Phosphorylation of mitophagic receptor protein Bcl2-like protein 13 plays an important role in maintaining cardiac function under pressure overload

Abstract

Abstract The most prominent role for mitochondria is to supply the cell with metabolic energy in the form of ATP generated by oxidative phosphorylation. Therefore, the quality control of mitochondria is essential for the maintenance of normal cellular functions. Accumulation of damaged mitochondria is characteristic of various diseases including heart failure. Damaged mitochondria are removed by a mitochondria-specific autophagy, called mitophagy. Recently, we identified Bcl2-like protein 13 (Bcl2-L-13) as a mammalian functional homolog of Atg32, which is an essential mitophagy receptor in yeast. Bcl2-L-13, localized in outer mitochondrial membrane, induces mitochondrial fragmentation and mitophagy in HEK293 cells. Bcl2-L-13 induces mitophagy through the binding with microtubule-associated protein 1A/1B-light chain 3 (LC3). We attempted to elucidate the activation mechanism of Bcl2-L-13-mediated mitophagy. Ser272 is close to the LC3 interacting motif in Bcl2-L-13. Overexpression of Bcl2-L-13 induced its Ser/Thr phosphorylation in HEK293 cells. The phosphorylation level of Bcl2-L-13 S272A was significantly attenuated compared with wild-type Bcl2-L-13, suggesting Ser272 is one of the phosphorylation sites. GST-pull down assay revealed that Bcl2-L-13 S272A showed less ability for binding with LC3, while immunocytochemical analysis using anti-ATP synthase and anti-LC3B antibodies showed that the mutant induced less mitophagic activity. Next, we generated sheep polyclonal antibody for the detection of Bcl2-L-13 phosphorylated at Ser272. The antibody recognized wild-type Bcl2-L-13 but not Bcl2-L-13 S272A mutant. The phosphorylation level of Bcl2-L-13 at Ser272 was upregulated in wild-type hearts 4 weeks after transverse aortic constriction (TAC). In order to examine the in vivo role of the phosphorylation, we generated Bcl2-L-13 S272A mutant knock-in mice. The mice showed no abnormal cardiac phenotypes at baseline. However, the knock-in mice showed significantly larger left ventricular end-diastolic and end-systolic dimensions and lower fractional shortening than wild-type mice 4 weeks after TAC. Taken together, the phosphorylation of Ser272 in Bcl2-L-13 regulates its mitophagic activity and plays an important role in maintaining cardiac function under pressure overload. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): British Heart Foundation