Phosphatidylinositol 4,5-bisphosphate mediates Arl4D self-interaction to promote Pak1 signaling

Self-association by small GTPases on membrane is critical for their signaling output and cellular function. However, a mechanistic understanding of how membrane components regulate this process remains incompletely understood. Here, we show that phosphatidylinositol 4,5-bisphosphate [PI(4,5)P 2 ] promotes Arl4D self-association to potentiate downstream Pak1 signaling. We first show that Arl4D self-association is GTP-dependent and occurs at the plasma membrane. Fibronectin stimulation increases this self-association through two cooperative mechanisms: i) direct binding of PI(4,5)P 2 by Arl4D via a conserved C-terminal polybasic motif, and ii) phosphorylation of Arl4D at Ser144 by its effector kinase Pak1. As a result, Arl4D membrane residency and protein stability are enhanced, with downstream signaling through Pak1 also amplified. Furthermore, pursuing structural prediction using AlphaFold, we generate an Arl4D mutant defective in self-association but retains GTP binding and membrane targeting, and find that this mutant fails to activate Pak1 for cell migration, while forced self-association of this mutant restores these downstream effects. Collectively, our findings reveal how an extracellular matrix cue leads to directional cell migration through Arl4D assembling into signaling-competent multimers at the plasma membrane, with cooperation between lipid recognition and kinase-mediated feedback playing critical roles.