Ubiquitination of the actin polymerase VASP

Filopodia are dynamic, actin-rich structures that extend outward from the cell to explore and respond to cues in the local environment. The actin polymerase VASP is a component of the filopodial tip complex, where it regulates actin polymerization and filopodial dynamics. Previously, we showed that VASP transiently co-localizes with the brain-enriched E3 ubiquitin ligase TRIM9 at the tips of neuronal filopodia. TRIM9 was required for the reversible, non-degradative ubiquitination of VASP and this modification was associated with decreased filopodia number and stability. Furthermore, the axon guidance cue netrin promoted deubiquitination of VASP. We hypothesize mono or multi-monoubiquitination of VASP is a mechanism to negatively regulate actin dynamics by blocking VASP and actin interactions. To determine the lysine residues that are ubiquitinated in VASP, we immunoprecipitated exogenously expressed human VASP from HEK293 cells. The immunopurified protein was analyzed by mass spectrometry to identify post-translational modifications. We identified numerous lysine residues that are ubiquitinated in VASP, including a high prevalence at K240 and K286.