Fig 1: ERK3 acts as a nucleation-promoting factor (NPF) in ARP2/3-dependent actin polymerization in vitro and in vivo.(A) Schematic overview of CDC42-WASP-stimulated ARP2/3-dependent actin polymerization based on the cited literature. The process involves ARP2/3 complex, WASP (VCA) as nucleation promoting factor, filamentous actin (F-actin), and monomeric actin (G-actin). In the initial step, CDC42 is activated by GEF-catalyzed exchange of GDP to GTP. Active CDC42 (CDC42-GTP) binds to the GTP-binding domain (GBD) on WASP, thereby displacing the VCA domain. While the V-verpolin-like motif binds actin monomer (G-actin), C-central and A-acidic domains bind and activate the ARP2/3 complex. Conformational changes induced by the binding of the ARP2/3 complex promote its binding to the actin filament, which is strengthened by the additional interaction of the ARP2/3 complex with WASP (VCA)-G-actin. Further conformational changes will secure the ARP2/3 complex on the filament and allow its binding to the actin monomer and the polymerization of the newly nucleated filament. Actin polymerizes at the fast-growing/barbed end, elongating toward the plasma membrane and the ARP2/3 complex would cross-link newly polymerizing filament to the existing filament. (B) ERK3 co-precipitates with active RAC1 and CDC42 in complex with ARP2/3. Active RAC1/CDC42 pull-down was performed using control and ERK3 knockdown human mammary epithelial cells (HMECs). Levels of the active RAC1 and CDC42 were assessed as well as the co-immunoprecipitation levels of ERK3, ARP2, ARP3, and ARPC1A. Levels of the total protein expression were evaluated in the total cell lysates (TCL) and Ponceau S staining was used as a loading control. (C–F) ERK3 regulates F-actin levels in vitro and in vivo. (C) Western blot analyses of control (CRISPR Co) and ERK3-depleted (CRISPR ERK3) HMECs are presented alongside with representative confocal images of F-actin staining. (D, E) In vivo analysis of F- and G-actin levels in HMECs upon ERK3 knockdown. (D) Representative western blot analyses of the enriched F- and G-actin fractions as well as the ERK3 knockdown validation and total actin levels in the TCL are presented. (E) F- and G-actin levels were quantified, and ratios were calculated from five (n = 5) independent experiments and are presented as mean ± SEM; *p<0.0332, **p<0.0021, ***p<0.0002, ****p<0.0001, unpaired t-test. Analyses of ERK3-dependent regulation of F-actin levels in cancerous MDA-MB231 cells is presented in Figure 5—figure supplement 1. Cellular colocalization between endogenous ERK3 and the ARP2/3 was assessed in the absence of CDC42 and is presented in Figure 5—figure supplement 2. (F) Effect of full-length ERK3 on ARP2/3-dependent pyrene actin polymerization was assessed using a pyrene actin polymerization assay. Polymerization induced by the VCA domain of WASP that served as a positive control (green) as well as the ARP2/3 (orange) and ERK3 protein alone (blue) are shown for reference. Actin alone (black) was used to establish a baseline of polymerization. Fluorescence at 360/415 was measured over time and is presented as mean fold change from at least three independent experiments after normalization to the first time point within the respective group. ARP2/3-dependent actin polymerization was measured in the presence of both ERK3 and WASP (VCA) domain, and the results are depicted in Figure 5—figure supplement 3. Figure 5—source data 1.Full membrane scans for western blot images for Figure 5B and C and Figure 4D. Figure 5—source data 2.Prism and Excel file for Figure 5E. Figure 5—source data 3.Prism and Excel file for Figure 5F.