The guanine nucleotide exchange factor Arhgef5 plays crucial roles in Src-induced podosome formation.

Podosomes and invadopodia are actin-rich membrane protrusions that play a crucial role in cell adhesion and migration, and extracellular matrix remodeling in normal and cancer cells. The formation of podosomes and invadopodia is promoted by upregulation of some oncogenic molecules and is closely related to the invasive potential of cancer cells. However, the molecular mechanisms underlying the podosome and invadopodium formation still remain unclear. Here, we show that a guanine nucleotide exchange factor (GEF) for Rho family GTPases (Arhgef5) is crucial for Src-induced podosome formation. Using an inducible system for Src activation, we found that Src-induced podosome formation depends upon the Src SH3 domain, and identified Arhgef5 as a Src SH3-binding protein. RNA interference (RNAi)-mediated depletion of Arhgef5 caused robust inhibition of Src-dependent podosome formation. Overexpression of Arhgef5 promoted actin stress fiber remodeling through activating RhoA, and the activation of RhoA or Cdc42 was required for Src-induced podosome formation. Arhgef5 was tyrosine-phosphorylated by Src and bound to Src to positively regulate its activity. Furthermore, the pleckstrin homology (PH) domain of Arhgef5 was required for podosome formation, and Arhgef5 formed a ternary complex with Src and phosphoinositide 3-kinase when Src and/or Arhgef5 were upregulated. These findings provide novel insights into the molecular mechanisms of podosome and invadopodium formation induced by Src upregulation.