Mechanism of IRSp53 inhibition by 14-3-3

David J Kast,Roberto Dominguez

doi:10.1038/s41467-019-08317-8

Abstract

Filopodia are precursors of dendritic spines and polarized cell migration. The I-BAR-domain protein IRSp53 is a key 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 the structural-functional basis for 14-3-3-dependent inhibition of IRSp53. Phosphoproteomics, quantitative binding and crystallographic studies demonstrate that 14-3-3 binds to two pairs of phosphorylation sites in IRSp53. Using bicistronic expression, we obtain an IRSp53 heterodimer in which only one subunit is phosphorylated, and show that each subunit of IRSp53 independently binds one 14-3-3 dimer. A FRET-sensor assay using natively phosphorylated IRSp53 reveals opposite conformational changes upon binding of activatory (Cdc42, Eps8) or inhibitory (14-3-3) inputs. Finally, we show that 14-3-3 inhibits IRSp53 binding to membranes. Collectively, our findings support a mechanism whereby phosphorylation-dependent inhibition of IRSp53 by 14-3-3 counters membrane binding and interactions with Cdc42 and downstream cytoskeletal effectors.

Highlights

Filopodia are precursors of dendritic spines and polarized cell migration
The I-BAR domain is immediately followed by a partial CRIB motif, which is interrupted by a proline-rich sequence and is referred to as the CRIB-PR domain[10]
Consistent with previous findings[35,41,42], these results indicate that AMPK phosphorylates IRSp53, and that the AMPK phosphorylation sites(s) are at least in part responsible for the binding of [-335]

Summary

Introduction

The I-BAR-domain protein IRSp53 is a key regulator of filopodia dynamics that couples Rho-GTPase signaling to cytoskeleton and membrane remodeling, playing essential roles in neuronal development and cell motility. Our findings support a mechanism whereby phosphorylation-dependent inhibition of IRSp53 by [-3] counters membrane binding and interactions with Cdc[42] and downstream cytoskeletal effectors. A tight correlation between plasma membrane and actin cytoskeleton dynamics is a common feature of many cellular functions, including cell migration, organelle trafficking and endo/exocytosis. Terminal to the membrane-binding BAR domain additional signaling and protein–protein or protein–membrane interaction modules, including SH3, PX, PH, RhoGEF, and RhoGAP domains[1,2]. IRSp53 features an N-terminal I-BAR domain (residues 1-231) implicated in membrane binding[32,33,34].

Methods

Results

Conclusion