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. 2017 Mar 4;13(3):592-607.
doi: 10.1080/15548627.2016.1269988. Epub 2017 Jan 6.

MTORC1-mediated NRBF2 phosphorylation functions as a switch for the class III PtdIns3K and autophagy

Xi Ma  1   2   3 , Shen Zhang  1   2   3 , Long He  1   2   3 , Yueguang Rong  2   3 , Livia Wilz Brier  4 , Qiming Sun  4 , Rong Liu  2   3 , Weiliang Fan  4 , She Chen  5 , Zhenyu Yue  6 , Joungmok Kim  7 , Kun-Liang Guan  7 , Defa Li  1 , Qing Zhong  2   3
Affiliations

MTORC1-mediated NRBF2 phosphorylation functions as a switch for the class III PtdIns3K and autophagy

Xi Ma et al. Autophagy. .

Abstract

NRBF2/Atg38 has been identified as the fifth subunit of the macroautophagic/autophagic class III phosphatidylinositol 3-kinase (PtdIns3K) complex, along with ATG14/Barkor, BECN1/Vps30, PIK3R4/p150/Vps15 and PIK3C3/Vps34. However, its functional mechanism and regulation are not fully understood. Here, we report that NRBF2 is a fine tuning regulator of PtdIns3K controlled by phosphorylation. Human NRBF2 is phosphorylated by MTORC1 at S113 and S120. Upon nutrient starvation or MTORC1 inhibition, NRBF2 phosphorylation is diminished. Phosphorylated NRBF2 preferentially interacts with PIK3C3/PIK3R4. Suppression of NRBF2 phosphorylation by MTORC1 inhibition alters its binding preference from PIK3C3/PIK3R4 to ATG14/BECN1, leading to increased autophagic PtdIns3K complex assembly, as well as enhancement of ULK1 protein complex association. Consequently, NRBF2 in its unphosphorylated form promotes PtdIns3K lipid kinase activity and autophagy flux, whereas its phosphorylated form blocks them. This study reveals NRBF2 as a critical molecular switch of PtdIns3K and autophagy activation, and its on/off state is precisely controlled by MTORC1 through phosphorylation.

Keywords: ATG14; BECN1; MTORC1; NRBF2; PI3KC3; PIK3C3; autophagy; phosphorylation.

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Figures

Figure 1.
Figure 1.
NRBF2 interacts with ATG14 as a stoichiometric component of the PtdIns3K complex. (A) NRBF2 is a stoichiometric component of the PtdIns3K complex. Silver staining of the tandem affinity-purified NRBF2 complex or vector alone in U2OS cells. All the marked bands were identified by mass spectrometry. (B-C) Mapping of the NRBF2-ATG14 binding. Different truncated fragments of Flag-NRBF2 or Flag-ATG14 were expressed with either HA-ATG14 or HA-NRBF2. Immunoprecipitation was performed using the anti-Flag resin. The minimum binding regions were marked in red. V, vector alone. (D-E) Identification of NRBF2 phosphorylation sites at S113 (D) and S120 (E) by mass spectrometry analysis.
Figure 2.
Figure 2.
NRBF2 phosphorylation is negatively regulated by autophagic stress. (A) Alignment of NRBF2 sequences in vertebrates. S113 and S120 are marked in red. (B) Phosphorylation of endogenous NRBF2 in mouse and human cells upon autophagic stress. Mouse embryonic fibroblasts (mouse, upper) and osteosarcoma U2OS cells (human, lower) were treated with EBSS (2 h) or rapamycin (Rapa, 50 nM, 2 h), then endogenous NRBF2 was detected using anti-NRBF2 antibody in western blotting. The phosphorylation of NRBF2 in human U2OS cells was detected by a phospho-specific antibody against human NRBF2 S120. Cell lysate from unstressed cells was also treated with CIP for 1 h as a control. (C) Phosphorylation sites in human and mouse NRBF2. NRBF2 knockout (KO) MEFs were transiently transfected with Flag-tagged human wild-type (WT), S113A or S120A NRBF2, as well as mouse WT or S112A NRBF2 mutants. NRBF2 was detected with anti-Flag antibody. (D) Generation of human NRBF2S113A,120A double mutant that eliminates NRBF2 phosphorylation. NRBF2 KO cells were transiently transfected with Flag-tagged wild-type (WT) NRBF2 (treated with or without CIP), NRBF2S113,120A (AA), and NRBF2S113,120D (DD) mutants. NRBF2 was detected using an anti-Flag antibody. (E) Characterization of the phospho-specific antibody against human NRBF2 S120 using overexpressed NRBF2. NRBF2 KO MEFs were transiently transfected with Flag-tagged WT NRBF2, NRBF2S113,120A (AA), or NRBF2S113,120D (DD) mutants. NRBF2 phosphorylation was detected using anti-p-S120 antibody, and NRBF2 were detected with anti-Flag antibody. (F) Human NRBF2 phosphorylation is negatively regulated by autophagic stress. Nrbf2 KO MEFs were transfected with a plasmid encoding Flag-tagged WT NRBF2 and treated with EBSS (2 h), rapamycin (50 nM, 2 h) or Torin 1 (100 nM, 2 h). NRBF2 phosphorylation was detected using the phospho-specific antibody against human NRBF2 S120 or mobility shift. NRBF2, SQSTM1 and LC3 were detected by western blotting.
Figure 3.
Figure 3.
NRBF2 is a substrate of MTORC1. (A-C) Immunoprecipitated AMPK (A), MTORC1-RPTOR (B) and ULK1 (C) were incubated with the indicated recombinant proteins in an in vitro kinase assay. Phosphorylation is detected by autoradiogram and input proteins were Coomassie Blue stained. TSC2 (aa 1300–1367), ATG13, and BECN1 (aa 1–85) served as positive controls for AMPK, MTORC1 and ULK1 kinase activities, respectively. WT, wild type; KD, kinase dead mutant. (D) Recombinant GST-tagged WT, S120A and S113A S120A NRBF2 proteins were used in the in vitro kinase assay with immunoprecipitated MTORC1. (E) Recombinant GST-tagged WT and S113A S120A NRBF2 proteins were used in the in vitro kinase assay with immunoprecipitated ULK1. (F) A schematic diagram to show that NRBF2 is phosphorylated by MTORC1.
Figure 4.
Figure 4.
NRBF2 S113 S120 phosphorylation differentiates its interaction with PtdIns3K subcomplexes. (A-H) Interactions between NRBF2 (unphosphorylated or phosphorylated mutants) and individual PtdIns3K complex components. HEK293T cells were transfected with MYC-PIK3C3 (A), HA-PIK3R4 (C), HA-ATG14 (E), or HA-BECN1 (G) together with Flag-tagged NRBF2 wild-type (WT), AA, or DD mutant, and treated with EBSS (2 h), rapamycin (50 nM, 2 h), or left untreated. Flag-NRBF2 and its associated proteins were immunoprecipitated uisng anti-Flag M2 resin and probed by individual antibodies in western blotting. The relative ratios of MYC-PIK3C3 (B), HA-PIK3R4 (D), HA-ATG14 (F), or HA-BECN1 (H) to NRBF2 were quantified and standardized. The error bars represent the standard error of the mean from 3 independent experiments within the same treatment group. *, P < 0.05. (I) A schematic showing that phosphorylated NRBF2 preferentially interacts with PIK3C3 and PIK3R4, while unphosphorylated NRBF2 preferentially interacts with ATG14 and BECN1.
Figure 5.
Figure 5.
NRBF2 S113 S120 phosphorylation regulates its interaction with the ULK1 protein complex. (A-F) Interaction between NRBF2 (unphosphorylated or phosphorylated forms) and individual ULK1 complex components. HEK293T cells were transfected with HA-ULK1 (A), MYC-ATG13 (C), or HA-RB1CC1 (E), respectively, together with NRBF2 wild-type (WT), AA, or DD mutant, and treated with EBSS (2 h), rapamycin (50 nM, 2 h) or left untreated. Flag-NRBF2 and its associated proteins were immunoprecipitated using anti-Flag M2 resin and probed with individual antibodies in western blotting. The relative ratios of HA-ULK1 (B), MYC-ATG13 (D), or HA-RB1CC1 (F) to NRBF2 were quantified and standardized. The error bars represent the standard error of the mean from 3 independent experiments within the same treatment group. *, P < 0.05. (G) A schematic showing the interaction between different forms of NRBF2 with the ULK1 protein complex ULK1, RB1CC1 and ATG13. Gray arrow means seldom interact, and red arrow means preferentially interact.
Figure 6.
Figure 6.
NRBF2 S113 S120 phosphorylation differentially regulates PtdIns3K complex and ULK1 complex assembly. (A) Generation of stable cell lines in Nrbf2 knockout (KO) mouse embryonic fibroblasts (MEFs) complemented with Flag-tagged NRBF2 wild-type (WT), AA or DD mutants. The expression of Flag-NRBF2 was detected with an anti-Flag antibody in western blotting. (B) A schematic showing how phosphorylation of NRBF2 affects PtdIns3K complex assembly. (C-F) NRBF2 S113 S120 phosphorylation negatively regulates PtdIns3K complex and ULK1 complex assembly. Nrbf2 KO MEFs stably complemented with Flag-tagged NRBF2 WT, AA, or DD mutant were treated with Torin 1 (50 nM, 2 h) or left untreated. Endogenous PtdIns3K complex and ULK1 complex were detected (C). (D-F) Endogenous immunoprecipitations were performed using anti-Flag antibody (D), mouse anti-ATG14 (E) or mouse anti-BECN1 primary antibodies (F), and analyzed by western blotting in the upper panels and quantitative analyses in the bottom panels. The error bars represent the standard error of the mean from 3 independent experiments within the same group. *, P < 0.05.
Figure 7.
Figure 7.
MTORC1-mediated NRBF2 phosphorylation regulates the assembly of the PIK3C3 complex and ULK1 complex in the endogenous co-immunoprecipitation. (A-D) WT and NRBF2 KD U2OS cells were treated with EBSS (2 h), rapamycin (Rapa, 50 nM, 2 h) or left untreated. Endogenous PtdIns3K complex and ULK1 complex were detected (A). (B-D) Endogenous immunoprecipitations were performed using mouse anti-ATG14 (B), mouse anti-BECN1 (C) or rabbit anti-NRBF2 primary antibodies (D). Endogenous NRBF2 and its associated proteins were probed with individual antibodies in western blotting. (E-G) Quantitative analysis of (B-D). The error bars represent the standard error of the mean from 3 independent experiments within the same treatment group. *, P < 0.05.
Figure 8.
Figure 8.
NRBF2 S113 S120 phosphorylation regulates PtdIns3K lipid kinase activity. (A) NRBF2 S113 S120 phosphorylation negatively regulates autophagic PtdIns3P production. Autophagic PtdIns3P binding protein GFP-ZFYVE1 was expressed and detected in Nrbf2 knockout (KO) mouse embryonic fibroblasts (MEFs) stably complemented with Flag-tagged NRBF2 WT, AA, or DD mutant. Scale bar, 10 µm. (B) Quantitative analysis of (A). The error bars represent the standard error of the mean from 3 independent experiments within the same group. *, P < 0.05. (C) NRBF2 S113 S120 phosphorylation regulates PtdIns3P production in vitro. Nrbf2 KO MEFs stably complemented with Flag-tagged NRBF2 WT, AA, or DD mutant were treated with Torin 1 (50 nM, 2 h) or left untreated, and then subjected to PtdIns3K lipid kinase assay. (D-E) Quantitative analysis of (C). The relative ratios of PtdIns3P production to NRBF2 (D) and PIK3C3 (E) were quantified and standardized. The error bars represent the standard error of the mean from 3 independent experiments within the same group. *, P < 0.05.
Figure 9.
Figure 9.
NRBF2 S113 S120 phosphorylation regulates autophagy. (A) NRBF2 S113 S120 phosphorylation regulates autophagy flux. Nrbf2 knockout (KO) mouse embryonic fibroblasts (MEFs) stably complemented with NRBF2 wild-type (WT), AA, or DD mutant were treated with rapamycin (Rapa, 50 nM, 2 h), CQ (20 µM, 2h) and rapamycin + CQ (50 nM rapamycin and 20 µM CQ, 2 h) or left untreated. Autophagy flux was analyzed by probing LC3-II and SQSTM1 in western blotting. The protein levels of NRBF2 and TUBB were also monitored. (B-C) Quantitative analysis of (A). The relative ratios of LC3-II (B) or SQSTM1 (C) to TUBB were standardized. The error bars represent the standard error of the mean from 3 independent experiments within the same group. *, P < 0.05. (D) Representative LC3 staining images of Nrbf2 KO MEFs stably complemented with NRBF2 WT, AA, or DD mutant, treated with rapamycin (Rapa, 50 nM, 2 h), CQ (20 µM, 2 h) and rapamycin + CQ (50 nM rapamycin and 20 µM CQ, 2 h) or left untreated. Endogenous LC3 staining was performed using an anti-LC3 antibody, and representative images were taken by 40× magnification. Scale bar: 10 µm. (E) Quantitative analysis of (D). LC3 puncta per cell were quantified in 100 cells for each panel. The error bars represent the standard error of the mean from 3 independent experiments within the same group. *, P < 0.05. (F) A working model of MTORC1-mediated NRBF2 S113 S120 phosphorylation as a switch to regulate PtdIns3K complex assembly, lipid kinase activity, ULK1 association and autophagy.
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References

    1. Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy. Dev Cell 2004; 6:463-77; PMID:15068787; http://dx.doi.org/10.1016/S1534-5807(04)00099-1 - DOI - PubMed
    1. Klionsky DJ. Autophagy: from phenomenology to molecular understanding in less than a decade. Nat Rev Mol Cell Biol 2007; 8:931-7; PMID:17712358; http://dx.doi.org/10.1038/nrm2245 - DOI - PubMed
    1. Mizushima N, Levine B, Cuervo AM, Klionsky DJ. Autophagy fights disease through cellular self-digestion. Nature 2008; 451:1069-75; PMID:18305538; http://dx.doi.org/10.1038/nature06639 - DOI - PMC - PubMed
    1. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Cell 2008; 132:27-42; PMID:18191218; http://dx.doi.org/10.1016/j.cell.2007.12.018 - DOI - PMC - PubMed
    1. Mathew R, Karantza-Wadsworth V, White E. Role of autophagy in cancer. Nat Rev Cancer 2007; 7:961-7; PMID:17972889; http://dx.doi.org/10.1038/nrc2254 - DOI - PMC - PubMed

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