Post-translational Modifications Of BNIP3 And The Regulation On Mitophagy Under Hypoxia

Mitochondria produce ATP using oxygen for cell growth and metabolism through oxidative phosphorylation.Thus changes in oxygen content easily cause mitochondrial dysfunction.When oxygen is scarce,the electron transfer chain generates reactive oxygen species?ROS?and the mitochondrial membrane potential?MTP?falls,which in turn impairs functions of mitochondria.On the other hand,various adaptive responses were activated to maintain cell homeostasis and normal functions under hypoxia.As an important hypoxia adaptive response,mitophagy is a process that eliminates damaged or unwanted mitochondria through the autophagy-lysosome pathway.Under hypoxia,mitophagy maintains redox homeostasis and prevents the programmed cell death by selectively removing and degrading damaged mitochondria.Many studies on mechanisms of hypoxia-induced mitophagy have revealed that some mitochondrial membrane proteins serve as autophagy receptors that recognize and interact with LC3 and mediate mitophagy.BNIP3 is not only an important receptor which is located on mitochondrial outer membrane,but also a best-known target of hypoxia-inducible factor 1?HIF-1?.BNIP3 has been identified at the transcriptional level as being upregulated that mediates mitophagy under hypoxia..However,whether or not that post-translational modifications of BNIP3 take participate in regulation of hypoxia-induced mitophagy remains unknown.In the present study,we investigate the post-translational modifications of BNIP3 and the regulation on mitophagy under hypoxia.1.BNIP3 mediates mitophagy under hypoxiaFirst,we observed the relationship between BNIP3 expression and mitophagy in PC12 and Hela cells under hypoxia via dynamic changes of related molecules.The results showed that BNIP3 was up-regulated and mitophagy was activated under hypoxia,just like previous studies.After exposed to long-term hypoxia,BNIP3 bands were remarkably shifted from 30 kDa to 21.5 kDa on the SDS-PAGE,which was consistent with the inhibition of mitophagy and the increase of cell death.Furthermore,we have proved that BNIP3 is required for the mediation of mitophagy under hypoxia in MEF cells from WT or BNIP3^-/-mutants.2.BNIP3 is regulated by phosphorylation and ubiquitination under hypoxiaBecause multiple bands of BNIP3,we firstly speculated that these bands might come from the fractions cleaved by caspases.Using a combination of TNF-?and CHX to induce apoptosis,we ruled out the presume that BNIP3 was cleaved by cysteine proteases.Unexpectedly,we found that BNIP3 was significantly down-regulated by CHX treatment.Moreover,BNIP3 was proved to be degradated by the ub-proteasome pathway through MG132 plus CHX treatment and BNIP3 ubiquitination was confirmed by the ub assays in vivo and in vitro.The ubiquitination sites of BNIP3were identified by site-directed mutants,and the KX7 and KX11 were determined as the main ubiquitination sites.Although BNIP3 can be ubiquitinated,the ubiquitination of BNIP3 did not affect the band shift.We suspected that phosphorylational modification might be the cause for BNIP3 band shift.,considering phosphorylation is a well-known modification to cause band shift.So,using?-PP or phosphatase inhibitor OA,we demonstrated that multiple bands of BNIP3 were different levelsof BNIP3 phosphorylation.Interestingly,when OA provoked BNIP3 phosphorylation,CHX and MG132 played little effects on BNIP3 expression.These results suggest that BNIP3 ubiquitination may be regulated by its phosphorylation.3.PP1 dephosphorylates BNIP3Because of OA is an inhibitor of phosphatase PP1 and PP2A,we focused on investigating the function of PP1 in regulating BNIP3.?1?First,we found that overexpression PP1A or PP1C resulted in the dephosphorylation of BNIP3.And the immunoprecipitation?IP?experiment showed that BNIP3 associated with PP1 directly.?2?Next,transfection with increasing concentrations of GFP-PP1A/PP1C plasmids caused BNIP3 dephosphorylation and inhibiton of mitophagy in a dose-dependent manner.?3?Finally,using CHX with or without MG132 treatment,we observed that BNIP3 dephosphorylation by PP1 was more easily degraded via the ub-proteasome pathway.4.S60/T66 are the main phosphosites on BNIP3We next searched the“PhosphositesPlus^?”website for the potential phosphorylation sites of BNIP3.Based on the reported proteomics data,We constructed the mutants in which the 12 S/T residues was changed to alanine,respectively,and similar S/T residues mutated to alanine together.Then plasmids were transfected and the migration of BNIP3 protein bands was detected by SDS-PAGE and Western blot.These results showed that S60/T66 were the main phosphosites of BNIP3.S60 residue is the critical and T66 residue is the synergistic phosphorylation site for BNIP3 function.The phosphorylation-disable S60/T66A mutants and the phosphorylation-mimicking S60D or S60E mutants were used to evaluate the effects on BNIP3-mediated mitophagy or the association with BNIP3 ubiquitination.Our results revealed that the phosphorylation of S60/T66 enhanced the stability of BNIP3and promoted the interaction of BNIP3 with LC3 and thus induced mitophagy.In the meanwhile,BNIP3 degradation via the ub-proteasome pathway was inhibited by BNIP3 phosphorylation at S60/T66.5.JNK phosphorylates BNIP3 at S60/T66We subsequently sought to identify which kinase is responsible for the phosphorylation of BNIP3 S60/T66.?1?Since S60/T66 resides within the consensus motif of MAPKs and CDKs,we screened the BNIP3-specific kinases using the inhibitors of MAPKs,CDKs,PKCs or CK2.The results indicated that JNK inhibitor SP600125 predominantly restrained BNIP3 phosphorylation and ERK inhibitor PD184352 suppressed faintly.?2?To confirmed the exact kinase response for BNIP3phosphorylation,we screened the ERK and JNK family using siRNAs targeting specific genes.The data showed that only JNK1 and JNK2 konckdown inhibited BNIP3 phosphorylation.?3?Further,both JNK1 and JNK2 were indentified an interaction with BNIP3 using immunoprecipitation assays,and JNK1 was shown a stronger interaction with BNIP3.?4?Finally,BNIP3 phosphorylation by JNK1/2 at S60/T66 was confirmed by JNK1 rescue experiment.6.Phosphorylation of BNIP3 by JNK1/2 mediates mitophagy under hypoxiaTo investigate the function and mechanism of BNIP3 phosphorylation,JNK1/2knockdown cells were exposed to hypoxia,and detected by SDS-PAGE and Western blot.The results indicated that JNK1/2 mediated mitophagy under hypoxia.Through JNK1 rescue assays,JNK1/2-dependent mitophagy was confirmed that is required for phosphorylation of BNIP3 S60/T66.Furthermore,through co-IP assays,we found that BNIP3 phosphorylation by JNK1/2 enhanced its stability and the interaction with LC3.7.Preliminary study on the role of BNIP3 phosphorylation in hypoxia-relateddiseases.After elucidating the mechanism of JNK phosphorylated BNIP3 mediates mitophagy under hypoxia,we explored the preliminary functions of JNK activity,BNIP3 phosphorylation and mitophagy in neuronal OGD model in vitro and MCAO model in vivo.Our results showed that both OGD and MCAO caused inhibition of JNK activity,reduced BNIP3 phosphorylation,and suppressed mitophagy simultaneously.Taken together,we investigated the phosphorylation and ubiquitination of BNIP3and the regulation of BNIP3 phosphorylation or ubiquitination on mitophagy under hypoxiaWe propose a hypothesis that under hypoxia,JNK is activated to phosphorylate BNIP3 at S60/T66,which enhances its stability and induces mitophagy via enhancing the interaction with LC3,to adapt to hypoxia.After exposed to long-term hypoxia,JNK activity is suppressed and BNIP3 cannot be phosphorylated,while PP1 is activated and dephosphorylates BNIP3 that results in the degradation of BNIP3 via the ub-proteasome pathway.In summary,this study revealed new regulatory mechanisms of BNIP3-mediated mitophagy under hypoxia,and provided a new clue hopefully for investigation of hypoxia associated diseases such as cardiovascular and cerebrovascular diseases or cancer.