfected into EGFP-LC3 stably expressing HT1080, HepG2 and Hep3B cells, and images were obtained by florescence microscopy
See this image and copyright information in PMC

References

    1. Mochly-Rosen D, Smith BL, Chen CH, Disatnik MH, Ron D. Interaction of protein kinase C with RACK1, a receptor for activated C-kinase: a role in beta protein kinase C mediated signal transduction. Biochem Soc Trans 1995; 23: 596–600. - PubMed
    1. McCahill A, Warwicker J, Bolger GB, Houslay MD, Yarwood SJ. The RACK1 scaffold protein: a dynamic cog in cell response mechanisms. Mol Pharmacol 2002; 62: 1261–1273. - PubMed
    1. Gandin V, Senft D, Topisirovic I, Ronai ZA. RACK1 function in cell motility and protein synthesis. Genes Cancer 2013; 4: 369–377. - PMC - PubMed
    1. Ron D, Jiang Z, Yao L, Vagts A, Diamond I, Gordon A. Coordinated movement of RACK1 with activated betaIIPKC. J Biol Chem 1999; 274: 27039–27046. - PubMed
    1. Buensuceso CS, Woodside D, Huff JL, Plopper GE, O'Toole TE. The WD protein Rack1 mediates protein kinase C and integrin-dependent cell migration. J Cell Sci 2001; 114(Pt 9): 1691–1698. - PubMed