TY - JOUR
T1 - GLIPR2 is a negative regulator of autophagy and the BECN1-ATG14-containing phosphatidylinositol 3-kinase complex
AU - Zhao, Yuting
AU - Zou, Zhongju
AU - Sun, Daxiao
AU - Li, Yue
AU - Sinha, Sangita C.
AU - Yu, Li
AU - Bennett, Lynda
AU - Levine, Beth
N1 - Funding Information:
This work was supported by NIH grants U19 AI109725 (B.L.), U19 AI142784 (B.L.), R15 GM122035 (S.S.), and a National Science Foundation grant MCB-1413525 (S.S.). We thank H.Wang and Y. Li for help on protein complex purification; X. Liu for helpful discussions on mass spectrometry analyses; N. Ktistakis, D.V. Kaloyanova and N. Mizushima for providing critical reagents; B. Ci for help with analyses of TCGA data; and H. Smith for assistance with manuscript preparation.
Publisher Copyright:
© 2020 The Author(s). Published by Informa UK Limited, trading as Taylor & Francis Group.
PY - 2021
Y1 - 2021
N2 - A key mediator of macroautophagy/autophagy induction is the class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) consisting of PIK3C3/VPS34, PIK3R4/VPS15, BECN1, and ATG14. Although several proteins are known to enhance or decrease PtdIns3K-C1 activity, our understanding of the molecular regulation of PtdIns3K-C1 is still incomplete. Previously, we identified a Golgi-associated protein, GLIPR2, in a screen for proteins that interact with amino acids 267–284 of BECN1, a region of BECN1 sufficient to induce autophagy when fused to a cell penetrating leader sequence. In this study, we used CRISPR-Cas9-mediated depletion of GLIPR2 in cells and mice to investigate the role of GLIPR2 in the regulation of autophagy and PtdIns3K-C1 activity. Depletion of GLIPR2 in HeLa cells increased autelophagic flux and generation of phosphatidylinositol 3-phosphate (PtdIns3P). GLIPR2 knockout resulted in less compact Golgi structures, which was also observed in autophagy-inducing conditions such as amino acid starvation or Tat-BECN1 peptide treatment. Importantly, the binding of GLIPR2 to purified PtdIns3K-C1 inhibited the in vitro lipid kinase activity of PtdIns3K-C1. Moreover, the tissues of glipr2 knockout mice had increased basal autophagic flux as well as increased recruitment of the PtdIns3P-binding protein, WIPI2. Taken together, our findings demonstrate that GLIPR2 is a negative regulator of PtdIns3K-C1 activity and basal autophagy. Abbreviations: ATG14: autophagy related 14; Baf A1: bafilomycin A1; BARA: β-α repeated, autophagy-specific; CQ: chloroquine; GFP: green fluorescent protein; GLIPR2: GLI pathogenesis related 2; HBSS: Hanks’ balanced salt solution; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PBS: phosphate-buffered saline; PtdIns3K-C1: phosphatidylinositol 3-kinase complex I; PtdIns3P: phosphatidylinositol-3-phosphate; SEM: standard error of the mean; WIPI2: WD repeat domain, phosphoinositide interacting 2.
AB - A key mediator of macroautophagy/autophagy induction is the class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1) consisting of PIK3C3/VPS34, PIK3R4/VPS15, BECN1, and ATG14. Although several proteins are known to enhance or decrease PtdIns3K-C1 activity, our understanding of the molecular regulation of PtdIns3K-C1 is still incomplete. Previously, we identified a Golgi-associated protein, GLIPR2, in a screen for proteins that interact with amino acids 267–284 of BECN1, a region of BECN1 sufficient to induce autophagy when fused to a cell penetrating leader sequence. In this study, we used CRISPR-Cas9-mediated depletion of GLIPR2 in cells and mice to investigate the role of GLIPR2 in the regulation of autophagy and PtdIns3K-C1 activity. Depletion of GLIPR2 in HeLa cells increased autelophagic flux and generation of phosphatidylinositol 3-phosphate (PtdIns3P). GLIPR2 knockout resulted in less compact Golgi structures, which was also observed in autophagy-inducing conditions such as amino acid starvation or Tat-BECN1 peptide treatment. Importantly, the binding of GLIPR2 to purified PtdIns3K-C1 inhibited the in vitro lipid kinase activity of PtdIns3K-C1. Moreover, the tissues of glipr2 knockout mice had increased basal autophagic flux as well as increased recruitment of the PtdIns3P-binding protein, WIPI2. Taken together, our findings demonstrate that GLIPR2 is a negative regulator of PtdIns3K-C1 activity and basal autophagy. Abbreviations: ATG14: autophagy related 14; Baf A1: bafilomycin A1; BARA: β-α repeated, autophagy-specific; CQ: chloroquine; GFP: green fluorescent protein; GLIPR2: GLI pathogenesis related 2; HBSS: Hanks’ balanced salt solution; KO: knockout; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PBS: phosphate-buffered saline; PtdIns3K-C1: phosphatidylinositol 3-kinase complex I; PtdIns3P: phosphatidylinositol-3-phosphate; SEM: standard error of the mean; WIPI2: WD repeat domain, phosphoinositide interacting 2.
KW - Autophagy
KW - BECN1
KW - GLIPR2
KW - Golgi
KW - PtdIns3k-C1 complex
KW - Tat-BECN1 peptide
UR - http://www.scopus.com/inward/record.url?scp=85096432073&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=85096432073&partnerID=8YFLogxK
U2 - 10.1080/15548627.2020.1847798
DO - 10.1080/15548627.2020.1847798
M3 - Article
C2 - 33222586
AN - SCOPUS:85096432073
SN - 1554-8627
VL - 17
SP - 2891
EP - 2904
JO - Autophagy
JF - Autophagy
IS - 10
ER -