Volume 41, Issue 6, Pages 661-671 (March 2011) ERK-MAPK Drives Lamellipodia Protrusion by Activating the WAVE2 Regulatory Complex  Michelle C. Mendoza, E. Emrah Er, Wenjuan Zhang, Bryan A. Ballif, Hunter L. Elliott, Gaudenz Danuser, John Blenis  Molecular Cell  Volume 41, Issue 6, Pages 661-671 (March 2011) DOI: 10.1016/j.molcel.2011.02.031 Copyright © 2011 Elsevier Inc. Terms and Conditions

Molecular Cell 2011 41, 661-671DOI: (10.1016/j.molcel.2011.02.031) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 1 ERK-MAPK Activity Is Required for Protrusion (A) Immunoblot of cell lysates shows the levels of activated phosphorylated ERK and Akt in HMECs expressing empty vector (V) or Rac1Q61L. (B) Graph of average EGF-stimulated HMEC protrusion. Error bars indicate SEM, n = 3, ten cells analyzed per experiment (∗p = 0.01 for RacQ61L cells untreated and pretreated with U0126). (C) Representative cells analyzed in (B). Scale bar indicates 10 μm. (D) Activity maps of PtK1 constitutive protrusion dynamics. Cells were pretreated with DMSO or U0126. The vertical axis indicates distance along the cell edge, which was subdivided into 24 segments of ∼1.7 μm width. The color indicates instantaneous protrusion or retraction velocity, as shown in the accompanying color scale. Molecular Cell 2011 41, 661-671DOI: (10.1016/j.molcel.2011.02.031) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 2 Rac Activity and the WRC Are Required for Lamellipodia Protrusion (A) Phospho-ERK immunoblot of lysates from HMECs expressing empty vector (V) or RacT17N, starved and stimulated with EGF for 5 min. (B) Average EGF-stimulated protrusion and retraction. Error bars indicate SEM, n = 3, ten cells analyzed per experiment (∗p < 0.001 for V versus RacT17N). (C) Immunoblot of WRC components in HMEC/EGFR cells transiently transfected with WRC siRNA, starved, and stimulated with EGF for 5 min. (D) Average EGF-stimulated protrusion and retraction in HMEC/EGFR cells. Error bars indicate SEM, n = 3, ten cells analyzed per experiment (∗p < 0.001 for scrambled versus erk2 and each wrc siRNA). Molecular Cell 2011 41, 661-671DOI: (10.1016/j.molcel.2011.02.031) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 3 Active ERK and WAVE2 Colocalize and Are Required for Lamellipodial Protrusions (A and B) Confocal images of indirect immunofluorescence on HMECs (A) and Cos-7 cells (B) starved and stimulated with EGF for 2.5 min. Phalloidin stained filamentous actin. Scale bar indicates 20 μm. Arrows indicate localization of WAVE2 and doubly phosphorylated ERK at protruding edges. (C) Phospho-ERK to WAVE2 nearest-neighbor distance probability relative to a random distribution. 1 indicates probability is identical to random. Values >1 indicate that finding a nearest point at this distance is more likely than would be seen by chance. Solid blue lines are means, n = 25 cells for both HMEC and Cos7. Highlighted blue band indicates ± 95% confidence interval of the mean. (D) TIRF images of indirect immunofluorescence of HMECs transfected with siRNA, starved, and stimulated with EGF for 5 min. Scale bars indicate 20 μm. Arrow indicates p-ERK and Paxillin staining within the lamellipodium. Arrowheads indicate p-ERK staining in large mature adhesions. Molecular Cell 2011 41, 661-671DOI: (10.1016/j.molcel.2011.02.031) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 4 ERK Phosphorylates WAVE2 and Abi1 (A) 32P-orthophosphate labeling of HMEC/EGFR cells. The same membrane was probed for total Abi1 by anti-Abi1 immunoblot. (B) In vitro ERK2 kinase assay with bacterially purified WRC components coexpressed in 293T cells and purified by FLAG-WAVE2 immunoprecipitation. (C) In vitro ERK2 kinase assay with WAVE2/Abi1 or WRC with variable levels of Nap1/Sra-1 cotransfected in 293T cells and purified by FLAG-WAVE2 immunoprecipitation. (D) Immunoblot of ERK2 immunoprecipitation and in vitro kinase assay using purified WRC as substrate. Molecular Cell 2011 41, 661-671DOI: (10.1016/j.molcel.2011.02.031) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 5 Identification of Multiple ERK Phosphorylation Sites on WAVE2 and Abi1 (A) Immunoblots of FLAG-WAVE2 proteins in growing 293T cells using phospho-WAVE2 antibodies. (B) Immunoblot of endogenous WAVE2 protein following EGF stimulation ± pretreatment with U0126. Aliquots of the same lysates were used for each immunoblot. (C) In vitro kinase assay with bacterially purified GST-ERK2 and GST-Abi1 point mutants. (D) 32P-orthophosphate labeling of Cos-7 cells cotransfected with Abi1-1 siRNA and T7-Abi1. The same membrane was probed for total Abi1 by anti-T7 immunoblot. (E) Immunoblot of T7-Abi1 in growing 293T cells using phospho-S225 Abi1 antibodies. (F) Immunoblot of endogenous Abi1 protein following EGF stimulation ± pretreatment with U0126. Molecular Cell 2011 41, 661-671DOI: (10.1016/j.molcel.2011.02.031) Copyright © 2011 Elsevier Inc. Terms and Conditions

Figure 6 ERK Phosphorylation of WAVE2 and Abi1 Regulates WRC Function (A–C) Immunoblots of coimmunoprecipitated Arp2/3 and actin in FLAG-WAVE2 immunoprecipitations from 293T cells coexpressing the WRC components. WCL denotes whole-cell lysate before immunoprecipitation. (D) Silver stain of purified WRC. (E) Average EGF-stimulated protrusion of microinjected HMEC/EGFR cells. Error bars indicate SEM for three independent experiments, with 15 cells analyzed per experiment (∗p = 0.004 for WT WRC versus A and 0.04 for WT WRC versus D/E-injected cells). Molecular Cell 2011 41, 661-671DOI: (10.1016/j.molcel.2011.02.031) Copyright © 2011 Elsevier Inc. Terms and Conditions