Abstract
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.
- DCC
- Deleted in Colorectal Cancer
- TRIM
- Tripartite Motif
- SFK
- src family kinase
- DCCKR
- 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