The Rho‐GTPase Rac1 stimulates actin remodelling at the cell periphery by relaying signals to Scar/WAVE proteins leading to activation of Arp2/3‐mediated actin polymerization. Scar/WAVE proteins do not interact with Rac1 directly, but instead assemble into multiprotein complexes, which was shown to regulate their activity in vitro . However, little information is available on how these complexes function in vivo . Here we show that the specifically Rac1‐associated protein‐1 (Sra‐1) and Nck‐associated protein 1 (Nap1) interact with WAVE2 and Abi‐1 (e3B1) in resting cells or upon Rac activation. Consistently, Sra‐1, Nap1, WAVE2 and Abi‐1 translocated to the tips of membrane protrusions after microinjection of constitutively active Rac. Moreover, removal of Sra‐1 or Nap1 by RNA interference abrogated the formation of Rac‐dependent lamellipodia induced by growth factor stimulation or aluminium fluoride treatment. Finally, microinjection of an activated Rac failed to restore lamellipodia protrusion in cells lacking either protein. Thus, Sra‐1 and Nap1 are constitutive and essential components of a WAVE2‐ and Abi‐1‐containing complex linking Rac to site‐directed actin assembly.

Class I phosphoinositide 3-kinases (PI3Ks) are implicated in many cellular responses controlled by receptor tyrosine kinases (RTKs), including actin cytoskeletal remodeling. Within this pathway, Rac is a key downstream target/effector of PI3K. However, how the signal is routed from PI3K to Rac is unclear. One possible candidate for this function is the Rac-activating complex Eps8–Abi1–Sos-1, which possesses Rac-specific guanine nucleotide exchange factor (GEF) activity. Here, we show that Abi1 (also known as E3b1) recruits PI3K, via p85, into a multimolecular signaling complex that includes Eps8 and Sos-1. The recruitment of p85 to the Eps8–Abi1–Sos-1 complex and phosphatidylinositol 3, 4, 5 phosphate (PIP3), the catalytic product of PI3K, concur to unmask its Rac-GEF activity in vitro. Moreover, they are indispensable for the activation of Rac and Rac-dependent actin remodeling in vivo. On growth factor stimulation, endogenous p85 and Abi1 consistently colocalize into membrane ruffles, and cells lacking p85 fail to support Abi1-dependent Rac activation. Our results define a mechanism whereby propagation of signals, originating from RTKs or Ras and leading to actin reorganization, is controlled by direct physical interaction between PI3K and a Rac-specific GEF complex.

Members of a family of intracellular molecular switches, the small GTPases, sense modifications of the extracellular environment and transduce them into a variety of homeostatic signals. Among small GTPases, Ras and the Rho family of proteins hierarchically and/or coordinately regulate signaling pathways leading to phenotypes as important as proliferation, differentiation and apoptosis. Ras and Rho‐GTPases are organized in a complex network of functional interactions, whose molecular mechanisms are being elucidated. Starting from the simple concept of linear cascades of events (GTPase→activator→ GTPase), the work of several laboratories is uncovering an increasingly complex scenario in which upstream regulators of GTPases also function as downstream effectors and influence the precise biological outcome. Furthermore, small GTPases assemble into macromolecular machineries that include upstream activators, downstream effectors, regulators and perhaps even final biochemical targets. We are starting to understand how these macromolecular complexes work and how they are regulated and targeted to their proper subcellular localization. Ultimately, the acquisition of a cogent picture of the various levels of integration and regulation in small GTPase‐mediated signaling should define the physiology of early signal transduction events and the pathological implication of its subversion.