E3 Ubiquitin Ligase Highwire/Phr1 Mediates Endocytic Control of JNK Signaling in Drosophila Neurons

Endocytosis is essential for synaptic membrane retrieval and the trafficking of growth-regulatory signaling components, yet the mechanistic links between endocytic dysfunction and synaptic overgrowth have remained elusive. Here, we uncover a previously unrecognized mechanism by which endocytosis constrains synaptic growth by regulating the neuronal E3 ubiquitin ligase Highwire (Hiw/Phr1), a key negative regulator of the c-Jun N-terminal Kinase (JNK) signaling pathway. We show that loss of endocytic proteins causes Hiw/Phr1 to accumulate in the neuronal cell bodies, leading to elevated MAP3K Wallenda (Wnd)/DLK levels and hyperactivation of JNK signaling. The accumulated Hiw/Phr1 assembles into dynamic punctate structures, as revealed by their rapid and reversible dissolution with 1,6-hexanediol (1,6-HD). Acute blockade of endocytosis using a conditional temperature-sensitive dynamin mutant Shibirets similarly triggered robust Hiw accumulation at restrictive temperatures. We further demonstrate that Rab11-positive recycling endosomes are essential for proper Hiw localization and turnover, directly linking endosomal trafficking to the control of JNK signaling. Finally, we show that both the Bone Morphogenetic Protein (BMP) pathway and JNK signaling are necessary and sufficient to guide synaptic morphogenesis at the Drosophila neuromuscular junction (NMJ), thereby integrating endocytic trafficking with synaptic growth signaling. Our findings establish endocytosis as a critical regulator of Hiw/Phr1-dependent JNK signaling, with implications that extend beyond synaptic morphogenesis to axon injury and degeneration pathways.