TRIM9-dependent ubiquitination of DCC constrains kinase signaling, exocytosis, and axon branching
AbstractExtracellular 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 paperDCCDeleted in Colorectal CancerTRIMTripartite MotifSFKsrc family kinaseDCCKRnon ubiquitinatable DCC mutantVAMPvesicle associated membrane proteinTRIM9ΔRINGTRIM9 lacking the ubiquitin ligase RING domainTRIM9ΔSPRYTRIM9 variant lacking the DCC-binding SPRY domainTIRFTotal Internal Reflection FluorescencepYphosphotyrosineFAKipharmacological FAK inhibitor 14FRNKFAK related non-kinaseSTX-1Asyntaxin 1AIPimmunoprecipitate