Mechanism of IRSp53 inhibition by 14-3-3

Filopodia are precursors of dendritic spines and polarized cell migration. The I-BAR-domain protein IRSp53 is an essential regulator of filopodia dynamics that couples Rho-GTPase signaling to cytoskeleton and membrane remodeling, playing essential roles in neuronal development and cell motility. Here, we describe a mechanism whereby phosphorylation-dependent inhibition of IRSp53 by 14-3-3 counters membrane binding and activation by Cdc42 or downstream cytoskeletal effectors. Phosphoproteomics, quantitative binding studies and crystal structures show that 14-3-3 binds to two pairs of phosphorylation sites in IRSp53. Using bicistronic expression we obtained a heterodimer of IRSp53 in which only one subunit is phosphorylated, and show that each subunit of the IRSp53 dimer independently binds a 14-3-3 dimer. A FRET-sensor assay developed using natively phosphorylated and 14-3-3-binding competent IRSp53 purified from mammalian cells reveals opposite conformational changes in IRSp53 upon binding of activatory (Cdc42, Eps8) vs. inhibitory (14-3-3) inputs.