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• W1992080421 abstract "Mitochondria play central roles in integrating pro- and antiapoptotic stimuli, and JNK is well known to have roles in activating apoptotic pathways. We establish a critical link between stress-induced JNK activation, mitofusin 2, which is an essential component of the mitochondrial outer membrane fusion apparatus, and the ubiquitin-proteasome system (UPS). JNK phosphorylation of mitofusin 2 in response to cellular stress leads to recruitment of the ubiquitin ligase (E3) Huwe1/Mule/ARF-BP1/HectH9/E3Histone/Lasu1 to mitofusin 2, with the BH3 domain of Huwe1 implicated in this interaction. This results in ubiquitin-mediated proteasomal degradation of mitofusin 2, leading to mitochondrial fragmentation and enhanced apoptotic cell death. The stability of a nonphosphorylatable mitofusin 2 mutant is unaffected by stress and protective against apoptosis. Conversely, a mitofusin 2 phosphomimic is more rapidly degraded without cellular stress. These findings demonstrate how proximal signaling events can influence both mitochondrial dynamics and apoptosis through phosphorylation-stimulated degradation of the mitochondrial fusion machinery. Mitochondria play central roles in integrating pro- and antiapoptotic stimuli, and JNK is well known to have roles in activating apoptotic pathways. We establish a critical link between stress-induced JNK activation, mitofusin 2, which is an essential component of the mitochondrial outer membrane fusion apparatus, and the ubiquitin-proteasome system (UPS). JNK phosphorylation of mitofusin 2 in response to cellular stress leads to recruitment of the ubiquitin ligase (E3) Huwe1/Mule/ARF-BP1/HectH9/E3Histone/Lasu1 to mitofusin 2, with the BH3 domain of Huwe1 implicated in this interaction. This results in ubiquitin-mediated proteasomal degradation of mitofusin 2, leading to mitochondrial fragmentation and enhanced apoptotic cell death. The stability of a nonphosphorylatable mitofusin 2 mutant is unaffected by stress and protective against apoptosis. Conversely, a mitofusin 2 phosphomimic is more rapidly degraded without cellular stress. These findings demonstrate how proximal signaling events can influence both mitochondrial dynamics and apoptosis through phosphorylation-stimulated degradation of the mitochondrial fusion machinery. Mitofusin 2 is degraded by the ubiquitin-proteasome system in response to stress Mitofusin 2 degradation is dependent on JNK-mediated phosphorylation Phosphorylation results in recruitment of the E3 Huwe1 leading to Mfn2 degradation This enhances mitochondrial fragmentation potentially linking it to cell death Mitochondria are highly dynamic organelles that continually undergo fusion and fission of both their outer and inner membranes. The equilibrium between these processes determines mitochondrial morphology (Bereiter-Hahn and Vöth, 1994Bereiter-Hahn J. Vöth M. Dynamics of mitochondria in living cells: shape changes, dislocations, fusion, and fission of mitochondria.Microsc. Res. Tech. 1994; 27: 198-219Crossref PubMed Scopus (718) Google Scholar, Sesaki and Jensen, 1999Sesaki H. Jensen R.E. Division versus fusion: Dnm1p and Fzo1p antagonistically regulate mitochondrial shape.J. Cell Biol. 1999; 147: 699-706Crossref PubMed Scopus (431) Google Scholar), which in general is a highly interconnected tubular network. The fidelity of mitochondrial membrane fusion and fission is required to maintain the electrochemical gradient necessary for oxidative phosphorylation, for a variety of anabolic processes, for proper DNA inheritance, and for appropriate cellular responses to apoptotic stimuli (Chen et al., 2005bChen H. Chomyn A. Chan D.C. Disruption of fusion results in mitochondrial heterogeneity and dysfunction.J. Biol. Chem. 2005; 280: 26185-26192Crossref PubMed Scopus (1008) Google Scholar, Chen et al., 2010Chen H. Vermulst M. Wang Y.E. Chomyn A. Prolla T.A. McCaffery J.M. Chan D.C. Mitochondrial fusion is required for mtDNA stability in skeletal muscle and tolerance of mtDNA mutations.Cell. 2010; 141: 280-289Abstract Full Text Full Text PDF PubMed Scopus (814) Google Scholar, Lee et al., 2004Lee Y.J. Jeong S.Y. Karbowski M. Smith C.L. Youle R.J. Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 in apoptosis.Mol. Biol. Cell. 2004; 15: 5001-5011Crossref PubMed Scopus (851) Google Scholar, Liesa et al., 2009Liesa M. Palacín M. Zorzano A. Mitochondrial dynamics in mammalian health and disease.Physiol. Rev. 2009; 89: 799-845Crossref PubMed Scopus (685) Google Scholar). Mitochondrial fission and fusion are carried out by distinct machineries, both of which employ large dynamin-like GTPases (Okamoto and Shaw, 2005Okamoto K. Shaw J.M. Mitochondrial morphology and dynamics in yeast and multicellular eukaryotes.Annu. Rev. Genet. 2005; 39: 503-536Crossref PubMed Scopus (594) Google Scholar). In higher eukaryotes, there are two such GTPases that are integral to mitochondrial outer membrane (MOM) fusion, referred to as mitofusin 1 and 2 (Mfn1 and Mfn2) (Rojo et al., 2002Rojo M. Legros F. Chateau D. Lombès A. Membrane topology and mitochondrial targeting of mitofusins, ubiquitous mammalian homologs of the transmembrane GTPase Fzo.J. Cell Sci. 2002; 115: 1663-1674Crossref PubMed Google Scholar). Mfn1 and Mfn2 are essential for mitochondrial fusion and function and form both homo- and heterotypic complexes (Chen et al., 2003Chen H. Detmer S.A. Ewald A.J. Griffin E.E. Fraser S.E. Chan D.C. Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development.J. Cell Biol. 2003; 160: 189-200Crossref PubMed Scopus (1783) Google Scholar, Hoppins et al., 2011Hoppins S. Edlich F. Cleland M.M. Banerjee S. McCaffery J.M. Youle R.J. Nunnari J. The soluble form of Bax regulates mitochondrial fusion via MFN2 homotypic complexes.Mol. Cell. 2011; 41: 150-160Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar, Detmer and Chan, 2007Detmer S.A. Chan D.C. Complementation between mouse Mfn1 and Mfn2 protects mitochondrial fusion defects caused by CMT2A disease mutations.J. Cell Biol. 2007; 176: 405-414Crossref PubMed Scopus (233) Google Scholar). Critical to their activity are their GTPase domains, as well as heptad repeat coiled-coil domains that interact in trans with mitofusin molecules on apposing mitochondria, thereby initiating early events leading to outer membrane fusion (Koshiba et al., 2004Koshiba T. Detmer S.A. Kaiser J.T. Chen H. McCaffery J.M. Chan D.C. Structural basis of mitochondrial tethering by mitofusin complexes.Science. 2004; 305: 858-862Crossref PubMed Scopus (659) Google Scholar). Mfn1 and Mfn2 exhibit ∼60% amino acid identity and appear to function in part through the generation of hetero-oligomers, with wild-type Mfn1 having the capacity to complement disease-associated mutations in Mfn2 (Detmer and Chan, 2007Detmer S.A. Chan D.C. Complementation between mouse Mfn1 and Mfn2 protects mitochondrial fusion defects caused by CMT2A disease mutations.J. Cell Biol. 2007; 176: 405-414Crossref PubMed Scopus (233) Google Scholar). Specific nonredundant roles have been identified, as exemplified by the finding that Mfn1 null embryos are smaller at earlier stages of development, while deletion of Mfn2 results in earlier lethality (Chen et al., 2003Chen H. Detmer S.A. Ewald A.J. Griffin E.E. Fraser S.E. Chan D.C. Mitofusins Mfn1 and Mfn2 coordinately regulate mitochondrial fusion and are essential for embryonic development.J. Cell Biol. 2003; 160: 189-200Crossref PubMed Scopus (1783) Google Scholar). Mfn1 appears to play the dominant role in mitochondrial fusion by effectively tethering mitochondria and exhibiting greater GTPase activity than Mfn2 (Ishihara et al., 2004Ishihara N. Eura Y. Mihara K. Mitofusin 1 and 2 play distinct roles in mitochondrial fusion reactions via GTPase activity.J. Cell Sci. 2004; 117: 6535-6546Crossref PubMed Scopus (509) Google Scholar); Mfn2 has been demonstrated to be more important in differentiated tissues. In particular, Mfn2 is crucial to mitochondrial function in Purkinje cells (Chen et al., 2007Chen H. McCaffery J.M. Chan D.C. Mitochondrial fusion protects against neurodegeneration in the cerebellum.Cell. 2007; 130: 548-562Abstract Full Text Full Text PDF PubMed Scopus (679) Google Scholar), and mutations of Mfn2, primarily in its GTPase domain, are causal for Charcot-Marie-Tooth 2A (CMT2A) neuropathy and can be associated with optic atrophy (Casasnovas et al., 2010Casasnovas C. Banchs I. Cassereau J. Gueguen N. Chevrollier A. Martinez-Matos J.A. Bonneau D. Volpini V. Phenotypic Spectrum of MFN2 Mutations in the Spanish Population.J. Med. Genet. 2010; 47: 249-256Crossref PubMed Scopus (43) Google Scholar, Züchner et al., 2004Züchner S. Mersiyanova I.V. Muglia M. Bissar-Tadmouri N. Rochelle J. Dadali E.L. Zappia M. Nelis E. Patitucci A. Senderek J. et al.Mutations in the mitochondrial GTPase mitofusin 2 cause Charcot-Marie-Tooth neuropathy type 2A.Nat. Genet. 2004; 36: 449-451Crossref PubMed Scopus (1233) Google Scholar). Mfn2 has also been established as having functions that are not obviously related to mitochondrial membrane fusion. These include roles in sequestering p21Ras (Chen et al., 2004Chen K.H. Guo X. Ma D. Guo Y. Li Q. Yang D. Li P. Qiu X. Wen S. Xiao R.P. Tang J. Dysregulation of HSG triggers vascular proliferative disorders.Nat. Cell Biol. 2004; 6: 872-883Crossref PubMed Scopus (327) Google Scholar), maintaining the mitochondrial electrochemical gradient (Pich et al., 2005Pich S. Bach D. Briones P. Liesa M. Camps M. Testar X. Palacín M. Zorzano A. The Charcot-Marie-Tooth type 2A gene product, Mfn2, up-regulates fuel oxidation through expression of OXPHOS system.Hum. Mol. Genet. 2005; 14: 1405-1415Crossref PubMed Scopus (338) Google Scholar), and generating contacts between mitochondria and the endoplasmic reticulum (de Brito and Scorrano, 2008de Brito O.M. Scorrano L. Mitofusin 2 tethers endoplasmic reticulum to mitochondria.Nature. 2008; 456: 605-610Crossref PubMed Scopus (1718) Google Scholar). The function and levels of mitofusins are interwoven with the role of the mitochondria in apoptosis. Results from studies based on Mfn2 overexpression support an antiapoptotic role for this protein (Jahani-Asl et al., 2007Jahani-Asl A. Cheung E.C. Neuspiel M. MacLaurin J.G. Fortin A. Park D.S. McBride H.M. Slack R.S. Mitofusin 2 protects cerebellar granule neurons against injury-induced cell death.J. Biol. Chem. 2007; 282: 23788-23798Crossref PubMed Scopus (160) Google Scholar, Sugioka et al., 2004Sugioka R. Shimizu S. Tsujimoto Y. Fzo1, a protein involved in mitochondrial fusion, inhibits apoptosis.J. Biol. Chem. 2004; 279: 52726-52734Crossref PubMed Scopus (288) Google Scholar). Mitochondrial integrity is critical for preventing the release of cytochrome C, and loss of mitochondrial fusion and generation of fragmented mitochondria correlates with apoptosis. However, the causal role of fragmentation in apoptosis remains unresolved in the literature (Autret and Martin, 2009Autret A. Martin S.J. Emerging role for members of the Bcl-2 family in mitochondrial morphogenesis.Mol. Cell. 2009; 36: 355-363Abstract Full Text Full Text PDF PubMed Scopus (159) Google Scholar, Jahani-Asl et al., 2007Jahani-Asl A. Cheung E.C. Neuspiel M. MacLaurin J.G. Fortin A. Park D.S. McBride H.M. Slack R.S. Mitofusin 2 protects cerebellar granule neurons against injury-induced cell death.J. Biol. Chem. 2007; 282: 23788-23798Crossref PubMed Scopus (160) Google Scholar, Lee et al., 2004Lee Y.J. Jeong S.Y. Karbowski M. Smith C.L. Youle R.J. Roles of the mammalian mitochondrial fission and fusion mediators Fis1, Drp1, and Opa1 in apoptosis.Mol. Biol. Cell. 2004; 15: 5001-5011Crossref PubMed Scopus (851) Google Scholar, Sheridan et al., 2008Sheridan C. Delivani P. Cullen S.P. Martin S.J. Bax- or Bak-induced mitochondrial fission can be uncoupled from cytochrome C release.Mol. Cell. 2008; 31: 570-585Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). An even more complex relationship exists between mitofusins and the proapoptotic members of the Bcl-2 family, Bax and Bak, which have been shown to associate with both Mfn1 and Mfn2 (Brooks et al., 2007Brooks C. Wei Q. Feng L. Dong G. Tao Y. Mei L. Xie Z.J. Dong Z. Bak regulates mitochondrial morphology and pathology during apoptosis by interacting with mitofusins.Proc. Natl. Acad. Sci. USA. 2007; 104: 11649-11654Crossref PubMed Scopus (289) Google Scholar, Karbowski et al., 2006Karbowski M. Norris K.L. Cleland M.M. Jeong S.Y. Youle R.J. Role of Bax and Bak in mitochondrial morphogenesis.Nature. 2006; 443: 658-662Crossref PubMed Scopus (527) Google Scholar, Neuspiel et al., 2005Neuspiel M. Zunino R. Gangaraju S. Rippstein P. McBride H. Activated mitofusin 2 signals mitochondrial fusion, interferes with Bax activation, and reduces susceptibility to radical induced depolarization.J. Biol. Chem. 2005; 280: 25060-25070Crossref PubMed Scopus (263) Google Scholar, Perumalsamy et al., 2010Perumalsamy L.R. Nagala M. Sarin A. Notch-activated signaling cascade interacts with mitochondrial remodeling proteins to regulate cell survival.Proc. Natl. Acad. Sci. USA. 2010; 107: 6882-6887Crossref PubMed Scopus (88) Google Scholar, Hoppins et al., 2011Hoppins S. Edlich F. Cleland M.M. Banerjee S. McCaffery J.M. Youle R.J. Nunnari J. The soluble form of Bax regulates mitochondrial fusion via MFN2 homotypic complexes.Mol. Cell. 2011; 41: 150-160Abstract Full Text Full Text PDF PubMed Scopus (169) Google Scholar). Transcription of Mfn2 is known to be upregulated by peroxisome proliferator-activated receptor γ coactivator-1β (PGC-1 β) in MEFs (Liesa et al., 2008Liesa M. Borda-d'Agua B. Medina-Gómez G. Lelliott C.J. Paz J.C. Rojo M. Palacín M. Vidal-Puig A. Zorzano A. Mitochondrial fusion is increased by the nuclear coactivator PGC-1beta.PLoS ONE. 2008; 3: e3613Crossref PubMed Scopus (142) Google Scholar). However, the posttranslational regulation of Mfn2 remains less clear. Over the last two years, several studies have provided evidence for ubiquitination of mitofusins by Parkin as part of the process leading to autophagy (Poole et al., 2010Poole A.C. Thomas R.E. Yu S. Vincow E.S. Pallanck L. The mitochondrial fusion-promoting factor mitofusin is a substrate of the PINK1/parkin pathway.PLoS ONE. 2010; 5: e10054Crossref PubMed Scopus (364) Google Scholar, Ziviani et al., 2010Ziviani E. Tao R.N. Whitworth A.J. Drosophila parkin requires PINK1 for mitochondrial translocation and ubiquitinates mitofusin.Proc. Natl. Acad. Sci. USA. 2010; 107: 5018-5023Crossref PubMed Scopus (589) Google Scholar, Glauser et al., 2011Glauser L. Sonnay S. Stafa K. Moore D.J. Parkin promotes the ubiquitination and degradation of the mitochondrial fusion factor mitofusin 1.J. Neurochem. 2011; 118: 636-645Crossref PubMed Scopus (193) Google Scholar, Gegg et al., 2010Gegg M.E. Cooper J.M. Chau K.Y. Rojo M. Schapira A.H. Taanman J.W. Mitofusin 1 and mitofusin 2 are ubiquitinated in a PINK1/parkin-dependent manner upon induction of mitophagy.Hum. Mol. Genet. 2010; 19: 4861-4870Crossref PubMed Scopus (682) Google Scholar). A recent study suggests that the role of Parkin at damaged mitochondria is more general and that it is involved in the remodeling of the mitochondrial outer membrane preceding autophagy by stimulating the ubiquitination and proteasomal degradation of proteins with diverse functions in mitochondrial fusion and fission as well as in solute and protein transport (Chan et al., 2011Chan N.C. Salazar A.M. Pham A.H. Sweredoski M.J. Kolawa N.J. Graham R.L. Hess S. Chan D.C. Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy.Hum. Mol. Genet. 2011; 20: 1726-1737Crossref PubMed Scopus (761) Google Scholar). In mammals, Mfn1 levels inversely correlate with expression of the ubiquitin ligase (E3) March V, although a direct link to degradation has not been established (Park et al., 2010Park Y.Y. Lee S. Karbowski M. Neutzner A. Youle R.J. Cho H. Loss of MARCH5 mitochondrial E3 ubiquitin ligase induces cellular senescence through dynamin-related protein 1 and mitofusin 1.J. Cell Sci. 2010; 123: 619-626Crossref PubMed Scopus (172) Google Scholar). Finally, MG132, which inhibits proteasome function, has been shown to increase Mfn2 steady-state levels (Karbowski et al., 2007Karbowski M. Neutzner A. Youle R.J. The mitochondrial E3 ubiquitin ligase MARCH5 is required for Drp1 dependent mitochondrial division.J. Cell Biol. 2007; 178: 71-84Crossref PubMed Scopus (359) Google Scholar). In this study we examine the relationship between Mfn2 and stress-induced apoptosis. We establish that Mfn2 is a substrate for Jun N-terminal kinase (JNK), which becomes activated in response to genotoxic stresses including doxorubicin (DR; Adriamycin) as well as other cellular stresses. Phosphorylation by JNK leads to enhanced ubiquitination and proteasomal degradation of Mfn2. Moreover, we demonstrate that the large HECT domain ubiquitin ligase (E3) Huwe1 is recruited to Mfn2 in response to this phosphorylation and that Huwe1 recruitment is responsible for the accelerated degradation of Mfn2. Further, while loss of Huwe1 expression has been shown to have an antiapoptotic role in response to genotoxic stress (Zhong et al., 2005Zhong Q. Gao W. Du F. Wang X. Mule/ARF-BP1, a BH3-only E3 ubiquitin ligase, catalyzes the polyubiquitination of Mcl-1 and regulates apoptosis.Cell. 2005; 121: 1085-1095Abstract Full Text Full Text PDF PubMed Scopus (673) Google Scholar), this is bypassed by loss of Mfn2 expression, consistent with a causal role for Huwe1-mediated Mfn2 ubiquitination and proteasomal targeting in stress-induced apoptosis. It is well known that treatment of many cell types, including the sarcoma U2OS, with the genotoxic chemotherapeutic agent doxorubicin results in apoptosis. We find that doxorubicin induces fragmentation of mitochondria (Figure 1A ), which first becomes detectable after ∼4 hr of treatment (not shown). To begin to assess whether critical components of the mitochondrial fusion machinery might be altered by this genotoxic stress, we examined changes in levels of Mfn1 and Mfn2. Doxorubicin treatment resulted in loss of Mfn2, but not of Mfn1 (Figure 1B). Similarly, neither the integral mitochondrial protein TOM40 nor the soluble dynamin-like component of the mitochondrial fission apparatus DRP1 decreased. This selectivity excludes mitophagy as an explanation for the loss of Mfn2. Pulse-chase experiments revealed that Mfn2 degradation increases in response to doxorubicin (Figures 1C and 1D). This, together with doxorubicin-induced increase in Mfn2 ubiquitination (Figure 1E), indicates that the decrease in Mfn2 (Figure 1B) is not simply due to known inhibitory effects of doxorubicin on protein expression (Momparler et al., 1976Momparler R.L. Karon M. Siegel S.E. Avila F. Effect of adriamycin on DNA, RNA, and protein synthesis in cell-free systems and intact cells.Cancer Res. 1976; 36: 2891-2895PubMed Google Scholar), but rather is consistent with accelerated degradation of Mfn2 by the ubiquitin-proteasome system (UPS). HeLa cells, which do not express endogenous Parkin, an E3 required for degradation of multiple mitochondrial proteins as part of the process leading to mitophagy (Chan et al., 2011Chan N.C. Salazar A.M. Pham A.H. Sweredoski M.J. Kolawa N.J. Graham R.L. Hess S. Chan D.C. Broad activation of the ubiquitin-proteasome system by Parkin is critical for mitophagy.Hum. Mol. Genet. 2011; 20: 1726-1737Crossref PubMed Scopus (761) Google Scholar), also show enhanced degradation of Mfn2 in response to doxorubicin (Figure S1A). The selective loss of Mfn2 suggests that this might play a causal role in apoptosis in this setting. Alternatively its loss could be secondary to apoptotic changes. To directly assess the role of Mfn2 in apoptosis, we examined caspase activation after Mfn2 knockdown. Loss of Mfn2 alone did not induce apoptosis as assessed by caspase 3 activation; however, it markedly sensitized cells to doxorubicin-induced apoptosis (Figures 1F and 1G; see Supplemental Experimental Procedures for complete list of RNAi reagents used in this study). This sensitivity is reversed by re-expression of Mfn2 (Figure S1B). Consistent with an antiapoptotic role for Mfn2, overexpression of Mfn2 protected cells from UV irradiation comparable to a dominant negative antiapoptotic DRP1 mutant (DRP1K38A) (Frank et al., 2001Frank S. Gaume B. Bergmann-Leitner E.S. Leitner W.W. Robert E.G. Catez F. Smith C.L. Youle R.J. The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis.Dev. Cell. 2001; 1: 515-525Abstract Full Text Full Text PDF PubMed Scopus (1441) Google Scholar) and to the apoptosis inhibitor Bcl-2 (Figure S1C). To explore the regulation of Mfn2, we generated a stable transfectant of U2OS that expresses Mfn2 with C-terminal HA tags (U2OSMfn2HA) at a level that approximates the endogenous protein (Figure S2A). Notably, the levels of endogenous Mfn2 are reduced in all cell lines stably expressing exogenous Mfn2, suggesting that levels of Mfn2 are tightly regulated (data not shown). We then purified Mfn2HA from U2OSMfn2HA cells treated with proteasome inhibitor for mass spectrometry (MS) analysis. Tandem MS analysis showed over 80% coverage of Mfn2, and identified multiple ubiquitination signatures throughout (data not shown). An unanticipated observation was phosphorylation of Ser27. Direct evidence of phosphorylation has not been previously described for components of the mitochondrial fusion apparatus. We found, using Group-based Prediction Software 2.1, that the peptide sequence 25NASPLK30 scored highest for several mitogen-activated protein (MAP) kinase family members involved in stress responses (Xue et al., 2008Xue Y. Ren J. Gao X. Jin C. Wen L. Yao X. GPS 2.0, a tool to predict kinase-specific phosphorylation sites in hierarchy.Mol. Cell. Proteomics. 2008; 7: 1598-1608Crossref PubMed Scopus (533) Google Scholar). To confirm Ser27 phosphorylation, we immunoprecipitated Mfn2 and immunoblotted it with antibody that recognizes S∗PXR/K (∗ denotes Ser phosphorylation) (Ritt et al., 2010Ritt D.A. Monson D.M. Specht S.I. Morrison D.K. Impact of feedback phosphorylation and Raf heterodimerization on normal and mutant B-Raf signaling.Mol. Cell. Biol. 2010; 30: 806-819Crossref PubMed Scopus (163) Google Scholar). A low level of reactivity was observed with proteasome inhibition (MG132) that increased markedly with doxorubicin, which activates multiple MAP kinases (Niiya et al., 2004Niiya M. Niiya K. Shibakura M. Asaumi N. Yoshida C. Shinagawa K. Teshima T. Ishimaru F. Ikeda K. Tanimoto M. Involvement of ERK1/2 and p38 MAP kinase in doxorubicin-induced uPA expression in human RC-K8 lymphoma and NCI-H69 small cell lung carcinoma cells.Oncology. 2004; 67: 310-319Crossref PubMed Scopus (16) Google Scholar) (Figure 2A ). To further validate phosphorylation of Mfn2 Ser27, we generated a Mfn2 phospho-Ser27 polyclonal antibody that was validated using a nonphosphorylatable (S27A) mutant, Mfn2HAS27A (Figure 2B). Using this antibody, we found that Mfn2 phosphorylation peaks at ∼2 hr of treatment with doxorubicin and decreases thereafter (not shown). We also found that stress-induced phosphorylation of Mfn2 Ser27 could be induced in response to other agents including etoposide, 17-AAG, cis-platinum, tunicamycin, and MG132 (Figure S2B). To assess which stress-activated kinases might be involved in Mfn2 phosphorylation, we examined the activation of JNK, ERK, and p38. Doxorubicin activates JNK and p38 (Figure S2C). In contrast, doxorubicin fails to increase the activity of ERKs, which are constitutively activated in these cells, making these less likely to be responsible for the observed phosphorylation of Mfn2. The role of JNK and p38 in doxorubicin-induced phosphorylation of Mfn2 Ser27 was assessed by knockdown of JNK (Figure 2C). JNK knockdown resulted in a loss of Mfn2 phosphorylation, while knockdown of p38 was without effect. The role of JNK was confirmed using distinct siRNAs directed against the 3′UTRs of JNK1 and JNK2 isoforms and re-expression of constitutively active JNK2α2 (Pimienta et al., 2007Pimienta G. Ficarro S.B. Gutierrez G.J. Bhoumik A. Peters E.C. Ronai Z. Pascual J. Autophosphorylation properties of inactive and active JNK2.Cell Cycle. 2007; 6: 1762-1771Crossref PubMed Scopus (13) Google Scholar), which results in Mfn2 phosphorylation (Figures S2D and S2E). These data establish that Mfn2 is phosphorylated on Ser27 in response to a variety of cellular stresses and implicate JNK in this process. Coimmunoprecipitation was carried out to assess the interaction of Mfn2. Mfn2HA specifically associated with FLAG-JNK2α2, whereas GFP, which served as a control, showed no specific binding (Figure 2D). The functional interaction between the two proteins was confirmed by the Ser27 phosphorylation of Mfn2 by recombinant JNK2α2 in vitro (Figures 2E and S2F). To determine whether JNK has a role in doxorubicin-induced Mfn2 degradation, we monitored degradation of Mfn2 after JNK knockdown. This abrogated the doxorubicin-induced accelerated degradation of Mfn2 (Figures 3A and 3B ). To investigate the significance of Ser27 phosphorylation in Mfn2 degradation, we assessed ubiquitination of wild-type (Mfn2HA), Mfn2HAS27A, and a Mfn2 phosphomimic (Mfn2HAS27D). The phosphomimic showed increased ubiquitination, in accord with a proteasome-targeting role for Ser27 phosphorylation (Figure 3C). Consistent with a destabilizing role for phospho-Ser27, Mfn2HAS27D exhibited a marked increase in the rate of degradation compared to Mfn2HAS27A, which was degraded with kinetics resembling Mfn2HA (Figure 3D). However, in response to doxorubicin, WT Mfn2 became less stable and degraded with kinetics resembling the phosphomimic. In contrast, Mfn2HAS27A degradation did not increase with doxorubicin (Figure 3E). Both the baseline degradation (WT and Mfn2HAS27A) and the accelerated degradation (Mfn2HAS27D) were largely prevented by the proteasome inhibitor lactacystin (Figure 3F), indicating UPS-mediated degradation. We next assessed the effect of expressing Mfn2 and its Ser27 mutants on mitochondrial morphology after doxorubicin treatment. Transfection of Mfn2 and mutants reduced mitochondrial fragmentation compared to vector (Figures 4A and 4B ), reinforcing the idea that doxorubicin-induced loss of Mfn2 promotes mitochondrial fission. Furthermore, consistent with its increased stability, Mfn2HAS27A reduced mitochondrial fragmentation more effectively than either wild-type Mfn2HA or the relatively unstable Mfn2HAS27D (Figures 4A and 4B). In accord with these findings, knockdown of JNK1 and JNK2 resulted in a >85% decrease in mitochondrial fragmentation to doxorubicin at 6 hr (data not shown). To determine the role of Ser27 phosphorylation in apoptosis, we assessed the effect of overexpressing Mfn2 Ser27 mutants on doxorubicin-induced cell death. Overexpression of Mfn2 and mutants reduced doxorubicin-induced apoptosis (Figure 4C; see Figure S3 for levels of Mfn2). Mfn2HAS27A was most protective in this assay, inhibiting apoptosis to a similar extent as the dominant negative antiapoptotic DRP1K38A (Frank et al., 2001Frank S. Gaume B. Bergmann-Leitner E.S. Leitner W.W. Robert E.G. Catez F. Smith C.L. Youle R.J. The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis.Dev. Cell. 2001; 1: 515-525Abstract Full Text Full Text PDF PubMed Scopus (1441) Google Scholar) and approaching that seen with overexpression of Bcl-2. In comparison, overexpression of Mfn2HAS27D was less effective (Figure 4C). A similar trend was observed with thapsigargin, which induces endoplasmic reticulum stress, activates JNK (Urano et al., 2000Urano F. Wang X. Bertolotti A. Zhang Y. Chung P. Harding H.P. Ron D. Coupling of stress in the ER to activation of JNK protein kinases by transmembrane protein kinase IRE1.Science. 2000; 287: 664-666Crossref PubMed Scopus (2311) Google Scholar), and can induce cell death. Overexpression of Mfn2HAS27D was ineffective in inhibiting cell death whereas overexpression of Mfn2HAS27A was protective to a similar degree as Bcl-2 and DRP1K38A (Figure 4D). To determine whether Ser27 phosphorylation and the consequent decrease in stability plays a role in sensitizing cells to apoptosis, we examined apoptosis in cells depleted of endogenous Mfn2 but re-expressing WT or Ser27 mutants of Mfn2. Cells expressing Mfn2HAS27D showed increased caspase 3 activation when treated with doxorubicin. In contrast, cells re-expressing Mfn2HAS27A exhibited resistance to doxorubicin-induced caspase 3 activation (Figure 4E), establishing a role for Ser27 phosphorylation in sensitization to stress-induced cell death. Specificity in ubiquitination is conferred by ubiquitin-protein ligases (E3s), which together with ubiquitin-conjugating enzymes (E2s) mediate the transfer of ubiquitin to substrates. Parkin targets mitofusins (Poole et al., 2010Poole A.C. Thomas R.E. Yu S. Vincow E.S. Pallanck L. The mitochondrial fusion-promoting factor mitofusin is a substrate of the PINK1/parkin pathway.PLoS ONE. 2010; 5: e10054Crossref PubMed Scopus (364) Google Scholar, Ziviani et al., 2010Ziviani E. Tao R.N. Whitworth A.J. Drosophila parkin requires PINK1 for mitochondrial translocation and ubiquitinates mitofusin.Proc. Natl. Acad. Sci. USA. 2010; 107: 5018-5023Crossref PubMed Scopus (589) Google Scholar, Glauser et al., 2011Glauser L. Sonnay S. Stafa K. Moore D.J. Parkin promotes the ubiquitination and degradation of the mitochondrial fusion factor mitofusin 1.J. Neurochem. 2011; 118: 636-645Crossref PubMed Scopus (193) Google Scholar, Gegg et al., 2010Gegg M.E. Cooper J.M. Chau K.Y. Rojo M. Schapira A.H. Taanman J.W. Mitofusin 1 and mitofusin 2 are ubiquitinated in a PINK1/parkin-dependent manner upon induction of mitophagy.Hum. Mol. Gen" @default.
• W1992080421 created "2016-06-24" @default.
• W1992080421 creator A5009679775 @default.
• W1992080421 creator A5019010663 @default.
• W1992080421 creator A5021545775 @default.
• W1992080421 creator A5035369750 @default.
• W1992080421 creator A5062953965 @default.
• W1992080421 creator A5068130062 @default.
• W1992080421 creator A5069825701 @default.
• W1992080421 creator A5083498586 @default.
• W1992080421 date "2012-08-01" @default.
• W1992080421 modified "2023-10-14" @default.
• W1992080421 title "Stress-Induced Phosphorylation and Proteasomal Degradation of Mitofusin 2 Facilitates Mitochondrial Fragmentation and Apoptosis" @default.
• W1992080421 cites W1508962729 @default.
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