1. Phosphorylation of the WASP-VCA Domain Increases Its Affinity for the Arp2/3 Complex and Enhances Actin Polymerization by WASP 
Giles O.C. Cory, Rainer Cramer, Laurent Blanchoin, Anne J. Ridley 
Molecular Cell 
Volume 11, Issue 5, Pages (May 2003)
DOI: /S (03)

2. Figure 1 Mass Spectrometric Analysis of WASP Phosphorylation
(A) IR-MALDI mass spectrum of 480AIHSSDEGEDQAGDEDEDDEWDDASYTDIEMNRLGK515 and 478SRAIHSSDEGEDQAGDEDEDDEWDDASYTDIEMNRLGK515 from micro-immuno-purified WASP-VSV showing the unmodified and singly- and doubly phosphorylated forms.
(B) Sequence alignment of WASP family proteins CA regions.
Molecular Cell  , DOI: ( /S (03) )

3. Figure 2 SK896 Specifically Recognizes Phospho-WASP and Phospho-N-WASP
(A) Cos-7 cells electroporated with WASP-VSV (W) or EGFp-N-WASP (N) expression vectors were immunoprecipitated (IP) with either anti-VSV or anti-EGFP antibodies. Immune complexes were treated with calf intestinal phosphatase where indicated (CIP) and, together with whole cell lysates (wcl), were resolved by SDS-PAGE and immunoblotted (IB) with anti-WASP antiserum. Membranes were stripped and reprobed with SK896 as indicated.
(B) Cos-7 cells were electroporated with full-length untagged WASP constructs that were either wild-type or mutated as shown (AA represents the double mutant S483A/S484A). Cell lysates were immunoblotted as indicated.
Molecular Cell  , DOI: ( /S (03) )

4. Figure 3
Phospho-WASP and Phospho-N-WASP Are Present in a Number of Cell Types
(A) The human hematopoietic cell lines indicated were grown under normal conditions (G) or were serum starved for 18 hr (S). Cell lysates were normalized for protein concentration, resolved by SDS-PAGE, and immunoblotted with anti-WASP antibody, stripped, and reprobed with SK896.
(B) Lysates from the indicated cell types were normalized for protein concentration and immunoblotted with SK896. Possible WASP degradation (deg.) products are indicated.
Molecular Cell  , DOI: ( /S (03) )

5. Figure 4 CK2 Phosphorylates S483/ S484 In Vivo and In Vitro
(A) U937 cells were treated with the inhibitors staurosporine (St.), 1 μM; GF109203X (GF′X), 1 μM; DRB, 50 μM; LY (LY), 50 μM; Wortmannin (Wort.), 50 nM; or DMSO control for the indicated times. Lysates were analyzed by immunoblotting with anti-WASP antibody, and the membranes stripped and reprobed with SK896.
(B) Bacterially produced GST-WASP-VCA fusion proteins or a GST control was incubated with recombinant CK2 and [γ-32P]ATP. Fusion proteins were resolved by SDS-PAGE, Coomassie blue-stained, dried, and exposed to X-ray film. Degraded GST-VCA fusion protein is indicated (deg.). AA represents the S483A/S484A double-mutant fusion protein.
(C) GST-WASP-VCA fusion proteins were purified from Cos-7 cells and untreated (WT) or incubated with CIP (WT/c). Fusion proteins on GS4B beads were incubated with U937 cell lysate or recombinant CK2 or a combination of both. A lysis buffer control (0) was also performed. Samples were resolved by SDS-PAGE and immunoblotted with SK896 before stripping and reprobing with anti-GST.
(D) Determination of stoichiometry of endogenous WASP phosphorylation. Lysates from the indicated number of U937 cells were separated by SDS-PAGE next to the indicated quantities of purified, phosphorylated GST-WASP. The membrane was probed with α-WASP antiserum, stripped, and reprobed with SK896.
Molecular Cell  , DOI: ( /S (03) )

6. Figure 5
Phosphorylation of the WASP-VCA Domain Enhances Its Binding to Components of the Arp2/3 Complex
(A) GST-WASP-VCA fusion proteins were purified from Cos-7 cells and untreated (WT, AA) or incubated with CIP (WT/c, AA/c). AA represents the S483A/S484A double-mutant fusion protein. Fusion proteins on GS4B beads were incubated with U937 cell lysate (20 × 106 cells in 0.5 ml LB), and complexes were resolved by SDS-PAGE followed by silver staining. Molecular masses of marker proteins are indicated on the right. Px represents protein species of apparent molecular mass x. Rec.GST, recombinant GST.
(B) MALDI-MS identification of VCA domain ligands. Silver stained protein bands were excised from the WT lane of a gel similar to that shown in (A). aProtein (P) mass calculated from protein markers (kDa); bprotein mass calculated from protein database sequence by ProteinProspector; cnumber of matched peptides and their combined sequence coverage for the identified protein of the first search (and second search following “/” for p45).
(C) GST-WASP-VCA fusion proteins were purified from Cos-7 cells and untreated (WT, closed circles; AA, closed squares) or incubated with CIP (WT CIP, open circles; AA CIP, open squares). Indicated amounts of fusion proteins on GS4B beads were incubated with 250 μl U937 cell lysate (2–2.5 mg protein/ml). Complexes were resolved by SDS-PAGE and immunoblotted with anti-Arp3 followed by densitometry. Solid lines are the best fits of Equation 1 to the data from triplicate samples.
(D) Untreated (WT, AA) or CIP treated (WT/c, AA/c) GST-VCA fusion proteins were incubated with lysates of Cos-7 cells expressing WASPΔC-VSV. Complexes were purified using GS4B beads and resolved by SDS-PAGE and immunoblotted as indicated.
Molecular Cell  , DOI: ( /S (03) )

7. Figure 6
Phosphorylation of WASP Enhances Its Ability to Stimulate the Arp2/3 Complex
(A) Effect of phosphorylation on GST-VCA on its ability to stimulate the Arp2/3 complex. Actin polymerization was measured using the change in fluorescence of pyrene-labeled actin with 4 μM actin alone; in presence of 50 nM Arp2/3 complex; or in the presence of 50 nM Arp2/3 complex and 25 nM untreated GST-VCA (WT), GST-VCA (AA), dephosphorylated GST-VCA (WT) CIP, or 25 nM dephosphorylated GST-VCA(AA) CIP.
(B) Effect of phosphorylation on full-length WASP on its ability to stimulate the Arp2/3 complex. Fluorescence was measured in the presence of 4 μM actin alone; in the presence of 50 nM Arp2/3 complex; or in the presence of 50 nM Arp2/3 complex and 15 nM GST-WASP (WT) treated with CK2, 15 nM GST-WASP (AA) CK2, 15 nM dephosphorylated GST-WASP (CIP), 15 nM GST-WASP (AA) CIP, and 200 nM GST-VCA (WT). AA represents the S483A/S484A double-mutant fusion protein.
Molecular Cell  , DOI: ( /S (03) )

8. Figure 7
Phosphorylation of S483/S484 Is Required for Actin Polymerization by WASP in Cell Extracts
GST-WASP-Y291E (E) or GST-WASP-Y291E-S483A/S484A (E/AA) fusion protein attached to GS4B beads was left untreated (−/−), phosphorylated with CK2 (−/CK2), dephosphorylated (CIP/−), or dephosphorylated and then phosphorylated with CK2 (CIP/CK2) before incubation with U937 cell lysate (5 × 106 cells/0.5 ml) supplemented with 2 mM MgCl2 for 6 hr at 4°C. Beads were washed and (A) bound proteins resolved by SDS-PAGE and silver staining or inmmunoblotting with SK896 (lower panel) or (B) stained with TRITC-phalloidin and visualized with fluorescence or phase microscopy. (C) Untreated GST-WASP-Y291E GS4B was incubated with U937 cell lysate as above before washing and incubation with CIP or a buffer control (−) followed by further washing. Left panel: silver staining of fusion protein and precipitated actin. Dephosphorylation was confirmed by an SK896 immunoblot. Right panel: beads were stained with TRITC phalloidin before fluorescence or phase microscopy.
Molecular Cell  , DOI: ( /S (03) )