biostudies-literatureQi YResearch Grants Council, University Grants Committee127https://www.ebi.ac.uk/biostudies/studies/S-EPMC11933580biostudies-literatureUnknown82(1)Podosomes are adhesion structures with densely-polymerized F-actin. While PI(3,4,5)P3 and Cdc42-GTP are known factors to trigger WASP-mediated actin polymerization at the macrophage podosome, their causal mechanism to activate WASP remains unclear. Here, we demonstrate that spatially elevated Cdc42-GTP is a downstream effector of local PI(3,4,5)P3 production at the podosome. We further examine the expression and distribution of 19 Cdc42 guanine exchange factors (GEFs) and identify VAV1 as the key PI(3,4,5)P3-dependent Cdc42 GEF. VAV1 is spatially enriched at the macrophage podosome, and the association of VAV1 with the membrane plays a critical role in upregulating its GEF activity. Reintroduction of wildtype VAV1, rather than the PI(3,4,5)P3-binding deficient or catalytically dead mutantsCellular and molecular life sciences : CMLSPI(3,4,5)P3-mediated Cdc42 activation regulates macrophage podosome assembly.PMC11933580CRF C7070-22EFGRF 17121022Yu CHQi YfalsePI(3,4,5)P3-mediated Cdc42 activation regulates macrophage podosome assembly.Podosomes are adhesion structures with densely-polymerized F-actin. While PI(3,4,5)P3 and Cdc42-GTP are known factors to trigger WASP-mediated actin polymerization at the macrophage podosome, their causal mechanism to activate WASP remains unclear. Here, we demonstrate that spatially elevated Cdc42-GTP is a downstream effector of local PI(3,4,5)P3 production at the podosome. We further examine the expression and distribution of 19 Cdc42 guanine exchange factors (GEFs) and identify VAV1 as the key PI(3,4,5)P3-dependent Cdc42 GEF. VAV1 is spatially enriched at the macrophage podosome, and the association of VAV1 with the membrane plays a critical role in upregulating its GEF activity. Reintroduction of wildtype VAV1, rather than the PI(3,4,5)P3-binding deficient or catalytically dead mutants2025-01-01T00:00:00Z2025 Mar2025-07-09T03:04:49.273Z2025-07-09T03:04:49.273ZS-EPMC119335804012669310.1007/s00018-025-05664-2