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. Author manuscript; available in PMC: 2018 May 7.
Published in final edited form as: Nat Cell Biol. 2016 Jul 11;18(8):851–863. doi: 10.1038/ncb3388

Figure 6.

Figure 6

Dab2 promotes CK2-mediated Beclin-1 phosphorylation resulting in Beclin-1–Vps34 dissociation. (a) Schematic representation of the three CK2 consensus phosphorylation sites in Beclin-1 (Ser64, Ser337 and Ser341). (b) Beclin-1 immunoprecipitation analysis using whole-cell lysates from cells treated with TGF-β for the times indicated. Immunoprecipitated complexes were immunoblotted to detect the expression of Dab2, CK2, Beclin-1, Atg14 and Vps34. Straight immunoblot analysis depicts the expression level of CK2 and Atg14 in whole-cell lysates. (c) Immunoblot analysis of whole-cell lysates from CTSB/OE + LVL Dab2 cells treated with TGF-β for the times indicated. The CK2 inhibitor apigenin (20 µM) was added 6 h before the preparation of whole-cell lysates. (d) In vitro kinase assay using recombinant CK2 and in vitro-synthesized WT Beclin-1 and Beclin-1 mutants S64A, S337A and S341A as substrates. Reactions were subjected to SDS–PAGE and autoradiography. Apigenin was used as a specific inhibitor of CK2. (e) Co-immunoprecipitation analysis using either anti-Flag or anti-Vps34 antibody of whole-cell lysates from NMuMG CTSB/OE cells transfected with Flag-tagged wild-type Beclin-1 and three phosphomimetic Beclin-1 mutants. Immunoprecipitated complexes were immunoblotted with anti-Vps34, anti-Atg14 and anti-Flag antibodies to determine the interaction between Vps34, Atg14 and Beclin-1. (f) Flag immunoprecipitation using whole-cell lysates from 7-day-TGF-β-treated CTSB/OE+LVL Dab2 cells transfected with Flag-tagged wild-type Beclin-1 and its mutants. Immunoprecipitated complexes were immunoblotted to determine the interaction between Dab2, CK2, Vps34, Atg14 and Flag-tagged-Beclin-1. (g) Immunoblot analysis of whole-cell lysates prepared from TGF-β-treated NMuMG LVL Dab2 cells silenced for endogenous Beclin-1 and re-expressing either Flag-tagged wild-type or the indicated Beclin-1 phosphomutants. Blots were probed with the autophagic markers (LC3B and p62) and apoptosis markers (Bim and cleaved caspase-3). Hsp90 was used as a loading control. (h) Immunoblot analysis of whole-cell lysates from NMuMG LVL Dab2 and Beclin-1 KD cells stably re-expressing Flag-tagged exogenous WT or the indicated Beclin-1 mutants. All experiments were repeated at least three times and similar results were observed. Unprocessed original scans of blots are available in Supplementary Fig. 9.