Cdc42hs Facilitates Cytoskeletal Reorganization and Neurite Outgrowth by Localizing the 58-Kd Insulin Receptor Substrate to Filamentous Actin

Abstract

Cdc42Hs is involved in cytoskeletal reorganization and is required for neurite outgrowth in N1E-115 cells. To investigate the molecular mechanism by which Cdc42Hs regulates these processes, a search for novel Cdc42Hs protein partners was undertaken by yeast two-hybrid assay. Here, we identify the 58-kD substrate of the insulin receptor tyrosine kinase (IRS-58) as a Cdc42Hs target. IRS-58 is a brain-enriched protein comprising at least four protein–protein interaction sites: a Cdc42Hs binding site, an Src homology (SH)3-binding site, an SH3 domain, and a tryptophan, tyrptophan (WW)-binding domain. Expression of IRS-58 in Swiss 3T3 cells leads to reorganization of the filamentous (F)-actin cytoskeleton, involving loss of stress fibers and formation of filopodia and clusters. In N1E-115 cells IRS-58 induces neurite outgrowth with high complexity. Expression of a deletion mutant of IRS-58, which lacks the SH3- and WW-binding domains, induced neurite extension without complexity in N1E-115 cells. In Swiss 3T3 cells and N1E-115 cells, IRS-58 colocalizes with F-actin in clusters and filopodia. An IRS-581267N mutant unable to bind Cdc42Hs failed to localize with F-actin to induce neurite outgrowth or significant cytoskeletal reorganization. These results suggest that Cdc42Hs facilitates cytoskeletal reorganization and neurite outgrowth by localizing protein complexes via adaptor proteins such as IRS-58 to F-actin.

RhoG Signals in Parallel with Rac1 and Cdc42

“…On the other hand, RhoG did not bind to a number of other Rac1/Cdc42 effectors that were tested (Pak1, Pak5, Pak6, WASP, Par6, POSH, and IRSp53). IRSp53, which was originally described to link Rac1 to WAVE/ Arp2/3 and lamellipodia formation (49), only bound Cdc42, in agreement with two recent reports (50,51). Our observation that RhoG can activate some Cdc42/Rac1 downstream pathways, but not others, provides further evidence that RhoG does not mediate its cellular effects solely through activation of Cdc42 and Rac1.…”

Dynamin1 Is a Novel Target for IRSp53 Protein and Works with Mammalian Enabled (Mena) Protein and Eps8 to Regulate Filopodial Dynamics

“…Cell Culture, Transfection, and siRNA-N1E-115 neuroblastoma cells were cultured in Dulbecco's modified Eagle's medium (4500 mg/ml glucose) supplemented with 10% fetal bovine serum in a humidified 37°C incubator with 5% CO 2 (25). For transient expression of plasmids, N1E-115 cells were transfected 1 day after seeding using Lipofectamine 2000 (Invitrogen), as described previously (11), or JetPRIME (Polyplus transfection) according to the manufacturer's instructions.…”

“…Neuroblastoma Cells-In our previous work on filopodial formation and neuronal morphology, we detailed the lengths and lifetimes of filopodia in N1E-115 neuroblastoma cells as a model neuronal cell (11,15,25). We took a similar approach using N1E-115 neuroblastoma cells in this study to investigate the role of Dyn in filopodial formation.…”

Functional interactions between phosphatase POPX2 and mDia modulate RhoA pathways

“…WASP interacts directly with CDC42 and activates the ARP2-ARP3 (ARP2/3) complexes to catalyze actin nucleation and polymerization (Miki et al, 1996). Other proteins such as IRSp53 (BAIAP2) and the diaphanous-related-formin DRF3 (DIAPH3) have also been implicated in promoting filopodia formation downstream of CDC42 (Govind et al, 2001;Krugmann et al, 2001;Peng et al, 2003). The WAVE proteins act downstream of RAC1 to modulate the formation of lamellipodia, using IRSp53 as an intermediate (Miki et al, 1998;Miki et al, 2000).…”