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. 2009 Nov 13;36(3):512-24.
doi: 10.1016/j.molcel.2009.10.024.

Activation of the WAVE complex by coincident signals controls actin assembly

Andres M Lebensohn  1 , Marc W Kirschner
Affiliations

Activation of the WAVE complex by coincident signals controls actin assembly

Andres M Lebensohn et al. Mol Cell. .

Abstract

WAVE proteins link upstream signals to actin nucleation by activating the Arp2/3 complex and are at the core of regulatory pathways driving membrane protrusion. They are found in heteropentameric complexes whose role in regulating WAVE function is presently unclear. Here we demonstrate that purified native WAVE complexes are basally inactive; previous reports of constitutive activity are artifacts of in vitro manipulation. Further, the native complexes are not activated by Rac alone. Activation of the WAVE2 complex requires simultaneous interactions with prenylated Rac-GTP and acidic phospholipids, as well as a specific state of phosphorylation. Together these signals promote full activation in a highly cooperative process on the membrane surface, by inducing an allosteric change in the complex rather than by simple recruitment or by dissociation of the subunits. These results explain how the WAVE complex can integrate coincident signals to promote localized actin nucleation during cell motility.

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Figures

Figure 1
Figure 1. Native WAVE2 and WAVE1 complexes are basally inactive and are not activated by Rac or Nck
(A) Scheme used to purify native WAVE2 complex from pig leukocyte extract and native WAVE1 complex from bovine brain extract. The sucrose gradient step indicated in brackets was only included during purification of the WAVE1 complex. (B) Native WAVE2 complex from pig leukocytes and native WAVE1 complex from bovine brain, resolved on 4–12% Bis-Tris polyacrylamide gels and stained with Coomassie (top panels) or silver (bottom panels). Constituent subunits, identified previously by mass spectrometry, are indicated. (C–G) The activity of WAVE complexes was tested in polymerization assays containing 1 μM actin/pyrene-actin and 30 nM Arp2/3 complex (C–F) or 2 μM actin/pyrene-actin and 60 nM Arp2/3 complex (G). (C) Native WAVE2 complex does not stimulate actin polymerization. The indicated concentrations of WAVE2 complex from pig leukocytes (“WAVE2c”) or isolated recombinant WAVE2 protein (“rWAVE2”) were tested. Recombinant WAVE2 protein activates the Arp2/3 complex as expected. (D) Native WAVE2 complex from pig leukocytes is not activated by Rac2 or Nck. 200 nM constitutively active prenylated Rac2-GTP (“pRac2-GTP”), 200 nM full length Nck or both were added to reactions containing 40 nM WAVE2 complex (“WAVE2c”) as indicated. A reaction containing 16 nM recombinant WAVE2 protein (“rWAVE2”) is shown for reference. (E) Thermal denaturation activates the WAVE2 complex. WAVE2 complex from pig leukocytes was heated for 10 minutes at the indicated temperature, cooled down to room temperature and 40 nM was immediately added to the assay. (F) Native WAVE1 complex is inactive and is not activated by Rac1 or Nck. 20 nM WAVE1 complex from bovine brain (“WAVE1c”) was tested alone or with 200 nM constitutively active unprenylated Rac1-GTP (“uRac1-GTP”), 200 nM full length Nck, or both. Thermal denaturation (57°C) revealed the activity of the WAVE1 complex. (G) Freezing and thawing without cryoprotectants activates the WAVE1 complex. WAVE1 complex from bovine brain was exchanged into buffer XB, freeze thawed directly or after addition of 10% glycerol, and assayed at a final concentration of 20 nM.
Figure 2
Figure 2. Prenylated Rac-GTP and acidic phospholipids activate the WAVE2 complex cooperatively
(A) Native WAVE2 complex immuno-purified from the cytosol of EGF stimulated A-431 cells, resolved on a 4–12% Bis-Tris polyacrylamide gel and stained with Coomassie. The main constituent subunits, identified by mass spectrometry (see Table S1), are indicated. The band labeled “Sra-1*” appears to be Sra-1 isoform 2 based on mass spectrometirc analysis and on its apparent molecular weight. (B–H) The activity of WAVE2 complex immuno-purified from the cytosol of EGF stimulated A-431 cells was tested in polymerization assays containing 1 μM actin/pyrene-actin and 30 nM Arp2/3 complex. (B) Native WAVE2 complex is fully activated by the combination of prenylated Rac1-GTP and PIP3 liposomes, but not by Rac or liposomes alone. Where indicated, the activity of 5 nM WAVE2 complex (“WAVE2c”) was measured in the presence of either 50 nM constitutively active prenylated Rac1-GTP (“pRac1-GTP”), 10 μM (total lipid) PIP3 liposomes composed of 45% (molar fraction) PC, 45% PI and 10% PIP3 (“PIP3”), or both. Actin polymerization induced by 5 nM isolated recombinant WAVE2 protein (“rWAVE2”) is shown for comparison. (C) Activation of WAVE2 complex depends on the nucleotide and prenylation state of Rac1. The activity of 5 nM WAVE2 complex was measured in the presence of 10 μM PIP3 liposomes and where indicated 50 nM of either prenylated Rac1-GDP, unprenylated Rac1-GTP or prenylated Rac1-GTP. (D) Prenylated Rac2-GTP, but not Cdc42, can activate the WAVE2 complex in conjunction with PIP3 liposomes. The activity of 5 nM WAVE2 complex was measured in the presence of 10 μM PIP3 liposomes and where indicated 50 nM of either prenylated Cdc42-GTPγS, prenylated Rac2-GDP, unprenylated Rac2-GTP, or prenylated Rac2-GTP. (E) Acidic phospholipids other than PIP3 can activate native WAVE2 complex in conjunction with prenylated Rac1-GTP. The activity of 5 nM WAVE2 complex was measured in the presence of 50 nM prenylated Rac1-GTP and where indicated 10 μM (total lipid) liposomes composed of either PC:PE, PC:PS (both 50:50 molar percentage), PC:PI (45:55 molar percentage), PC:PI:PI(3,5)P2, PC:PI:PI(4,5)P2 or PC:PI:PI(3,4,5)P3 (all 45:45:10 molar percentage). (F) Activation of native WAVE2 complex depends on acidic lipid concentration. Blue squares: maximum actin polymerization rate was measured in reactions containing 5 nM WAVE2 complex, 12.5 nM prenylated Rac1-GTP (“pRac1-GTP”) and 10 μM (total lipid) liposomes containing varying molar fractions of PIP3 (percentage PIP3 is indicated on the x-axis; all liposomes contain 25% PI and the remainder is PC). The data was fitted by least squares to the Langmuir isotherm to obtain the PIP3 concentration required for half maximal activation, 6.1%. Green circle: in the absence of Rac, no activity was observed with 10 μM liposomes containing 30% PIP3. Red triangle: a reaction containing 5 nM WAVE2 complex but no agonists is shown for reference. (G) Rac alone is not sufficient to activate native WAVE2 complex; activation requires acidic phospholipids and depends on the concentration of prenylated Rac-GTP. Maximum actin polymerization rate was measured in reactions containing 5 nM WAVE2 complex and various concentrations of prenylated Rac1-GTP (“pRac1-GTP”), unprenylated Rac1-GTP (“uRac1-GTP”) or unprenylated Rac1-GMPPNP (“uRac1-GMPPNP”) in the absence of lipids. For prenylated Rac1-GTP, titrations were also done together with 10 μM (total lipid) liposomes containing 30% (molar fraction) PIP3, 45% PC and 25% PI (“30% PIP3 liposomes”), or 10% PIP3, 45% PC and 45% PI (“10% PIP3 liposomes”). To the right of the hatched mark the x-axis is in log 10 scale. (H) Activation of native WAVE2 complex by prenylated Rac1-GTP and PIP3 liposomes is highly cooperative. Varying concentrations of WAVE2 complex in combination with 50 nM prenylated Rac1-GTP and 10 μM PIP3 liposomes were assayed for stimulation of actin polymerization. The maximum polymerization rate was plotted as a function of the concentration of WAVE2 complex (duplicates are shown for some concentrations) and the data was fitted to the Hill equation as described in supplemental experimental procedures to obtain the Hill coefficient.
Figure 3
Figure 3. Phosphorylation is required to activate the WAVE2 complex
(A, E–G) The activity of native WAVE2 complex from A-431 cells was tested in polymerization assays containing 1 μM actin/pyrene-actin and 30 nM Arp2/3 complex. (A) Native WAVE2 complexes (5 nM) purified from either serum starved (SS) or EGF stimulated A-431 cells are inactive and can be activated equally by the combination of prenylated Rac1-GTP (50 nM) and PIP3 liposomes (10 μM). (B) Native WAVE2 complexes immuno-purified from serum starved (SS) or EGF stimulated A431 cells, resolved on a 4–12% Bis-Tris polyacrylamide gel and stained with Coomassie. The reduced electrophoretic mobility of WAVE2 in the EGF lane is indicated with an arrow. (C) Native WAVE2 complex immuno-purified from EGF stimulated cells was treated with phosphatase buffer (“---“), lamda protein phosphatase (“λ”) or PP2A. The eluted complexes were resolved on a 4–12% Bis-Tris polyacrylamide gel and stained with Coomassie. (D) Diagram of human WAVE2, Abi-1 and Abi-2 indicating phosphorylated serine (pS) and threonine (pT) residues identified by mass spectrometric analysis of complexes purified from A-431 cells. Residues in yellow boxes were identified in both serum starved and EGF stimulated conditions, residues in orange boxes were identified only in serum starved conditions and residues in green boxes were identified only in EGF stimulated conditions. Residues marked by * could not be assigned unequivocally based on peptide fragmentation data, and equally likely alternative assignments are indicated in Table S2. Abbreviations for protein domains are as follows: WHD, WAVE homology domain; B, basic region; Pro-rich, proline rich region; V, verprolin homology motif; C, central motif; A, acidic motif; WAB, WAVE binding domain; PP, polyproline stretch; SH3, src homology 3 domain. (E) Phosphatase treatment abolishes the ability of prenylated Rac2-GTP and PIP3 liposomes to activate native WAVE2 complex purified from serum starved A-431 cells. WAVE2 complex was treated with phosphatase buffer (control), 0.5 units of PP2A, 1 unit of PP2A, or 1 unit of PP2A pre-incubated with okadaic acid (OA) (see supplemental experimental procedures for details) as indicated and activation of 5 nM WAVE2 complex was tested in the presence of prenylated Rac2-GTP (50 nM) and PIP3 liposomes (10 μM). Control treated WAVE2 complex in the absence of activators is shown for reference. (F) Treatment with PP2A does not affect the intrinsic capacity of the WAVE2 protein to stimulate Arp2/3 dependent actin polymerization. Native WAVE2 complexes purified from serum starved A-431 cells were treated as in (D), heated for 10 minutes at 57°C, and 2 nM was tested in the absence of activators. (G) Treatment with PP2A has no effect on the WAVE2 complex in the absence of activators. Native WAVE2 complexes purified from serum starved A-431 cells were treated as in Figure 3E, and 5 nM was tested in the absence of activators.
Figure 4
Figure 4. Activation of the WAVE2 complex takes place on the membrane surface without dissociation of constituent subunits
(A) Actin polymerization induced by WAVE2 complex activation takes place on the membrane surface. Fluorescence images of a reaction where 5 nM WAVE2 complex from serum starved A-431 cells was activated by 50 nM prenylated Rac2-GTP and 2.5 μM liposomes composed of PC, PI, PIP3 and lissamine rhodamine labeled phosphatidylethanolamine (RhPE) (44:45:10:1 molar percentage) in an assay containing 0.7 μM actin, 0.2 μM pyrene-actin, 0.1 μM Alexa 488 actin and 30 nM Arp2/3 complex. The two left panels show maximum intensity projections of confocal z-stacks, and the right panel shows a pseudo-colored, merged image. Image acquisition and processing details are described in supplemental experimental procedures. Scale bar = 5 μm. (B–C) Liposome co-sedimentation assays contain 30 nM Arp2/3 complex, 5 nM WAVE2 complex from serum starved A-431 cells, 20 μM PIP3 liposomes composed of PC:PI:PIP3:RhPE (44:45:10:1 molar percentage) and 50 nM prenylated Rac1-GTP. (B) Activation of the WAVE2 complex does not lead to dissociation of constituent subunits. Liposome co-sedimentation assays performed under activating conditions in the presence of PIP3 liposomes and prenylated Rac1-GTP were analyzed by Western blot for Pir121, Nap 1, WAVE2, Abi-1, Arp2 and Rac1. Sequential two-fold dilutions of the input, supernatant and pellet were loaded (the sequential dilutions of the pellet are 2× concentrated relative to the input and supernatant). The stoichiometry (relative to WAVE2) of constituent subunits was calculated from two independent experiments based on the relative amounts of the input signal present in the pellets, and is shown to the right of each blot. MW denotes a lane used for molecular weight standards. (C) Solubilization of the membrane during activation releases intact WAVE2 complex. The liposome pellet from an assay like the one in part (B) was resuspended and solubilized with 1% Triton X-100, clarified by centrifugation, and WAVE2 was immunoprecipitated. Sequential two-fold dilutions of the IP input, supernatant and beads were analyzed by Western blot for Pir121, Nap 1, WAVE2, Abi-1, HSPC300 and Rac1. The stoichiometry of constituent subunits was determined as in part (B) and is shown to the right of each blot (* the stoichiometry of Nap 1 could not be determined due to the low signal to background ratio).
Figure 5
Figure 5. Model for WAVE2 function
Counterclockwise from top right, the WAVE2 complex is intrinsically inactive, and must be phosphorylated before it can be activated. However, phosphorylation by itself does not activate the complex. Phosphorylated WAVE2 complex can bind acidic phospholipids, including PIP3, or prenylated Rac-GTP, but binding to either is not sufficient for activation. Binding to both acidic phospholipids and prenylated Rac-GTP activates the WAVE2 complex, most likely though allosteric changes, leading to Arp2/3 dependent actin polymerization. Although PIP3 is most potent, other acidic phospholipids can also activate the complex together with prenylated Rac-GTP. Cooperative association of multiple complexes on the membrane results in greatly enhanced actin nucleation. Key is at the top left corner of the diagram.
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