ERK-MAPK drives lamellipodia protrusion by activating the WAVE2 regulatory complex

Abstract

Cell movement begins with a leading edge protrusion, which is stabilized by nascent adhesions and retracted by mature adhesions. The ERK-MAPK (extracellular signal-regulated kinase-mitogen-activated protein kinase) localizes to protrusions and adhesions, but how it regulates motility is not understood. We demonstrate that ERK controls protrusion initiation and protrusion speed. Lamellipodial protrusions are generated via the WRC (WAVE2 regulatory complex), which activates the Arp2/3 actin nucleator for actin assembly. The WRC must be phosphorylated to be activated, but the sites and kinases that regulate its intermolecular changes and membrane recruitment are unknown. We show that ERK colocalizes with the WRC at lamellipodial leading edges and directly phosphorylates two WRC components: WAVE2 and Abi1. The phosphorylations are required for functional WRC interaction with Arp2/3 and actin during cell protrusion. Thus, ERK coordinates adhesion disassembly with WRC activation and actin polymerization to promote productive leading edge advancement during cell migration.

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 Rac1^Q61L. (B) Graph of average EGF-stimulated HMEC protrusion. Error bars indicate SEM, n=3, 10 cells analyzed per experiment (* p=0.01 for Rac^Q61L cells untreated and pre-treated with U0126). (C) Representative cells analyzed in (B). Scale bar indicates 10 μm. (D) Activity maps of PtK₁ constitutive protrusion dynamics. Cells were pre-treated with DMSO or U0126. The vertical axis indicates distance along the cell edge, which was sub-divided into 24 segments of ~1.7 μm width. The color indicates instantaneous protrusion or retraction velocity, as shown in the accompanying color scale.

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 Rac^T17N, starved and stimulated with EGF for 5 min. (B) Average EGF-stimulated protrusion and retraction. Error bars indicate SEM, n=3, 10 cells analyzed per experiment (* p<0.001 for V versus Rac^T17N). (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, 10 cells analyzed per experiment (* p<0.001 for scrambled versus erk2 and each wrc siRNA).

Figure 3. Active ERK and WAVE2 Co-localize and Are Required for Lamellipodial Protrusions

Confocal images of indirect immunofluorescence on (A) HMECs and (B) Cos-7 cells 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 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 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.

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 co-expressed 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 co-transfected in 293T cells and purified by FLAG-WAVE2 immunoprecipitation. (D) Immunoblot of ERK2 immunoprecipitation and in vitro kinase assay using purified WRC as substrate.

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 +/− pre-treatment 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 co-transfected 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 +/− pre-treatment with U0126.

Figure 6. ERK Phosphorylation of WAVE2 and Abi1 Regulates WRC Function

(A) (B) and (C) Immunoblots of co-immunoprecipitated Arp2/3 and actin in FLAG-WAVE2 immunoprecipitations from 293T cells co-expressing 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 3 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).