Mitophagy selectively eliminates damaged mitochondria through distinct molecular pathways (C. Chen et al., 2024). The PINK1-Parkin pathway, activated by PINK1 accumulation on depolarized mitochondria, involves Parkin recruitment and amplified ubiquitination signals recognized by autophagy receptors (Sarraf et al., 2013, Shimura et al., 2000). Alternative pathways include receptor-mediated mitophagy (e.g., BNIP3/NIX binding to microtubule-associated protein 1 light chain 3 (LC3)) and lipid-mediated mechanisms utilizing externalized cardiolipin (Daido et al., 2004, Kanki, 2010). AMP-activated protein kinase (AMPK) acts as an upstream regulator, phosphorylating ULK1, modulating mitochondrial dynamics, and stabilizing PINK1 (Egan et al., 2011, Pickrell and Youle, 2015). Together, these studies highlight that selective mitochondrial clearance is coordinated by interconnected ubiquitin-, receptor-, and lipid-mediated pathways that are tightly controlled by energy-sensing kinases such as AMPK.

Mitofusin 2 (MFN2), a GTPase on the outer mitochondrial membrane, is a key multifunctional protein whose role in mitophagy extends beyond mitochondrial fusion (Koshiba et al., 2004, Santel and Fuller, 2001). For instance, MFN2 serves as a direct substrate for both PINK1-mediated phosphorylation and Parkin-mediated ubiquitination, key steps that commit the mitochondrion to degradation through the canonical pathway (Y. Chen & Dorn, 2013). Furthermore, as a key component of mitochondria-associated ER membranes (MAMs), MFN2 helps create platforms for autophagosome formation and physically links mitochondrial stress to the autophagy machinery (de Brito and Scorrano, 2008, Hamasaki et al., 2013). These diverse functions establish MFN2 as a pivotal coordinator of mitochondrial fate. However, mechanisms by which MFN2 relays upstream signals remain incompletely understood, particularly in relation to AMPK-dependent signaling. We therefore asked whether MFN2 functions only as a downstream AMPK substrate or can also act upstream to promote AMPK Thr172 phosphorylation and mitochondrial recruitment during mitophagy.

AMPK is a master regulator of cellular energy homeostasis and initiates a core autophagic cascade upon activation. Canonically, AMPK directly phosphorylates the autophagy-initiating kinase ULK1 while simultaneously suppressing the process's key inhibitor, mTORC1, thereby ensuring autophagosome formation (Egan et al., 2011, Roczniak-Ferguson et al., 2012). However, this global activation mechanism does not explain how AMPK specifically targets damaged organelles for degradation. Recent evidence indicates that AMPK's functional specificity is dictated by spatial regulation (Drake et al., 2021, Laker et al., 2017). The targeted translocation of AMPK to the mitochondrial surface, for instance, is emerging as a critical step in distinguish a selective mitophagic response from general autophagy, thus preventing widespread cellular degradation and precisely linking metabolic stress to mitochondrial quality control (Khan and Frigo, 2017, Nichenko et al., 2022). These observations raise the possibility that mitochondrial outer membrane proteins, including MFN2, may help define organelle-specific pools of AMPK that execute selective mitophagy.

A direct link between AMPK's mitochondrial localisation and MFN2 has begun to emerge, establishing an AMPK-MFN2 regulatory axis where activated AMPK phosphorylates MFN2 to prime mitochondria for clearance (Hu et al., 2021, Schmitt et al., 2022). Such a unidirectional model, however, positions MFN2 as a passive downstream substrate, a role inconsistent with the protein's function as a dynamic multifunctional protein. These observations raise the key question of whether the regulation is truly one-way or whether MFN2 can, in turn, regulate AMPK by mediating mitochondrial recruitment and subsequent activation of the kinase. Clues from pharmacological agents like Leflunomide, which concurrently enhance MFN2 expression and AMPK activation, suggest a more intricate relationship may exist, but the underlying molecular mechanism remains unresolved (Ashraf and Kumar, 2022, Yu et al., 2019). We therefore hypothesized that MFN2 is not merely a substrate but also an upstream regulator of AMPK during mitophagy.

Here, we tested the hypothesis in MCF-7 breast cancer cells by combining genetic and pharmacological approaches to modulate MFN2 and AMPK. Our findings identify MFN2 as essential for AMPK phosphorylation and subsequent recruitment to mitochondria, revealing the specific MFN2-p-AMPK interaction as a prerequisite for efficient mitophagy—a process that AMPK activation alone fails to initiate.