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Cell adhesion molecule IGPR-1 activates AMPK connecting cell adhesion to autophagy

2020; Elsevier BV; Volume: 295; Issue: 49 Linguagem: Inglês

10.1074/jbc.ra120.014790

ISSN

1083-351X

Autores

Razie Amraei, Tooba Alwani, Rachel Ho, Zahra Aryan, Shawn Wang, Nader Rahimi,

Tópico(s)

Metabolism, Diabetes, and Cancer

Resumo

Autophagy plays critical roles in the maintenance of endothelial cells in response to cellular stress caused by blood flow. There is growing evidence that both cell adhesion and cell detachment can modulate autophagy, but the mechanisms responsible for this regulation remain unclear. Immunoglobulin and proline-rich receptor-1 (IGPR-1) is a cell adhesion molecule that regulates angiogenesis and endothelial barrier function. In this study, using various biochemical and cellular assays, we demonstrate that IGPR-1 is activated by autophagy-inducing stimuli, such as amino acid starvation, nutrient deprivation, rapamycin, and lipopolysaccharide. Manipulating the IκB kinase β activity coupled with in vivo and in vitro kinase assays demonstrated that IκB kinase β is a key serine/threonine kinase activated by autophagy stimuli and that it catalyzes phosphorylation of IGPR-1 at Ser220. The subsequent activation of IGPR-1, in turn, stimulates phosphorylation of AMP-activated protein kinase, which leads to phosphorylation of the major pro-autophagy proteins ULK1 and Beclin-1 (BECN1), increased LC3-II levels, and accumulation of LC3 punctum. Thus, our data demonstrate that IGPR-1 is activated by autophagy-inducing stimuli and in response regulates autophagy, connecting cell adhesion to autophagy. These findings may have important significance for autophagy-driven pathologies such cardiovascular diseases and cancer and suggest that IGPR-1 may serve as a promising therapeutic target. Autophagy plays critical roles in the maintenance of endothelial cells in response to cellular stress caused by blood flow. There is growing evidence that both cell adhesion and cell detachment can modulate autophagy, but the mechanisms responsible for this regulation remain unclear. Immunoglobulin and proline-rich receptor-1 (IGPR-1) is a cell adhesion molecule that regulates angiogenesis and endothelial barrier function. In this study, using various biochemical and cellular assays, we demonstrate that IGPR-1 is activated by autophagy-inducing stimuli, such as amino acid starvation, nutrient deprivation, rapamycin, and lipopolysaccharide. Manipulating the IκB kinase β activity coupled with in vivo and in vitro kinase assays demonstrated that IκB kinase β is a key serine/threonine kinase activated by autophagy stimuli and that it catalyzes phosphorylation of IGPR-1 at Ser220. The subsequent activation of IGPR-1, in turn, stimulates phosphorylation of AMP-activated protein kinase, which leads to phosphorylation of the major pro-autophagy proteins ULK1 and Beclin-1 (BECN1), increased LC3-II levels, and accumulation of LC3 punctum. Thus, our data demonstrate that IGPR-1 is activated by autophagy-inducing stimuli and in response regulates autophagy, connecting cell adhesion to autophagy. These findings may have important significance for autophagy-driven pathologies such cardiovascular diseases and cancer and suggest that IGPR-1 may serve as a promising therapeutic target. Autophagy (also called macroautophagy), the lysosomal degradation of cytoplasmic organelles or cytosolic components, is an evolutionarily conserved cytoprotective mechanism that is induced in response to cellular stress, such as nutrient withdrawal, loss of cell adhesion, and flow shear stress, or by therapeutic genotoxic agents and others (1Kroemer G. Mariño G. Levine B. Autophagy and the integrated stress response.Mol. Cell. 2010; 40 (20965422): 280-29310.1016/j.molcel.2010.09.023Abstract Full Text Full Text PDF PubMed Scopus (2460) Google Scholar, 2Vlahakis A. Debnath J. 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The serine/threonine protein kinase mTOR complex 1 (mTORC1) is a key regulator of autophagy in response to nutrient availability. In the presence of amino acids, mTORC1 is activated and suppresses autophagy through phosphorylation of ULK1 and ATG13. However, upon nutrient deprivation, mTORC1 activity is inhibited, leading to the activation of ULK1 that induces the autophagy program (8Jung C.H. Jun C.B. Ro S.H. Kim Y.M. Otto N.M. Cao J. Kundu M. Kim D.H. ULK–Atg13–FIP200 complexes mediate mTOR signaling to the autophagy machinery.Mol. Biol. Cell. 2009; 20 (19225151): 1992-200310.1091/mbc.e08-12-1249Crossref PubMed Scopus (1421) Google Scholar, 9Hosokawa N. Hara T. Kaizuka T. Kishi C. Takamura A. Miura Y. Iemura S. Natsume T. Takehana K. Yamada N. Guan J.L. Oshiro N. Mizushima N. Nutrient-dependent mTORC1 association with the ULK1–Atg13–FIP200 complex required for autophagy.Mol. Biol. 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Kundu M. et al.Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy.Science. 2011; 331 (21205641): 456-46110.1126/science.1196371Crossref PubMed Scopus (1731) Google Scholar). Commonly known autophagy-inducing conditions or agents such as nutrient withdrawal including amino acid and serum starvation, immunosuppressant rapamycin/Sirolimus, and LPS all activate several key kinases such as the IκB kinase (IKK) complex (1Kroemer G. Mariño G. Levine B. Autophagy and the integrated stress response.Mol. Cell. 2010; 40 (20965422): 280-29310.1016/j.molcel.2010.09.023Abstract Full Text Full Text PDF PubMed Scopus (2460) Google Scholar). The IKK complex is composed of at least three proteins, including two catalytic subunits (IKKα and IKKβ) and the scaffold protein NF-κB essential modulator (NEMO; also called IKKγ) (12Perkins N.D. Integrating cell-signalling pathways with NF-κB and IKK function.Nat. Rev. Mol. Cell Biol. 2007; 8 (17183360): 49-6210.1038/nrm2083Crossref PubMed Scopus (1887) Google Scholar). In addition to its pivotal role in mediating phosphorylation of IκB (12Perkins N.D. Integrating cell-signalling pathways with NF-κB and IKK function.Nat. Rev. Mol. Cell Biol. 2007; 8 (17183360): 49-6210.1038/nrm2083Crossref PubMed Scopus (1887) Google Scholar), IKKβ can regulate autophagy in IκB-independent manner (13Criollo A. Senovilla L. Authier H. Maiuri M.C. Morselli E. Vitale I. Kepp O. Tasdemir E. Galluzzi L. Shen S. Tailler M. Delahaye N. Tesniere A. De Stefano D. Younes A.B. et al.The IKK complex contributes to the induction of autophagy.EMBO J. 2010; 29 (19959994): 619-63110.1038/emboj.2009.364Crossref PubMed Scopus (235) Google Scholar, 14Comb W.C. Cogswell P. Sitcheran R. Baldwin A.S. IKK-dependent, NF-κB–independent control of autophagic gene expression.Oncogene. 2011; 30 (21151171): 1727-173210.1038/onc.2010.553Crossref PubMed Scopus (88) Google Scholar) by mechanisms that are not fully understood. IGPR-1 was identified as a novel cell adhesion molecule expressed in various human cell types, including endothelial and epithelial cells, and it mediates cell–cell adhesion (15Rahimi N. Rezazadeh K. Mahoney J.E. Hartsough E. Meyer R.D. Identification of IGPR-1 as a novel adhesion molecule involved in angiogenesis.Mol. Biol. Cell. 2012; 23 (22419821): 1646-165610.1091/mbc.E11-11-093410.1091/mbc.e11-11-0934Crossref PubMed Scopus (41) Google Scholar). IGPR-1 regulates angiogenesis and endothelial barrier function (15Rahimi N. Rezazadeh K. Mahoney J.E. Hartsough E. Meyer R.D. Identification of IGPR-1 as a novel adhesion molecule involved in angiogenesis.Mol. Biol. Cell. 2012; 23 (22419821): 1646-165610.1091/mbc.E11-11-093410.1091/mbc.e11-11-0934Crossref PubMed Scopus (41) Google Scholar, 16Wang Y.H.W. Meyer R.D. Bondzie P.A. Jiang Y. Rahimi I. Rezazadeh K. Mehta M. Laver N.M.V. Costello C.E. Rahimi N. IGPR-1 is required for endothelial cell–cell adhesion and barrier function.J. Mol. Biol. 2016; 428 (27838321): 5019-503310.1016/j.jmb.2016.11.003Crossref PubMed Scopus (16) Google Scholar), decreases sensitivity of tumor cells to the genotoxic agent doxorubicin, and supports tumor cell survival in response to anoikis (17Woolf N. Pearson B.E. Bondzie P.A. Meyer R.D. Lavaei M. Belkina A.C. Chitalia V. Rahimi N. Targeting tumor multicellular aggregation through IGPR-1 inhibits colon cancer growth and improves chemotherapy.Oncogenesis. 2017; 6 (28920928): e37810.1038/oncsis.2017.77Crossref PubMed Google Scholar). IGPR-1 is localized to adherens junctions and is activated through transhomophilic dimerization (16Wang Y.H.W. Meyer R.D. Bondzie P.A. Jiang Y. Rahimi I. Rezazadeh K. Mehta M. Laver N.M.V. Costello C.E. Rahimi N. IGPR-1 is required for endothelial cell–cell adhesion and barrier function.J. Mol. Biol. 2016; 428 (27838321): 5019-503310.1016/j.jmb.2016.11.003Crossref PubMed Scopus (16) Google Scholar). Additionally, IGPR-1 responds to various cellular stresses, because its phosphorylation (i.e. Ser220) is significantly increased by flow shear stress (18Ho R.X. Tahboub R. Amraei R. Meyer R.D. Varongchayakul N. Grinstaff M. Rahimi N. The cell adhesion molecule IGPR-1 is activated by, and regulates responses of endothelial cells to shear stress.J. Biol. Chem. 2019; 294 (31341021): 13671-1368010.1074/jbc.RA119.008548Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar) and exposure to doxorubicin (17Woolf N. Pearson B.E. Bondzie P.A. Meyer R.D. Lavaei M. Belkina A.C. Chitalia V. Rahimi N. Targeting tumor multicellular aggregation through IGPR-1 inhibits colon cancer growth and improves chemotherapy.Oncogenesis. 2017; 6 (28920928): e37810.1038/oncsis.2017.77Crossref PubMed Google Scholar, 18Ho R.X. Tahboub R. Amraei R. Meyer R.D. Varongchayakul N. Grinstaff M. Rahimi N. The cell adhesion molecule IGPR-1 is activated by, and regulates responses of endothelial cells to shear stress.J. Biol. Chem. 2019; 294 (31341021): 13671-1368010.1074/jbc.RA119.008548Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar). Curiously, both shear stress (19Liu J. Bi X. Chen T. Zhang Q. Wang S.X. Chiu J.J. Liu G.S. Zhang Y. Bu P. Jiang F. Shear stress regulates endothelial cell autophagy via redox regulation and Sirt1 expression.Cell Death Dis. 2015; 6 (26181207)e1827 10.1038/cddis.2015.193Crossref PubMed Scopus (98) Google Scholar) and doxorubicin (20Chen H. Zhao C. He R. Zhou M. Liu Y. Guo X. Wang M. Zhu F. Qin R. Li X. Danthron suppresses autophagy and sensitizes pancreatic cancer cells to doxorubicin.Toxicol In Vitro. 2019; 54 (30389604): 345-35310.1016/j.tiv.2018.10.019Crossref PubMed Scopus (23) Google Scholar, 21Sui X. Chen R. Wang Z. Huang Z. Kong N. Zhang M. Han W. Lou F. Yang J. Zhang Q. Wang X. He C. Pan H. Autophagy and chemotherapy resistance: a promising therapeutic target for cancer treatment.Cell Death Dis. 2013; 4 (24113172): e83810.1038/cddis.2013.350Crossref PubMed Scopus (819) Google Scholar) are well-known potent inducers of autophagy, raising a possibility for the involvement of IGPR-1 in autophagy. In this study, we demonstrate that upon the induction of autophagy, IGPR-1 is phosphorylated at Ser220 via a mechanism that involves activation of IKKβ. IKKβ-dependent phosphorylation of IGPR-1 stimulates phosphorylation of AMPK, leading to activation of BECN1 and ULK1, connecting cell adhesion and energy sensing to autophagy. Homophilic transdimerization of IGPR-1 regulates its phosphorylation at Ser220 (16Wang Y.H.W. Meyer R.D. Bondzie P.A. Jiang Y. Rahimi I. Rezazadeh K. Mehta M. Laver N.M.V. Costello C.E. Rahimi N. IGPR-1 is required for endothelial cell–cell adhesion and barrier function.J. Mol. Biol. 2016; 428 (27838321): 5019-503310.1016/j.jmb.2016.11.003Crossref PubMed Scopus (16) Google Scholar). Additionally, genotoxic agents such as doxorubicin (17Woolf N. Pearson B.E. Bondzie P.A. Meyer R.D. Lavaei M. Belkina A.C. Chitalia V. Rahimi N. Targeting tumor multicellular aggregation through IGPR-1 inhibits colon cancer growth and improves chemotherapy.Oncogenesis. 2017; 6 (28920928): e37810.1038/oncsis.2017.77Crossref PubMed Google Scholar) and flow shear stress (18Ho R.X. Tahboub R. Amraei R. Meyer R.D. Varongchayakul N. Grinstaff M. Rahimi N. The cell adhesion molecule IGPR-1 is activated by, and regulates responses of endothelial cells to shear stress.J. Biol. Chem. 2019; 294 (31341021): 13671-1368010.1074/jbc.RA119.008548Abstract Full Text Full Text PDF PubMed Scopus (9) Google Scholar) also simulate phosphorylation of IGPR-1at Ser220. Because both shear stress and genotoxic agents are well-known for their roles in autophagy, we asked whether IGPR-1 is activated in response to autophagy. We used human embryonic kidney epithelial-293 (HEK-293) cells ectopically expressing IGPR-1 as a model system to study the role of IGPR-1 in autophagy, because these cells do not express IGPR-1 endogenously at a detectable level (16Wang Y.H.W. Meyer R.D. Bondzie P.A. Jiang Y. Rahimi I. Rezazadeh K. Mehta M. Laver N.M.V. Costello C.E. Rahimi N. IGPR-1 is required for endothelial cell–cell adhesion and barrier function.J. Mol. Biol. 2016; 428 (27838321): 5019-503310.1016/j.jmb.2016.11.003Crossref PubMed Scopus (16) Google Scholar). To this end, we first, tested whether amino acid starvation, the best-known inducer of autophagy, can stimulate phosphorylation of IGPR-1. The cells were lysed, and whole cell lysates were subjected to Western blotting analysis followed by immunoblotting with pSer220 and total IGPR-1 antibodies. Phosphorylation of IGPR-1 at Ser220 was significantly increased by brief amino acid starvation of HEK-293 cells. The increase in phosphorylation of Ser220 peaked after 1 min with amino acid starvation and remained highly phosphorylated until 15 min (Fig. 1A). Furthermore, in an additional set of experiments we subjected HEK-293 cells expressing IGPR-1 to various other autophagy-inducing conditions or factors such as serum-starvation, rapamycin, and LPS treatments and measured phosphorylation of Ser220. Both rapamycin and LPS are known to induce autophagy (1Kroemer G. Mariño G. Levine B. Autophagy and the integrated stress response.Mol. Cell. 2010; 40 (20965422): 280-29310.1016/j.molcel.2010.09.023Abstract Full Text Full Text PDF PubMed Scopus (2460) Google Scholar, 22Xu Y. Jagannath C. Liu X.-D. Sharafkhaneh A. Kolodziejska K.E. Eissa N.T. Toll-like receptor 4 is a sensor for autophagy associated with innate immunity.Immunity. 2007; 27 (17658277): 135-14410.1016/j.immuni.2007.05.022Abstract Full Text Full Text PDF PubMed Scopus (711) Google Scholar, 23Dunlop E.A. Tee A.R. mTOR and autophagy: a dynamic relationship governed by nutrients and energy.Semin. Cell Dev. Biol. 2014; 36 (25158238): 121-12910.1016/j.semcdb.2014.08.006Crossref PubMed Scopus (313) Google Scholar). Phosphorylation of IGPR-1 at Ser220 was significantly increased by brief serum starvation of HEK-293 cells (Fig. 1B). Furthermore, both rapamycin and LPS treatments of HEK-293 cells stimulated phosphorylation of IGPR-1 at Ser220 (Fig. 1, C and D). To demonstrate whether IGPR-1 is phosphorylated by autophagy in biologically relevant human endothelial cells in which IGPR-1 is expressed endogenously, we subjected primary human microvascular endothelial cells (HMVECs) to amino acid starvation. The result showed that IGPR-1 is phosphorylated at Ser220 in HMVECs (Fig. 1E). Taken together, the data demonstrate that IGPR-1 is activated by autophagy in HEK-293 cells and human primary endothelial cells. Activation of serine/threonine kinases represents a salient mechanistic feature of autophagy. Particularly, activation of IKKβ by nutrient deprivation (24Comb W.C. Hutti J.E. Cogswell P. Cantley L.C. Baldwin A.S. p85α SH2 domain phosphorylation by IKK promotes feedback inhibition of PI3K and Akt in response to cellular starvation.Mol. Cell. 2012; 45 (22342344): 719-73010.1016/j.molcel.2012.01.010Abstract Full Text Full Text PDF PubMed Scopus (55) Google Scholar), LPS (25Yang F. Tang E. Guan K. Wang C.Y. IKKβ plays an essential role in the phosphorylation of RelA/p65 on serine 536 induced by lipopolysaccharide.J. Immunol. 2003; 170 (12759443): 5630-563510.4049/jimmunol.170.11.5630Crossref PubMed Scopus (329) Google Scholar, 26Dauphinee S.M. Karsan A. Lipopolysaccharide signaling in endothelial cells.Lab. Invest. 2006; 86 (16357866): 9-2210.1038/labinvest.3700366Crossref PubMed Scopus (427) Google Scholar), and rapamycin (27Dan H.C. Cooper M.J. Cogswell P.C. Duncan J.A. Ting J.P.Y. Baldwin A.S. Akt-dependent regulation of NF-κB is controlled by mTOR and Raptor in association with IKK.Genes Dev. 2008; 22 (18519641): 1490-150010.1101/gad.1662308Crossref PubMed Scopus (450) Google Scholar) is known to play a central role in autophagy. Therefore, we asked whether activation of IKKβ by serum starvation, LPS, or rapamycin can mediate phosphorylation of IGPR-1 at Ser220. To this end, we overexpressed WT IKKβ or kinase inactive IKKβ (IKKβ-A44) in IGPR-1/HEK-293 cells. After 48 h of transfection, the cells were either kept in 10% FBS or serum-starved for 15, 30, or 60 min. The cells were lysed, and whole cell lysates were immunoblotted for pSer220, total IGPR-1, and phospho-IKKβ. Expression of WT IKKβ in IGPR-1/HEK-293 cells resulted in a robust phosphorylation of IGPR-1 in the presence of 10% FBS, and this was further increased in response to serum starvation (Fig. 2A). In contrast, overexpression of kinase inactive IKKβ-A44 in IGPR-1/HEK-293 cells markedly reduced phosphorylation of IGPR-1 at Ser220 (Fig. 2A), indicating that IKKβ kinase activity is required for the serum starvation–dependent phosphorylation of IGPR-1 at Ser220. Similarly, LPS-induced phosphorylation of Ser220 on IGPR-1 was inhibited by a selective IKKβ inhibitor, IKK inhibitor III/BMS-345541 (28Burke J.R. Pattoli M.A. Gregor K.R. Brassil P.J. MacMaster J.F. McIntyre K.W. Yang X. Iotzova V.S. Clarke W. Strnad J. Qiu Y. Zusi F.C. BMS-345541 is a highly selective inhibitor of IκB kinase that binds at an allosteric site of the enzyme and blocks NF-κB–dependent transcription in mice.J. Biol. Chem. 2003; 278 (12403772): 1450-145610.1074/jbc.M209677200Abstract Full Text Full Text PDF PubMed Scopus (442) Google Scholar) (Fig. 2B). Furthermore, LPS stimulated phosphorylation of IKKβ and IKK inhibitor blocked its phosphorylation (Fig. 2B). Conversely, overexpression of a constitutively active IKKβ (IKKβ-S177E/S181E) in IGPR-1/HEK293 cells augmented LPS-induced phosphorylation of Ser220 (Fig. 2C), indicating that IKKβ activity also is required for LPS-induced phosphorylation of IGPR-1 at Ser220. Ser220 and the surrounding amino acids in IGPR-1 are strongly conserved both in human and nonhuman primates (Fig. 3A), suggesting an evolutionarily conserved mechanism for the phosphorylation of Ser220. IKKβ phosphorylates peptides with aromatic residues at the −2 position, hydrophobic residues at the +1 position, and acidic residues at the +3 position (29Hutti J.E. Turk B.E. Asara J.M. Ma A. Cantley L.C. Abbott D.W. IκB kinase β phosphorylates the K63 deubiquitinase A20 to cause feedback inhibition of the NF-κB pathway.Mol. Cell Biol. 2007; 27 (17709380): 7451-746110.1128/MCB.01101-07Crossref PubMed Scopus (133) Google Scholar), suggesting that IKKβ is a likely candidate kinase involved in the phosphorylation of IGPR-1 at Ser220 (Fig. 3B). Therefore, we asked whether IKKβ can phosphorylate IGPR-1 at Ser220 independent of the autophagy-inducing factors like serum starvation or LPS and rapamycin. We overexpressed WT IKKβ or kinase inactive IKKβ-A44 in IGPR-1/HEK-293, and 48 h after transfection, the cells were lysed, and phosphorylation of IGPR-1 was determined by Western blotting analysis. Overexpression of WT IKKβ increased phosphorylation of IGPR-1, whereas kinase inactive IKKβ-A44 inhibited phosphorylation of IGPR-1 at Ser220 (Fig. 3C). Similarly, IKKβ inhibitor inhibited both phosphorylation of IGPR-1 and IKKβ (Fig. 3D). In an additional approach, we knocked out IKKβ via CRISPR-Cas9 system and examined the effect of loss of IKKβ in IGPR-1 phosphorylation. To this end, cells were either treated with a control vehicle or AMPK activator, oligomycin and cells were lysed, and phosphorylation of IGPR-1 at Ser220 was determined. Stimulation of IGPR-1/Ctr.sgRNA/HEK-293 cells with oligomycin stimulated AMPK activation and phosphorylation of IGPR-1 at Ser220 (Fig. 3E). However, in IGPR-1/IKKβ.sgRNA/HEK-293 cells, phosphorylation of Ser220 was not detected, and treatment with oligomycin also did not stimulate phosphorylation of IGPR-1 at Ser220 (Fig. 3E). IKKβ.sgRNA-mediated knockout of IKKβ is shown (Fig. 3E). We next asked whether IKKβ can directly phosphorylates IGPR-1 at Ser220. To examine the direct involvement of IKKβ in catalyzing the phosphorylation of IGPR-1, we carried out an in vitro kinase assay using a purified recombinant GST–IGPR-1 protein that only encompasses the cytoplasmic domain of IGPR-1 and demonstrated that IKKβ phosphorylates IGPR-1 at Ser220 (Fig. 3F). Taken together, our data identify IGPR-1 as a novel substrate of IKKβ. Furthermore, we carried additional experiments to demonstrate the selectively of IGPR-1 phosphorylation at Ser220 by IKKβ. To this end, we immunoprecipitated IGPR-1 proteins from HEK-293 cells expressing either WT IGPR-1, A220–IGPR-1, or D220–IGPR-1. The immunoprecipitated proteins were subjected to calf intestinal alkaline phosphatase treatment, which removes phosphorylation. The removal of phosphorylation on IGPR-1 was confirmed by Western blotting analysis using pSer220 specific antibody (Fig. S1A). The dephosphorylated proteins were subjected to an in vitro kinase assay using a recombinant IKKβ. The result showed that IKKβ selectively phosphorylates IGPR-1 at Ser220 (Fig. S1B). We sought to determine whether IGPR-1 could activate AMPK, a widely considered master regulator of autophagy (10Kim J. Kundu M. Viollet B. Guan K.-L. AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1.Nat. Cell Biol. 2011; 13 (21258367): 132-14110.1038/ncb2152Crossref PubMed Scopus (4214) Google Scholar). HEK-293 cells expressing an empty vector (EV), IGPR-1, or A220–IGPR-1 were either maintained in 10% FBS or serum-starved for 30 min or 12 h. The pThr172-AMPK immunoblot of whole cell lysates showed that expression of IGPR-1 in HEK-293 cells bypassed the requirement for serum starvation–dependent activation of AMPK because AMPK was strongly phosphorylated at Thr172 in IGPR-1/HEK-293 cells in 10% FBS compared with EV/HEK-293 cells (Fig. 4A). Phosphorylation of AMPK was further augmented in IGPR-1/HEK-293 cells, particularly at 30 min, compared with control EV/HEK-293 cells (Fig. 4A). Interestingly, A220–IGPR-1/HEK-293 cells showed also an increase in phosphorylation of AMPK, notwithstanding significantly less than the WT IGPR-1, but more than the control EV/HEK-293 cells (Fig. 4A). Phosphorylation of AMPKα at Thr172 in the activation loop is required for AMPK activation (30Lizcano J.M. Göransson O. Toth R. Deak M. Morrice N.A. Boudeau J. Hawley S.A. Udd L. Mäkelä T.P. Hardie D.G. Alessi D.R. LKB1 is a master kinase that activates 13 kinases of the AMPK subfamily, including MARK/PAR-1.EMBO J. 2004; 23 (14976552): 833-84310.1038/sj.emboj.7600110Crossref PubMed Scopus (1030) Google Scholar, 31Hawley S.A. Davison M. Woods A. Davies S.P. Beri R.K. Carling D. Hardie D.G. Characterization of the AMP-activated protein kinase kinase from rat liver and identification of threonine 172 as the major site at which it phosphorylates AMP-activated protein kinase.J. Biol. Chem. 1996; 271 (8910387): 27879-2788710.1074/jbc.271.44.27879Abstract Full Text Full Text PDF PubMed Scopus (987) Google Scholar), indicating that expression of IGPR-1 in HEK-293 cells activated AMPK. We next asked whether IGPR-1 induced AMPK activation also stimulates phosphorylation of BECN1 and ULK1, which are the best-known substrates of AMPK and play central roles in autophagy (32Menon M.B. Dhamija S. Beclin 1 phosphorylation: at the center of autophagy regulation.Front. Cell Dev. Biol. 2018; 6 (30370269): 13710.3389/fcell.2018.00137Crossref PubMed Scopus (132) Google Scholar). Similar to AMPK activation, phosphorylation of BECN1 at Ser93 was significantly increased in IGPR-1/HEK-293 cells compared with EV/HEK-293 or A220–IGPR-1 cells (Fig. 4A), indicating that IGPR-1–induced activation of AMPK in HEK-293 cells also induced phosphorylation of BECN1 (Fig. 4A) as previously reported (33Kim J. Kim Y.C. Fang C. Russell R.C. Kim J.H. Fan W. Liu R. Zhong Q. Guan K.L. Differential regulation of distinct Vps34 complexes by AMPK in nutrient stress and autophagy.Cell. 2013; 152 (23332761): 290-30310.1016/j.cell.2012.12.016Abstract Full Text Full Text PDF PubMed Scopus (519) Google Scholar). Furthermore, phosphorylation of ULK1 at Ser555 was also increased in IGPR-1/HEK-293 cells compared with control EV/HEK-293 cells. Interestingly, ULK1 phosphorylation was significantly reduced in A220–IGPR-1 cells, indicating that phosphorylation of Ser220, in part, is required for phosphorylation of ULK-1 (Fig. 4A). The data indicate that phosphorylation of Ser220 on IGPR-1 plays an important role in the phosphorylation of AMPK, BECN1, and ULK1. We next examined the role of IGPR-1 in the autophagosome formation by measuring expression of LC3-phosphatidylethanolamine conjugate (LC3-II) and p62 endogenously expressed in HEK-293 cells, which are required for autophagosome development during autophagy (34Schaaf M.B. Keulers T.G. Vooijs M.A. Rouschop K.M. LC3/GABARAP family proteins: autophagy-(un)related functions.FASEB J. 2016; 30 (27601442): 3961-397810.1096/fj.201600698RCrossref PubMed Scopus (288) Google Scholar), by Western blotting analysis. HEK-293 cells expressing EV or IGPR-1 were either kept at 10% FBS or serum-starved for overnight. The cells were lysed, and expression of LC3 and p62 levels was determined by immunoblotting with LC3 and p62 antibodies. Although expression of LC3II was increased in response to serum starvation in EV/HEK-293 cells, expression of LC3II was significantly higher both in 10% FBS and in serum-starved conditions in IGPR-1/HEK-293 cells (Fig. 5A), indicating that IGPR-1 through increased in expression of LC3II regulates autophagosome formation. Additionally, as expected, p62 level was markedly decreased in response to serum starvation (Fig. 5A). To further elucidate the role of IGPR-1 in induction of autophagy, we established autophagic flux reporter cell lines by creating GFP-LC3 (microtubule-associated protein 1 light chain 3β)–RFP/HEK-293 and IGPR-1/GFP-LC3-RFP/HEK-293 cell lines via a retroviral expression system as previously reported (35Kaizuka T. Morishita H. Hama Y. Tsukamoto S. Matsui T. Toyota Y. Kodama A. Ishihara T. Mizushima T. Mizushima N. An autophagic flux probe that releases an internal control.Mol. Cell. 2016; 64 (27818143): 835-84910.1016/j.molcel.2016.09.037Abstract Full Text Full Text PDF PubMed Scopus (255) Google Scholar). During autophagy, GFP-LC3-RFP labeled autophagosomes fuse with lysosomes. Although the GFP signals are quenched because of the acidic environment (GFP is acid-sensitive) in the autolysosomes, the RFP signals remain stable because RFP is acid-stable, and hence an increase in the number of RFP-LC3 (red only) puncta is considered a reflection of autophagic flux (36Ni H.-M. Bockus A. Wozniak A.L. Jones K. Weinman S. Yin X.-M. Ding W.-X. Dissecting the dynamic turnover of GFP-LC3 in the autolysosome.Autophagy. 2011; 7 (21107021): 188-20410.4161/auto.7.2.14181Crossref PubMed Scopus (273) Google Scholar). In the presence of 10% FBS, only a few RFP-LC3 positive puncta were observed in GFP-LC3-RFP/HEK-293 cells (Fig. 5, B and D). However, we observed a significantly higher bas

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