TRIM9-dependent ubiquitination of DCC constrains kinase signaling, exocytosis, and axon branching

Extracellular netrin-1 and its receptor DCC promote axon branching in developing cortical neurons. Netrin-dependent morphogenesis is preceded by multimerization of DCC, activation of FAK and Src family kinases, and increases in exocytic vesicle fusion, yet how these occurrences are linked is unknown. Here we demonstrate that TRIM9-dependent ubiquitination of DCC blocks the interaction with and phosphorylation of FAK. Upon netrin-1 stimulation TRIM9 promotes DCC multimerization, but TRIM9-dependent ubiquitination of DCC is reduced, which promotes an interaction with FAK and subsequent FAK activation. We found that inhibition of FAK activity blocks elevated frequencies of exocytosis in vitro and elevated axon branching in vitro and in vivo. Although FAK inhibition decreased SNARE-mediated exocytosis, assembled SNARE complexes and vesicles adjacent to the plasma membrane were increased, suggesting a novel role for FAK in the progression from assembled SNARE complexes to vesicle fusion in developing murine neurons. Abbreviations used in this paper DCC Deleted in Colorectal Cancer TRIM Tripartite Motif SFK src family kinase DCC KR non ubiquitinatable DCC mutant VAMP vesicle associated membrane protein TRIM9ΔRING TRIM9 lacking the ubiquitin ligase RING domain TRIM9ΔSPRY TRIM9 variant lacking the DCC-binding SPRY domain TIRF Total Internal Reflection Fluorescence pY phosphotyrosine FAKi pharmacological FAK inhibitor 14 FRNK FAK related non-kinase STX-1A syntaxin 1A IP immunoprecipitate