Ubiquitylation-dependent Rap2 activation regulates lamellipodia dynamics during cell migration

Cell migration is a complex process hallmarked by front-to-back cell polarity that is established by the highly dynamic actin cytoskeleton. Branched actin polymerization creates a lamellipodium at the leading edge of the cell, while the contractile acto-myosin cytoskeleton is present at the lagging edge. Rap2, a Ras GTPase family member, has previously been reported to localize to the lamellipodium as a result of ubiquitylation by a Rab40-Cullin5 E3 ubiquitin ligase complex (Rab40/CRL5). However, how Rap2 functions and how ubiquitylation targets Rap2 to the lamellipodium remained unclear. Here, we demonstrate that Rap2 is recruited to retracting lamellipodia ruffles where it inhibits RhoA, likely through interactions with ARHGAP29, and regulates lamellipodia dynamics, thus facilitating cell migration. Furthermore, using a variety of genetic and pharmacological techniques, we show that Rab40/CRL5-dependent ubiquitylation is required for guanine-nucleotide-exchange factor (GEF)-dependent Rap2 activation, a necessary step for Rap2 targeting to the lamellipodium membrane. As such, we demonstrate how this unique ubiquitylation and activation of Rap2 regulates lamellipodia actin dynamics during cell migration.