- Email: [email protected]
CCCP induces autophagy in an AMPK-independent manner
Biochemical and Biophysical Research Communications 416 (2011) 343–348
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
CCCP induces autophagy in an AMPK-independent manner Kyum-Yil Kwon a, Benoit Viollet b,c,d, Ook Joon Yoo a,⇑ a
Laboratory of Molecular Biology, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea Inserm, U1016, Institut Cochin, Paris, France c Cnrs, UMR8104, Paris, France d Universite Paris Descartes, Sorbonne Paris cite, Paris, France b
a r t i c l e
i n f o
Article history: Received 7 November 2011 Available online 15 November 2011 Keywords: AMPK CCCP Autophagy Mitophagy mTORC1
a b s t r a c t AMP-activated protein kinase (AMPK) is an important sensor of cellular energy status, and is involved in cell growth and autophagy through mammalian target of rapamycin complex 1 (mTORC1). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, leads to AMPK activation and Parkin-dependent mitophagy, respectively. However, the detailed biochemical mechanism of how CCCP induces autophagy or mitophagy has not been investigated yet. Here, we showed that CCCP inhibits mTORC1 independently of AMPK, although CCCP induces AMPK activation. Using wild type (WT) and AMPKa1/a2 double knockout (DKO) MEFs, we observed that CCCP promotes endogenous LC3 lipidation and formation of RFPLC3 puncta, indicating autophagosome or autolysosome, in an AMPK-independent manner. Finally, we also revealed that the percentage of CCCP-dependent colocalization between mitochondria and RFP-LC3 puncta is similar both in WT and AMPKa1/a2 DKO MEFs. Based on these data, we concluded that AMPK is not essential in regulation of CCCP-induced autopahgy including mitophagy. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction AMP-activated protein kinase (AMPK) plays crucial roles in cell growth and energy homeostasis including autophagy and metabolism [1]. The most well-documented mechanism by which AMPK controls cell growth is through downregulation of mTORC1 (mammalian target of rapamycin complex 1) activity [2]. One mechanism of AMPK-dependent mTORC1 inhibition is by phosphorylation of TSC2 [3] and the other is directly by phosphorylation of Raptor, one of mTORC1 subunits [4]. AMPK also controls autophagy either indirectly by negatively regulating mTORC1 or directly by phosphorylating ULK1, a downstream target of mTORC1 and a crucial inducer of autophagy [5–7]. Autophagy includes non-selective autophagy and selective autophagy such as mitophagy known for the removal of depolarized mitochondria [8]. However, at present, the precise relationship between autophagy and mitophagy remains to be further investigated. It has been shown that carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial protonophore, leads to Parkin-dependent mitophagy [9,10]. Interestingly a recent study showed that CCCP promotes mitophagy through ULK1 [11]. However, the detailed molecular mechanism on CCCP-induced mitophagy is little revealed. In addition, as CCCP enhances activation ⇑ Corresponding author. Fax: +82 42 350 8160. E-mail address: [email protected] (O.J. Yoo). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.11.038
of AMPK and inhibition of mTORC1, respectively [12,13], we hypothesized that CCCP-dependent AMPK signaling affects autophagy including mitophagy. In this study, we examined whether CCCP-induced alteration of mTORC1 activity is mediated by AMPK. Moreover, we evaluated whether CCCP treatment triggers autophagy and/or mitophagy in an AMPK-dependent manner. Our results unexpectedly suggested that CCCP inhibits mTORC1 activity and induces autophagy including mitophagy in an AMPK-independent manner. 2. Materials and methods 2.1. Reagents and antibodies CCCP and DMSO were purchased from Calbiocam and Sigma– Aldrich, respectively. Anti-c-myc antibody (9E10) and beta-tubulin mouse antibody (E7) were from Developmental Studies Hybridoma Bank. Most phospho-specific antibodies were from Cell Signaling Technology. Anti-LC3 antibody was from Abgent. 2.2. Cell culture and transfection Wild type (WT) and AMPKa1/a2 double knockout (DKO) mouse embryonic fibroblasts (MEFs) have been described previously [14]. HEK293 and MEF cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen) at
344
K.-Y. Kwon et al. / Biochemical and Biophysical Research Communications 416 (2011) 343–348
37 °C in a humidified atmosphere with 5% CO2, and were transiently transfected using Lipofectamine Plus reagent (Invitrogen).
3. Results and discussion 3.1. Effect of CCCP on mTORC1
2.3. Preparation of cell lysates, immunoprecipitation, and immunoblotting Cell stimulation by DMSO or CCCP treatment was terminated by washing cells with ice-cold PBS. Cell lysates were prepared in Lysis buffer A (20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 2 mM EGTA, 50 mM beta-glycerophosphate, 50 mM NaF, 1 mM sodium vanadate, 2 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 lg/ml leupeptin, 1 lg/ml pepstatin A, and 1% Triton X-100) and subjected to immunoprecipitation and immunoblot analysis according to standard procedures [15]. 2.4. Immunocytochemistry After the indicated treatments, the cells were fixed with 2% paraformaldehyde in PBS, and were subjected to immunofluorescence staining according to a standard protocol [15]. All images were obtained using a laser scanning confocal microscope LSM 710 from Carl Zeiss. 2.5. Quantification and statistical analysis Quantification of RFP-LC3 punctuation and colocalization assay with indicated markers were based on enumeration of the number of RFP-LC3 dots in each cell or the number of co-localized cells in each condition. The statistical analyses were performed using Student’s t-test.
The upstream regulator of CCCP-dependent autophagy or mitophagy remains uncertain [9–11]. Therefore, we investigated whether CCCP affects mTORC1, a crucial upstream regulator of autophagy [16]. To evaluate acute responses of mTORC1, CCCP was treated for maximum 1 h. We checked not only mTORC1 but also mTORC2 activity to distinguish selective inhibition of mTORC1 from non-selective inhibition of PI3K/Akt pathway, and to trace the upstream regulator by CCCP-treatment. In overexpression of Myc-tagged S6 kinase (Myc-S6K) or MycAkt, CCCP treatment of HEK293 cells reduced the phosphorylation of S6K at Thr 389 representing mTORC1 activity, while sustained the phosphorylation of Akt at Ser 473 representing mTORC2 activity (Fig. 1A and B). Similarly, we also investigated the endogenous activities of mTORC1 and mTORC2 (Fig. 1C and D). As a result, CCCP strongly decreased the phosphorylation of S6K at Thr 389 and 4EBP at Thr 37/46, also representing mTORC1 activity, in dose- and timedependent manners. However, CCCP did not affect the phosphorylation of Akt at Ser 473. These data indicated that CCCP downregulates mTORC1, but not mTORC2 activity. Notably, CCCP rather increased the phosphorylation of Akt at Thr 308 representing PDK1 activity. It is consistent with the previous report suggesting that inactivated S6K, via CCCP-dependent mTORC1 inhibition, reduces the phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser 270, thereby increasing the activity of PDK1 [17]. Based on these results, we concluded that CCCP treatment can selectively block mTORC1 activity.
Fig. 1. CCCP decreases mTORC1 activity, but not mTORC2 activity. (A, B) HEK293 cells were transfected with Myc-tagged S6K (A) or Myc-tagged Akt (B) for 24 h, and incubated with DMSO or CCCP (10 lM) for 1 h. Then, the cells were subjected to immunoprecipitation with anti-Myc antibody followed by immunoblot analysis with the indicated antibodies. (C) HEK293 cells were cultured with indicated doses of CCCP for 1 h before cell lysis, and analyzed by immunoblot analysis with the indicated antibodies. (D) HEK293 cell were treated with CCCP (10 lM) for the indicated times before cell lysis, and followed by immunoblot analysis with the indicated antibodies. ⁄ Indicates immunoglobulin heavy chain.
K.-Y. Kwon et al. / Biochemical and Biophysical Research Communications 416 (2011) 343–348
3.2. Requirement of AMPK on CCCP-dependent inhibition of mTORC1 Although our data showed that CCCP selectively inhibits mTORC1, but it has never been determined whether CCCP-dependent AMPK activation is essential for regulating mTORC1 activity. Besides, the biochemical mechanism of how CCCP activates AMPK remains unknown. Therefore we investigated whether CCCP decreases mTORC1 activity in an AMPK-dependent manner using three different methods. Firstly, we used compound C, an AMPK inhibitor, to block CCCPdependent activation of AMPK (Fig. 2A). As expected, CCCP increased the phosphorylation of AMPKa at Thr 172, which indicates its activation, as well as its downstream targets, the phosphorylation of ACC at Ser 79 and Raptor at Ser 792. These AMPK activities were strongly inhibited by co-treatment of compound C with CCCP. However, surprisingly, the inhibited phosphorylation of S6K at Thr 389 and 4EBP at Thr 37/46 by CCCP was not restored by compound C. This unexpected data indicated that compound C inhibits CCCPinduced AMPK activation, but does not affect CCCP-induced
345
mTORC1 inactivation. Secondly, a dominant negative mutant of AMPK (AMPK-DN) was utilized to block AMPK signaling induced by CCCP (Fig. 2B). Similar to the above result (Fig. 2A), expression of AMPK-DN did not affect the reduced phosphorylation of S6K at Thr 389 by CCCP, compared with AMPK–WT expression controls (Fig. 2B). These pharmacological and molecular biological data consistently supported that CCCP-dependent AMPK activation is not enough to downregulate mTORC1 activity, although the inhibitory phosphorylation of Raptor at Ser 792 was strongly induced. Lastly, we evaluated CCCP-induced mTORC1 activity using wild type (WT) and AMPKa1/a2 double knockout (DKO) MEF cells (Fig. 2C). Since AMPKa has two isoforms (AMPKa1 and a2), it is necessary to utilize AMPKa1/a2 DKO cells for blocking AMPK signaling completely [14]. CCCP treatment potently suppressed the phosphorylation of S6K at Thr 389 and 4EBP at Thr 37/46 in AMPKa1/a2 DKO MEFs as well as WT MEFs. Taken together, our data demonstrated that CCCP-dependent inhibition of mTORC1 is in an AMPK-independent manner. Notably, the phosphorylation of Raptor at Ser 792 was abolished by
Fig. 2. CCCP-dependent activation of AMPK is not enough to inhibit mTORC1. (A) HEK293 cells were treated with 20 lM compound C and 10 lM CCCP as indicated for 1 h, and examined by immunoblot analysis with the indicated antibodies. (B) HEK293 cells were cotransfected with Myc-S6K and wild type (WT) or dominant negative (DN) mutant AMPK for 24 h, incubated with DMSO or CCCP (10 lM) for 10 min, and subjected to immunoprecipitation (IP) with anti-Myc antibody. Immunoprecipitates or whole cell lysates (WCL) were immunoblotted as indicated. (C) WT or AMPKa1/a2 DKO MEFs were treated with DMSO or CCCP (10 lM) for 1 h before cell lysis, and immunoblotted with indicated antibodies. ⁄Indicates immunoglobulin heavy chain.
346
K.-Y. Kwon et al. / Biochemical and Biophysical Research Communications 416 (2011) 343–348
CCCP treatment in AMPKa1/a2 DKO MEFs. This result indicated that Raptor phosphorylation by AMPK is not crucial to regulate mTORC1 activity. In addition, we observed that CCCP enhances the phosphorylation of AMPKa at Thr 172, a direct regulation site by LKB1 or CaMKK2 [1], implying that CCCP positively affects the upstream activation process of AMPK. 3.3. Requirement of AMPK on CCCP-dependent autophagy induction Recent evidence suggested that AMPK directly stimulates ULK1 to induce autophagy under various physiologic conditions [4,5]. We sought for the definite evidence whether AMPK activation by CCCP treatment could indeed require autophagy. We first examined autophagy formation in WT and AMPKa1/a2 DKO MEF cells using LC3 as one of reliable markers for autophagy [18]. We found that CCCP treatment increased LC3 lipidation (LC3I to LC3II) and RFP-LC3 punctuation both in WT and AMPKa1/a2 DKO MEFs (Fig. 3A and B). Furthermore, the accumulation level of RFP-LC3 punctuation following CCCP treatment in WT MEFs was not different from that in AMPKa1/a2 DKO MEFs (Fig. 3C). Collectively, these observations demonstrated that CCCP treatment is able to induce autophagy independently of AMPK.
3.4. Requirement of AMPK on CCCP-dependent mitophagy induction It has been proposed that CCCP induces mitophagy in a Parkin-dependent manner [8,9], which is considered to be mediated by autophagy machinery [11,19,20]. Although our data suggested that AMPK is not crucial for CCCP-induced autophagy (Fig. 3), we focused on an interesting report that CCCP treatment induces mitophagy in an ULK1-dependent manner [11]. Because, up to date, the known regulators of ULK1 are mTORC1 and AMPK [5–7], we could not exclude the possibility that AMPK mediates CCCP-dependent mitophagy. Immunostaining with an antibody recognizing MnSOD, a mitochondrial marker, showed that the pattern of CCCP-induced RFPLC3 puncta colocalized with MnSOD in WT MEFs was not different from that in AMPKa1/a2 DKO MEFs (Fig. 4A and C). Because we observed massive cell death following long time CCCP treatment over 12 h, the data showing complete mitochondrial clearance could not be obtained in CCCP-treated WT or AMPKa1/a2 DKO MEFs (data not shown). However, colocalization of intracellular organelles and LC3 puncta definitely indicated autophagosome or autolysosome [18]. Therefore, we could conclude that CCCP-induced colocalization of mitochondria and RFP-LC3 puncta in WT or AMPKa1/a2 DKO MEFs
Fig. 3. CCCP induces autophagy in an AMPK-independent manner. (A) WT or AMPKa1/a2 DKO MEFs were treated with DMSO or CCCP (20 lM) for 4 h as indicated, and analyzed by immunoblotting with indicated antibodies. (B) RFP-LC3 was transfected for 24 h in WT or AMPKa1/a2 DKO MEFs. Then, the cells were incubated with DMSO or CCCP (20 lM) for 4 h, and subjected to immunocytochemistry as described in Section 2. RFP-LC3, red; Hoechst, blue. Original magnification, 1000. (C) The number of RFPLC3 puncta/RFP-positive cell treated with DMSO or CCCP was quantified in WT or AMPKa1/a2 DKO MEFs. The data shown are the means ± SD (n > 100 cells/each condition). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
K.-Y. Kwon et al. / Biochemical and Biophysical Research Communications 416 (2011) 343–348
347
Fig. 4. CCCP induces mitophagy in an AMPK-independent manner. (A) YFP-Parkin and RFP-LC3 were transfected for 24 h in WT or AMPKa1/a2 DKO MEFs. Then, the cells were incubated with DMSO or CCCP (20 lM) for 8 h, and subjected to immunocytochemistry as described in Section 2. YFP-Parkin, green; RFP-LC3, red; MnSOD, yellow; Hoechst, blue. Original magnification, 1000. (B) Percentages of colocalization between mitochondria and YFP-Parkin with DMSO or CCCP treatment were quantified in WT or AMPKa1/a2 DKO MEFs. (C) Percentages of colocalization between mitochondria and RFP-LC3 punata with DMSO or CCCP treatment were quantified in WT or AMPKa1/a2 DKO MEFs. The data shown are the means ± SD of four different experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
were undergoing mitophagy. Furthermore, CCCP-induced YFPParkin translocation at mitochondria made no difference between in WT and AMPKa1/a2 DKO MEFs (Fig. 4B). Taken together, our data strongly supported that CCCP-dependent AMPK activation is not crucial for the induction of mitophagy. ULK1 stimulation by AMPK affects autophagy in vitro and in vivo [5,7]. However, our data indicated that AMPK is dispensable for CCCP-induced autophagy. In addition, we observed that CCCP-induced ULK1 phosphorylation at Ser 555, recently known as a direct regulation site by AMPK [7], is not crucial to induce LC3 lipidation (data not shown). This discrepancy is maybe, at least in cellular level, due to AMPK-independent effects by CCCP. On the other hand, one group reported that CCCP induces mitophagy in an ULK1-dependent manner [11]. In this regard, it is
highly possible that CCCP stimulates ULK1-dependent mitophagy through another unknown regulator, not through AMPK. Recent results suggested that metformin or phenformin, independent of AMPK, inhibited mTORC1 through Rag GTPases [21] or REDD1 [22]. However, the data on AMPK-dependent mTORC1 modulation by metformin or phenformin are still conflicting [4,7,21,22], and the mediator between such pharmacological AMPK stimulators and ULK1 activation has been little revealed. Therefore, more detailed and comprehensive studies are required to find the upstream regulator of ULK1 in CCCP-induced mitophagy. In summary, we demonstrated that mTORC1 inhibition by CCCP is independently of AMPK. We also revealed that CCCP induces autophagy including mitophagy in an AMPK-independent manner. These findings will contribute to the expansion of our knowledge
348
K.-Y. Kwon et al. / Biochemical and Biophysical Research Communications 416 (2011) 343–348
on mitochondrial defects-dependent intracellular signaling pathways for autophagy or mitophagy. Acknowledgments We are grateful to Dr. K.L. Guan for wild type and dominant negative mutant pCDNA3-HA-AMPK. YFP-Parkin and RFP-LC3 were from Dr. R. Youle and Dr. T. Yoshimori through Addgene (www.addgene.org), respectively. References [1] M.M. Mihaylova, R.J. Shaw, The AMPK signalling pathway coordinates cell growth, autophagy and metabolism, Nat. Cell Biol. 13 (2011) 1016–1023. [2] R. Zoncu, A. Efeyan, D.M. Sabatini, MTOR: from growth signal integration to cancer, diabetes and ageing, Nat. Rev. Mol. Cell Biol. 12 (2011) 21–35. [3] K. Inoki, T. Zhu, K.L. Guan, TSC2 mediates cellular energy response to control cell growth and survival, Cell 115 (2003) 577–590. [4] D.M. Gwinn, D.B. Shackelford, D.F. Egan, M.M. Mihaylova, A. Mery, D.S. Vasquez, B.E. Turk, R.J. Shaw, AMPK phosphorylation of Raptor mediates a metabolic checkpoint, Mol. Cell 30 (2008) 214–226. [5] J. Kim, M. Kundu, B. Viollet, K.L. Guan, AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1, Nat. Cell Biol. 13 (2011) 132–141. [6] J.W. Lee, S. Park, Y. Takahashi, H.G. Wang, The association of AMPK with ULK1 regulates autophagy, PLoS ONE 5 (2010) e15394. [7] D.F. Egan, D.B. Shackelford, M.M. Mihaylova, S. Gelino, R.A. Kohnz, W. Mair, D.S. Vasquez, A. Joshi, D.M. Gwinn, R. Taylor, J.M. Asara, J. Fitzpatrick, A. Dillin, B. Viollet, M. Kundu, M. Hansen, R.J. Shaw, Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy, Science 331 (2011) 456–461. [8] R.J. Youle, D.P. Narendra, Mechanisms of mitophagy, Nat. Rev. Mol. Cell Biol. 12 (2011) 9–14. [9] D. Narendra, A. Tanaka, D.F. Suen, R.J. Youle, Parkin is recruited selectively to impaired mitochondria and promotes their autophagy, J. Cell Biol. 183 (2008) 795–803. [10] S. Geisler, K.M. Holmstrom, D. Skujat, F.C. Fiesel, O.C. Rothfuss, P.J. Kahle, W. Springer, PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/ SQSTM1, Nat. Cell Biol. 12 (2010) 119–131.
[11] J.H. Joo, F.C. Dorsey, A. Joshi, K.M. Hennessy-Walters, K.L. Rose, K. McCastlain, J. Zhang, R. Iyengar, C.H. Jung, D.F. Suen, M.A. Steeves, C.Y. Yang, S.M. Prater, D.H. Kim, C.B. Thompson, R.J. Youle, P.A. Ney, J.L. Cleveland, M. Kundu, Hsp90– cdc37 chaperone complex regulates ulk1- and atg13-mediated mitophagy, Mol. Cell 43 (2011) 572–585. [12] L.E. McLeod, C.G. Proud, ATP depletion increases phosphorylation of elongation factor eEF2 in adult cardiomyocytes independently of inhibition of mTOR signalling, FEBS Lett. 531 (2002) 448–452. [13] B. Thors, H. Halldorsson, G. Thorgeirsson, Thrombin and histamine stimulate endothelial nitric-oxide synthase phosphorylation at Ser1177 via an AMPK mediated pathway independent of PI3K-Akt, FEBS Lett. 573 (2004) 175–180. [14] K.R. Laderoute, K. Amin, J.M. Calaoagan, M. Knapp, T. Le, J. Orduna, M. Foretz, B. Viollet, 50 -AMP-activated protein kinase (AMPK) is induced by low-oxygen and glucose deprivation conditions found in solid-tumor microenvironments, Mol. Cell. Biol. 26 (2006) 5336–5347. [15] Y. Kim, J. Park, S. Kim, S. Song, S.K. Kwon, S.H. Lee, T. Kitada, J.M. Kim, J. Chung, PINK1 controls mitochondrial localization of Parkin through direct phosphorylation, Biochem. Biophys. Res. Commun. 377 (2008) 975–980. [16] G. Kroemer, G. Marino, B. Levine, Autophagy and the integrated stress response, Mol. Cell 40 (2010) 280–293. [17] J. Zhang, Z. Gao, J. Yin, M.J. Quon, J. Ye, S6K directly phosphorylates IRS-1 on Ser-270 to promote insulin resistance in response to TNF-(alpha) signaling through IKK2, J. Biol. Chem. 283 (2008) 35375–35382. [18] N. Mizushima, T. Yoshimori, B. Levine, Methods in mammalian autophagy research, Cell 140 (2010) 313–326. [19] S. Kawajiri, S. Saiki, S. Sato, F. Sato, T. Hatano, H. Eguchi, N. Hattori, PINK1 is recruited to mitochondria with parkin and associates with LC3 in mitophagy, FEBS Lett. 584 (2010) 1073–1079. [20] D. Narendra, L.A. Kane, D.N. Hauser, I.M. Fearnley, R.J. Youle, P62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both, Autophagy 6 (2010) 1090–1106. [21] A. Kalender, A. Selvaraj, S.Y. Kim, P. Gulati, S. Brule, B. Viollet, B.E. Kemp, N. Bardeesy, P. Dennis, J.J. Schlager, A. Marette, S.C. Kozma, G. Thomas, Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPasedependent manner, Cell Metab. 11 (2010) 390–401. [22] I. Ben Sahra, C. Regazzetti, G. Robert, K. Laurent, Y. Le Marchand-Brustel, P. Auberger, J.F. Tanti, S. Giorgetti-Peraldi, F. Bost, Metformin, independent of AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1, Cancer Res. 71 (2011) 4366–4372.
Contents lists available at SciVerse ScienceDirect
Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc
CCCP induces autophagy in an AMPK-independent manner Kyum-Yil Kwon a, Benoit Viollet b,c,d, Ook Joon Yoo a,⇑ a
Laboratory of Molecular Biology, Graduate School of Medical Science and Engineering, Korea Advanced Institute of Science and Technology, Daejeon 305-701, Republic of Korea Inserm, U1016, Institut Cochin, Paris, France c Cnrs, UMR8104, Paris, France d Universite Paris Descartes, Sorbonne Paris cite, Paris, France b
a r t i c l e
i n f o
Article history: Received 7 November 2011 Available online 15 November 2011 Keywords: AMPK CCCP Autophagy Mitophagy mTORC1
a b s t r a c t AMP-activated protein kinase (AMPK) is an important sensor of cellular energy status, and is involved in cell growth and autophagy through mammalian target of rapamycin complex 1 (mTORC1). Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, leads to AMPK activation and Parkin-dependent mitophagy, respectively. However, the detailed biochemical mechanism of how CCCP induces autophagy or mitophagy has not been investigated yet. Here, we showed that CCCP inhibits mTORC1 independently of AMPK, although CCCP induces AMPK activation. Using wild type (WT) and AMPKa1/a2 double knockout (DKO) MEFs, we observed that CCCP promotes endogenous LC3 lipidation and formation of RFPLC3 puncta, indicating autophagosome or autolysosome, in an AMPK-independent manner. Finally, we also revealed that the percentage of CCCP-dependent colocalization between mitochondria and RFP-LC3 puncta is similar both in WT and AMPKa1/a2 DKO MEFs. Based on these data, we concluded that AMPK is not essential in regulation of CCCP-induced autopahgy including mitophagy. Ó 2011 Elsevier Inc. All rights reserved.
1. Introduction AMP-activated protein kinase (AMPK) plays crucial roles in cell growth and energy homeostasis including autophagy and metabolism [1]. The most well-documented mechanism by which AMPK controls cell growth is through downregulation of mTORC1 (mammalian target of rapamycin complex 1) activity [2]. One mechanism of AMPK-dependent mTORC1 inhibition is by phosphorylation of TSC2 [3] and the other is directly by phosphorylation of Raptor, one of mTORC1 subunits [4]. AMPK also controls autophagy either indirectly by negatively regulating mTORC1 or directly by phosphorylating ULK1, a downstream target of mTORC1 and a crucial inducer of autophagy [5–7]. Autophagy includes non-selective autophagy and selective autophagy such as mitophagy known for the removal of depolarized mitochondria [8]. However, at present, the precise relationship between autophagy and mitophagy remains to be further investigated. It has been shown that carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial protonophore, leads to Parkin-dependent mitophagy [9,10]. Interestingly a recent study showed that CCCP promotes mitophagy through ULK1 [11]. However, the detailed molecular mechanism on CCCP-induced mitophagy is little revealed. In addition, as CCCP enhances activation ⇑ Corresponding author. Fax: +82 42 350 8160. E-mail address: [email protected] (O.J. Yoo). 0006-291X/$ - see front matter Ó 2011 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2011.11.038
of AMPK and inhibition of mTORC1, respectively [12,13], we hypothesized that CCCP-dependent AMPK signaling affects autophagy including mitophagy. In this study, we examined whether CCCP-induced alteration of mTORC1 activity is mediated by AMPK. Moreover, we evaluated whether CCCP treatment triggers autophagy and/or mitophagy in an AMPK-dependent manner. Our results unexpectedly suggested that CCCP inhibits mTORC1 activity and induces autophagy including mitophagy in an AMPK-independent manner. 2. Materials and methods 2.1. Reagents and antibodies CCCP and DMSO were purchased from Calbiocam and Sigma– Aldrich, respectively. Anti-c-myc antibody (9E10) and beta-tubulin mouse antibody (E7) were from Developmental Studies Hybridoma Bank. Most phospho-specific antibodies were from Cell Signaling Technology. Anti-LC3 antibody was from Abgent. 2.2. Cell culture and transfection Wild type (WT) and AMPKa1/a2 double knockout (DKO) mouse embryonic fibroblasts (MEFs) have been described previously [14]. HEK293 and MEF cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% fetal bovine serum (Invitrogen) at
344
K.-Y. Kwon et al. / Biochemical and Biophysical Research Communications 416 (2011) 343–348
37 °C in a humidified atmosphere with 5% CO2, and were transiently transfected using Lipofectamine Plus reagent (Invitrogen).
3. Results and discussion 3.1. Effect of CCCP on mTORC1
2.3. Preparation of cell lysates, immunoprecipitation, and immunoblotting Cell stimulation by DMSO or CCCP treatment was terminated by washing cells with ice-cold PBS. Cell lysates were prepared in Lysis buffer A (20 mM Tris, pH 7.5, 100 mM NaCl, 1 mM EDTA, 2 mM EGTA, 50 mM beta-glycerophosphate, 50 mM NaF, 1 mM sodium vanadate, 2 mM dithiothreitol (DTT), 1 mM phenylmethylsulfonyl fluoride (PMSF), 10 lg/ml leupeptin, 1 lg/ml pepstatin A, and 1% Triton X-100) and subjected to immunoprecipitation and immunoblot analysis according to standard procedures [15]. 2.4. Immunocytochemistry After the indicated treatments, the cells were fixed with 2% paraformaldehyde in PBS, and were subjected to immunofluorescence staining according to a standard protocol [15]. All images were obtained using a laser scanning confocal microscope LSM 710 from Carl Zeiss. 2.5. Quantification and statistical analysis Quantification of RFP-LC3 punctuation and colocalization assay with indicated markers were based on enumeration of the number of RFP-LC3 dots in each cell or the number of co-localized cells in each condition. The statistical analyses were performed using Student’s t-test.
The upstream regulator of CCCP-dependent autophagy or mitophagy remains uncertain [9–11]. Therefore, we investigated whether CCCP affects mTORC1, a crucial upstream regulator of autophagy [16]. To evaluate acute responses of mTORC1, CCCP was treated for maximum 1 h. We checked not only mTORC1 but also mTORC2 activity to distinguish selective inhibition of mTORC1 from non-selective inhibition of PI3K/Akt pathway, and to trace the upstream regulator by CCCP-treatment. In overexpression of Myc-tagged S6 kinase (Myc-S6K) or MycAkt, CCCP treatment of HEK293 cells reduced the phosphorylation of S6K at Thr 389 representing mTORC1 activity, while sustained the phosphorylation of Akt at Ser 473 representing mTORC2 activity (Fig. 1A and B). Similarly, we also investigated the endogenous activities of mTORC1 and mTORC2 (Fig. 1C and D). As a result, CCCP strongly decreased the phosphorylation of S6K at Thr 389 and 4EBP at Thr 37/46, also representing mTORC1 activity, in dose- and timedependent manners. However, CCCP did not affect the phosphorylation of Akt at Ser 473. These data indicated that CCCP downregulates mTORC1, but not mTORC2 activity. Notably, CCCP rather increased the phosphorylation of Akt at Thr 308 representing PDK1 activity. It is consistent with the previous report suggesting that inactivated S6K, via CCCP-dependent mTORC1 inhibition, reduces the phosphorylation of insulin receptor substrate-1 (IRS-1) at Ser 270, thereby increasing the activity of PDK1 [17]. Based on these results, we concluded that CCCP treatment can selectively block mTORC1 activity.
Fig. 1. CCCP decreases mTORC1 activity, but not mTORC2 activity. (A, B) HEK293 cells were transfected with Myc-tagged S6K (A) or Myc-tagged Akt (B) for 24 h, and incubated with DMSO or CCCP (10 lM) for 1 h. Then, the cells were subjected to immunoprecipitation with anti-Myc antibody followed by immunoblot analysis with the indicated antibodies. (C) HEK293 cells were cultured with indicated doses of CCCP for 1 h before cell lysis, and analyzed by immunoblot analysis with the indicated antibodies. (D) HEK293 cell were treated with CCCP (10 lM) for the indicated times before cell lysis, and followed by immunoblot analysis with the indicated antibodies. ⁄ Indicates immunoglobulin heavy chain.
K.-Y. Kwon et al. / Biochemical and Biophysical Research Communications 416 (2011) 343–348
3.2. Requirement of AMPK on CCCP-dependent inhibition of mTORC1 Although our data showed that CCCP selectively inhibits mTORC1, but it has never been determined whether CCCP-dependent AMPK activation is essential for regulating mTORC1 activity. Besides, the biochemical mechanism of how CCCP activates AMPK remains unknown. Therefore we investigated whether CCCP decreases mTORC1 activity in an AMPK-dependent manner using three different methods. Firstly, we used compound C, an AMPK inhibitor, to block CCCPdependent activation of AMPK (Fig. 2A). As expected, CCCP increased the phosphorylation of AMPKa at Thr 172, which indicates its activation, as well as its downstream targets, the phosphorylation of ACC at Ser 79 and Raptor at Ser 792. These AMPK activities were strongly inhibited by co-treatment of compound C with CCCP. However, surprisingly, the inhibited phosphorylation of S6K at Thr 389 and 4EBP at Thr 37/46 by CCCP was not restored by compound C. This unexpected data indicated that compound C inhibits CCCPinduced AMPK activation, but does not affect CCCP-induced
345
mTORC1 inactivation. Secondly, a dominant negative mutant of AMPK (AMPK-DN) was utilized to block AMPK signaling induced by CCCP (Fig. 2B). Similar to the above result (Fig. 2A), expression of AMPK-DN did not affect the reduced phosphorylation of S6K at Thr 389 by CCCP, compared with AMPK–WT expression controls (Fig. 2B). These pharmacological and molecular biological data consistently supported that CCCP-dependent AMPK activation is not enough to downregulate mTORC1 activity, although the inhibitory phosphorylation of Raptor at Ser 792 was strongly induced. Lastly, we evaluated CCCP-induced mTORC1 activity using wild type (WT) and AMPKa1/a2 double knockout (DKO) MEF cells (Fig. 2C). Since AMPKa has two isoforms (AMPKa1 and a2), it is necessary to utilize AMPKa1/a2 DKO cells for blocking AMPK signaling completely [14]. CCCP treatment potently suppressed the phosphorylation of S6K at Thr 389 and 4EBP at Thr 37/46 in AMPKa1/a2 DKO MEFs as well as WT MEFs. Taken together, our data demonstrated that CCCP-dependent inhibition of mTORC1 is in an AMPK-independent manner. Notably, the phosphorylation of Raptor at Ser 792 was abolished by
Fig. 2. CCCP-dependent activation of AMPK is not enough to inhibit mTORC1. (A) HEK293 cells were treated with 20 lM compound C and 10 lM CCCP as indicated for 1 h, and examined by immunoblot analysis with the indicated antibodies. (B) HEK293 cells were cotransfected with Myc-S6K and wild type (WT) or dominant negative (DN) mutant AMPK for 24 h, incubated with DMSO or CCCP (10 lM) for 10 min, and subjected to immunoprecipitation (IP) with anti-Myc antibody. Immunoprecipitates or whole cell lysates (WCL) were immunoblotted as indicated. (C) WT or AMPKa1/a2 DKO MEFs were treated with DMSO or CCCP (10 lM) for 1 h before cell lysis, and immunoblotted with indicated antibodies. ⁄Indicates immunoglobulin heavy chain.
346
K.-Y. Kwon et al. / Biochemical and Biophysical Research Communications 416 (2011) 343–348
CCCP treatment in AMPKa1/a2 DKO MEFs. This result indicated that Raptor phosphorylation by AMPK is not crucial to regulate mTORC1 activity. In addition, we observed that CCCP enhances the phosphorylation of AMPKa at Thr 172, a direct regulation site by LKB1 or CaMKK2 [1], implying that CCCP positively affects the upstream activation process of AMPK. 3.3. Requirement of AMPK on CCCP-dependent autophagy induction Recent evidence suggested that AMPK directly stimulates ULK1 to induce autophagy under various physiologic conditions [4,5]. We sought for the definite evidence whether AMPK activation by CCCP treatment could indeed require autophagy. We first examined autophagy formation in WT and AMPKa1/a2 DKO MEF cells using LC3 as one of reliable markers for autophagy [18]. We found that CCCP treatment increased LC3 lipidation (LC3I to LC3II) and RFP-LC3 punctuation both in WT and AMPKa1/a2 DKO MEFs (Fig. 3A and B). Furthermore, the accumulation level of RFP-LC3 punctuation following CCCP treatment in WT MEFs was not different from that in AMPKa1/a2 DKO MEFs (Fig. 3C). Collectively, these observations demonstrated that CCCP treatment is able to induce autophagy independently of AMPK.
3.4. Requirement of AMPK on CCCP-dependent mitophagy induction It has been proposed that CCCP induces mitophagy in a Parkin-dependent manner [8,9], which is considered to be mediated by autophagy machinery [11,19,20]. Although our data suggested that AMPK is not crucial for CCCP-induced autophagy (Fig. 3), we focused on an interesting report that CCCP treatment induces mitophagy in an ULK1-dependent manner [11]. Because, up to date, the known regulators of ULK1 are mTORC1 and AMPK [5–7], we could not exclude the possibility that AMPK mediates CCCP-dependent mitophagy. Immunostaining with an antibody recognizing MnSOD, a mitochondrial marker, showed that the pattern of CCCP-induced RFPLC3 puncta colocalized with MnSOD in WT MEFs was not different from that in AMPKa1/a2 DKO MEFs (Fig. 4A and C). Because we observed massive cell death following long time CCCP treatment over 12 h, the data showing complete mitochondrial clearance could not be obtained in CCCP-treated WT or AMPKa1/a2 DKO MEFs (data not shown). However, colocalization of intracellular organelles and LC3 puncta definitely indicated autophagosome or autolysosome [18]. Therefore, we could conclude that CCCP-induced colocalization of mitochondria and RFP-LC3 puncta in WT or AMPKa1/a2 DKO MEFs
Fig. 3. CCCP induces autophagy in an AMPK-independent manner. (A) WT or AMPKa1/a2 DKO MEFs were treated with DMSO or CCCP (20 lM) for 4 h as indicated, and analyzed by immunoblotting with indicated antibodies. (B) RFP-LC3 was transfected for 24 h in WT or AMPKa1/a2 DKO MEFs. Then, the cells were incubated with DMSO or CCCP (20 lM) for 4 h, and subjected to immunocytochemistry as described in Section 2. RFP-LC3, red; Hoechst, blue. Original magnification, 1000. (C) The number of RFPLC3 puncta/RFP-positive cell treated with DMSO or CCCP was quantified in WT or AMPKa1/a2 DKO MEFs. The data shown are the means ± SD (n > 100 cells/each condition). (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
K.-Y. Kwon et al. / Biochemical and Biophysical Research Communications 416 (2011) 343–348
347
Fig. 4. CCCP induces mitophagy in an AMPK-independent manner. (A) YFP-Parkin and RFP-LC3 were transfected for 24 h in WT or AMPKa1/a2 DKO MEFs. Then, the cells were incubated with DMSO or CCCP (20 lM) for 8 h, and subjected to immunocytochemistry as described in Section 2. YFP-Parkin, green; RFP-LC3, red; MnSOD, yellow; Hoechst, blue. Original magnification, 1000. (B) Percentages of colocalization between mitochondria and YFP-Parkin with DMSO or CCCP treatment were quantified in WT or AMPKa1/a2 DKO MEFs. (C) Percentages of colocalization between mitochondria and RFP-LC3 punata with DMSO or CCCP treatment were quantified in WT or AMPKa1/a2 DKO MEFs. The data shown are the means ± SD of four different experiments. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this paper.)
were undergoing mitophagy. Furthermore, CCCP-induced YFPParkin translocation at mitochondria made no difference between in WT and AMPKa1/a2 DKO MEFs (Fig. 4B). Taken together, our data strongly supported that CCCP-dependent AMPK activation is not crucial for the induction of mitophagy. ULK1 stimulation by AMPK affects autophagy in vitro and in vivo [5,7]. However, our data indicated that AMPK is dispensable for CCCP-induced autophagy. In addition, we observed that CCCP-induced ULK1 phosphorylation at Ser 555, recently known as a direct regulation site by AMPK [7], is not crucial to induce LC3 lipidation (data not shown). This discrepancy is maybe, at least in cellular level, due to AMPK-independent effects by CCCP. On the other hand, one group reported that CCCP induces mitophagy in an ULK1-dependent manner [11]. In this regard, it is
highly possible that CCCP stimulates ULK1-dependent mitophagy through another unknown regulator, not through AMPK. Recent results suggested that metformin or phenformin, independent of AMPK, inhibited mTORC1 through Rag GTPases [21] or REDD1 [22]. However, the data on AMPK-dependent mTORC1 modulation by metformin or phenformin are still conflicting [4,7,21,22], and the mediator between such pharmacological AMPK stimulators and ULK1 activation has been little revealed. Therefore, more detailed and comprehensive studies are required to find the upstream regulator of ULK1 in CCCP-induced mitophagy. In summary, we demonstrated that mTORC1 inhibition by CCCP is independently of AMPK. We also revealed that CCCP induces autophagy including mitophagy in an AMPK-independent manner. These findings will contribute to the expansion of our knowledge
348
K.-Y. Kwon et al. / Biochemical and Biophysical Research Communications 416 (2011) 343–348
on mitochondrial defects-dependent intracellular signaling pathways for autophagy or mitophagy. Acknowledgments We are grateful to Dr. K.L. Guan for wild type and dominant negative mutant pCDNA3-HA-AMPK. YFP-Parkin and RFP-LC3 were from Dr. R. Youle and Dr. T. Yoshimori through Addgene (www.addgene.org), respectively. References [1] M.M. Mihaylova, R.J. Shaw, The AMPK signalling pathway coordinates cell growth, autophagy and metabolism, Nat. Cell Biol. 13 (2011) 1016–1023. [2] R. Zoncu, A. Efeyan, D.M. Sabatini, MTOR: from growth signal integration to cancer, diabetes and ageing, Nat. Rev. Mol. Cell Biol. 12 (2011) 21–35. [3] K. Inoki, T. Zhu, K.L. Guan, TSC2 mediates cellular energy response to control cell growth and survival, Cell 115 (2003) 577–590. [4] D.M. Gwinn, D.B. Shackelford, D.F. Egan, M.M. Mihaylova, A. Mery, D.S. Vasquez, B.E. Turk, R.J. Shaw, AMPK phosphorylation of Raptor mediates a metabolic checkpoint, Mol. Cell 30 (2008) 214–226. [5] J. Kim, M. Kundu, B. Viollet, K.L. Guan, AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1, Nat. Cell Biol. 13 (2011) 132–141. [6] J.W. Lee, S. Park, Y. Takahashi, H.G. Wang, The association of AMPK with ULK1 regulates autophagy, PLoS ONE 5 (2010) e15394. [7] D.F. Egan, D.B. Shackelford, M.M. Mihaylova, S. Gelino, R.A. Kohnz, W. Mair, D.S. Vasquez, A. Joshi, D.M. Gwinn, R. Taylor, J.M. Asara, J. Fitzpatrick, A. Dillin, B. Viollet, M. Kundu, M. Hansen, R.J. Shaw, Phosphorylation of ULK1 (hATG1) by AMP-activated protein kinase connects energy sensing to mitophagy, Science 331 (2011) 456–461. [8] R.J. Youle, D.P. Narendra, Mechanisms of mitophagy, Nat. Rev. Mol. Cell Biol. 12 (2011) 9–14. [9] D. Narendra, A. Tanaka, D.F. Suen, R.J. Youle, Parkin is recruited selectively to impaired mitochondria and promotes their autophagy, J. Cell Biol. 183 (2008) 795–803. [10] S. Geisler, K.M. Holmstrom, D. Skujat, F.C. Fiesel, O.C. Rothfuss, P.J. Kahle, W. Springer, PINK1/Parkin-mediated mitophagy is dependent on VDAC1 and p62/ SQSTM1, Nat. Cell Biol. 12 (2010) 119–131.
[11] J.H. Joo, F.C. Dorsey, A. Joshi, K.M. Hennessy-Walters, K.L. Rose, K. McCastlain, J. Zhang, R. Iyengar, C.H. Jung, D.F. Suen, M.A. Steeves, C.Y. Yang, S.M. Prater, D.H. Kim, C.B. Thompson, R.J. Youle, P.A. Ney, J.L. Cleveland, M. Kundu, Hsp90– cdc37 chaperone complex regulates ulk1- and atg13-mediated mitophagy, Mol. Cell 43 (2011) 572–585. [12] L.E. McLeod, C.G. Proud, ATP depletion increases phosphorylation of elongation factor eEF2 in adult cardiomyocytes independently of inhibition of mTOR signalling, FEBS Lett. 531 (2002) 448–452. [13] B. Thors, H. Halldorsson, G. Thorgeirsson, Thrombin and histamine stimulate endothelial nitric-oxide synthase phosphorylation at Ser1177 via an AMPK mediated pathway independent of PI3K-Akt, FEBS Lett. 573 (2004) 175–180. [14] K.R. Laderoute, K. Amin, J.M. Calaoagan, M. Knapp, T. Le, J. Orduna, M. Foretz, B. Viollet, 50 -AMP-activated protein kinase (AMPK) is induced by low-oxygen and glucose deprivation conditions found in solid-tumor microenvironments, Mol. Cell. Biol. 26 (2006) 5336–5347. [15] Y. Kim, J. Park, S. Kim, S. Song, S.K. Kwon, S.H. Lee, T. Kitada, J.M. Kim, J. Chung, PINK1 controls mitochondrial localization of Parkin through direct phosphorylation, Biochem. Biophys. Res. Commun. 377 (2008) 975–980. [16] G. Kroemer, G. Marino, B. Levine, Autophagy and the integrated stress response, Mol. Cell 40 (2010) 280–293. [17] J. Zhang, Z. Gao, J. Yin, M.J. Quon, J. Ye, S6K directly phosphorylates IRS-1 on Ser-270 to promote insulin resistance in response to TNF-(alpha) signaling through IKK2, J. Biol. Chem. 283 (2008) 35375–35382. [18] N. Mizushima, T. Yoshimori, B. Levine, Methods in mammalian autophagy research, Cell 140 (2010) 313–326. [19] S. Kawajiri, S. Saiki, S. Sato, F. Sato, T. Hatano, H. Eguchi, N. Hattori, PINK1 is recruited to mitochondria with parkin and associates with LC3 in mitophagy, FEBS Lett. 584 (2010) 1073–1079. [20] D. Narendra, L.A. Kane, D.N. Hauser, I.M. Fearnley, R.J. Youle, P62/SQSTM1 is required for Parkin-induced mitochondrial clustering but not mitophagy; VDAC1 is dispensable for both, Autophagy 6 (2010) 1090–1106. [21] A. Kalender, A. Selvaraj, S.Y. Kim, P. Gulati, S. Brule, B. Viollet, B.E. Kemp, N. Bardeesy, P. Dennis, J.J. Schlager, A. Marette, S.C. Kozma, G. Thomas, Metformin, independent of AMPK, inhibits mTORC1 in a rag GTPasedependent manner, Cell Metab. 11 (2010) 390–401. [22] I. Ben Sahra, C. Regazzetti, G. Robert, K. Laurent, Y. Le Marchand-Brustel, P. Auberger, J.F. Tanti, S. Giorgetti-Peraldi, F. Bost, Metformin, independent of AMPK, induces mTOR inhibition and cell-cycle arrest through REDD1, Cancer Res. 71 (2011) 4366–4372.