A Human Recombinant Antibody Against DRP1 Serine 616 Phosphorylation Enables Detection of BRAFV600E-Associated Mitochondrial Division in Cancer

Abstract

Mitochondria are dynamic organelles that continuously undergo balanced cycles of fusion and division to meet cellular demands. Mitochondrial division is mediated by Dynamin-Related Protein 1 (DRP1), a cytosolic large GTPase whose phosphorylation at serine 616 (DRP1-S616) promotes its translocation to the outer mitochondrial membrane and organelle division. Dysregulated, mitochondrial division disrupts cellular homeostasis and contributes to disease pathogenesis, including cancer. Our prior work demonstrated that the oncogene-induced mitogen-activated protein kinase (MAPK) pathway constitutively phosphorylates DRP1 at serine 616 (DRP1-S616), which is essential to cellular transformation and correlates with oncogene status in patient tissues. Similarly, DRP1-S616 is subject to pharmacologic control by targeted therapies against oncogenic MAPK signaling. Building upon this foundation, we developed a human recombinant monoclonal antibody with high specificity for DRP1-S616, referred to as 3G11. Using diverse biochemical platforms, we demonstrate the robust utility of 3G11 to detect DRP1-S616 in melanoma cell extracts and isolated organelles. Immunofluorescence revealed that pharmacologic inhibition of oncogenic MAPK signaling reduces DRP1-S616 levels which correlates with mitochondrial hyperfusion; while immunohistochemistry showed that elevated DRP1-S616 expression in human tissues correlates with BRAFV600E disease. Together, these findings establish 3G11 as a specific, versatile, renewable, and cost-effective tool for studying mitochondrial division, with strong potential for clinical applications.