GRAF1 integrates PINK1-Parkin signaling and actin dynamics to mediate cardiac mitochondrial homeostasis

Abstract

The serine/threonine kinase, PINK1, and the E3 ubiquitin ligase, Parkin, are known to facilitate LC3-dependent autophagosomal encasement and lysosomal clearance of dysfunctional mitochondria, and defects in this process contribute to a variety of cardiometabolic and neurological diseases. Although recent evidence indicates that dynamic actin remodeling plays an important role in PINK1/Parkin-mediated mitochondrial autophagy (mitophagy), the underlying signaling mechanisms remain unknown. Here, we identify the RhoGAP GRAF1 (Arhgap26) as a PINK1 substrate that regulates mitophagy. GRAF1 promotes the release of damaged mitochondria from F-actin anchors, regulates mitochondrial-associated Arp2/3-mediated actin remodeling and facilitates Parkin-LC3 interactions to enhance mitochondria capture by autophagosomes. Graf1 phosphorylation on PINK1-dependent sites is dysregulated in human heart failure, and cardiomyocyte-restricted Graf1 depletion in mice blunts mitochondrial clearance and attenuates compensatory metabolic adaptations to stress. Overall, we identify GRAF1 as an enzyme that coordinates cytoskeletal and metabolic remodeling to promote cardioprotection.

GRAF1 Acts as a Downstream Mediator of Parkin to Regulate Mitophagy in Cardiomyocytes

“…We recently showed that GRAF1 promotes the release of damaged mitochondria from F-actin anchors, regulates mitochondrial-associated Arp2/3-mediated actin remodeling, and facilitates Parkin-LC3 interactions to enhance mitochondria capture by autophagosomes [ 39 ]. Our initial insight into GRAF1′s function largely centered on its ability to serve as a RhoA GAP to regulate actin remodeling-dependent mitophagy in Hela cells.…”

“…As noted above, we observed a distinct GRAF1 band shift following OA treatment, suggesting a post-translational modification ( Figure 3 A, green arrow). Our previous study identified S668, T670, and S671 as the major GRAF1 phosphorylation sites mediated by the PINK1-Parkin pathway [ 39 ]. Notably, these amino acids exhibit partial conservation in other GRAF family members.…”

“…In accordance with the methodology detailed in the provided reference [ 39 ], GRAF1flox/+ mice were initially generated. Subsequently, crossbreeding was carried out between GRAF1flox/+ mice and hemizygous αMHC-MerCreMer (MCM) mice, resulting in the production of αMHC-MCM/+: GRAF1flox/+ mice.…”

“…The purification of anti-phospho-peptide antibodies involved the use of two peptide columns—a non-phospho-peptide column to eliminate antibodies recognizing total protein and a phospho-peptide column for the affinity purification of phospho-specific antibodies. The purification was performed as described in references [ 39 , 40 , 41 ].…”

“…The N-terminal BAR and PH domains induce membrane curvature and determine binding specificity, and the C-terminal SH3 domain mediates protein-protein interactions with partners that control endocytic recycling, adhesion, and/or branched actin remodeling (e.g., dynamin, focal adhesion kinase, Abi2) [ 34 , 35 , 36 , 37 , 38 ]. Interestingly, we recently reported that GRAF1 plays a critical role in regulating PINK1/Parkin-dependent mitophagy by regulating precise spatial and temporal control of actin remodeling [ 39 ]. Notably, we demonstrated using a Hela cell model that GRAF1 plays a role in facilitating F-actin de-polymerization induced by mitochondrial poisons.…”