Protein kinase D1-mediated phosphorylations regulate vasodilator-stimulated phosphoprotein (VASP) localization and cell migration

Abstract

Enabled/Vasodilator-stimulated phosphoprotein (Ena/VASP) protein family members link actin dynamics and cellular signaling pathways. VASP localizes to regions of dynamic actin reorganization such as the focal adhesion contacts, the leading edge or filopodia, where it contributes to F-actin filament elongation. Here we identify VASP as a novel substrate for protein kinase D1 (PKD1). We show that PKD1 directly phosphorylates VASP at two serine residues, Ser-157 and Ser-322. These phosphorylations occur in response to RhoA activation and mediate VASP re-localization from focal contacts to the leading edge region. The net result of this PKD1-mediated phosphorylation switch in VASP is increased filopodia formation and length at the leading edge. However, such signaling when persistent induced membrane ruffling and decreased cell motility.

Keywords: Kinase; Phosphorylation; Protein Kinase C (PKC); Protein Kinase D (PKD); Signal Transduction; Signaling; VASP.

FIGURE 1.

PKD1 phosphorylates VASP at Ser-157 and Ser-322 in vitro. A, HeLa cells (0.65 × 10⁶ cells, 6-cm dish) were transfected with vector control, HA-tagged constitutively active PKD1 (PKD1.CA) and FLAG-tagged VASP as indicated. VASP was immunoprecipitated (anti-FLAG), and precipitates were analyzed with the pMOTIF antibody that recognizes PKD-mediated phosphorylation. Samples were re-stained for VASP (anti-FLAG) and lysates were control stained for expressed PKD1.CA (anti-PKD1). B, depicted is the PKD consensus phosphorylation motif with arginine or lysine at −3 and leucine, valine, or isoleucine at −5 relative to the serine or threonine phosphorylation site. Also shown are known phosphorylation sites in human VASP (Ser-157, Ser-239, Thr-278) and potential PKD1 phosphorylation motifs at Ser-322 and Ser-379 in VASP of different species. C, PKD1 phosphorylates VASP at Ser-157 in an in vitro assay. Bacterially expressed and purified GST (negative control) or GST-VASP was incubated in a kinase reaction with purified active PKD1. Substrate phosphorylation was detected using the phosphosite-specific antibodies anti-pS157-VASP or anti-pS239-VASP as indicated. Control blots were performed for protein input (anti-PKD1, anti-GST). D, Ser-157 is not the only PKD1 phosphorylation site in VASP. Bacterially expressed and purified GST (negative control), GST-VASP, or GST-VASP.S157A was incubated in a kinase reaction with purified active PKD1. Substrate phosphorylation was detected using the pMOTIF antibody, which recognizes the phosphorylated PKD motif in PKD substrates. Control blots were performed for protein input (anti-PKD1, anti-GST). E, bacterially expressed and purified GST (negative control), GST-VASP, GST-VASP.S157A, GST-VASP.S322A, GST-VASP.S157A.S322A were incubated in a kinase reaction with purified active PKD1. Substrate phosphorylation was detected using the pMOTIF antibody, which recognizes the phosphorylated PKD motif in PKD substrates. Shown are short and long exposures. Control blots were performed for protein input (anti-PKD1, anti-VASP). F, PKD1 phosphorylates VASP at Ser-322 in an in vitro assay. Bacterially-expressed and purified GST-VASP or GST-VASP.S322A was incubated in a kinase reaction with purified active PKD1. Substrate phosphorylation was detected using the novel anti-pS322-VASP antibody specifically generated for this site. Control blots were performed for protein input (anti-PKD1, anti-GST).

FIGURE 2.

PKD1-mediated phosphorylations of VASP are independent of each other. A, mimicking Ser-157 phosphorylation does not prime for Ser-322 phosphorylation. HeLa cells were transfected with GFP-VASP, GFP-VASP.S157E, or vector control as indicated. Cells were lysed and analyzed by Western blot for phosphorylation at Ser-322 using a phosphospecific antibody. VASP expression was controlled by re-probing with anti-GFP. B, mimicking Ser-322 phosphorylation does not prime for Ser-157 phosphorylation. HeLa cells were transfected with GFP-VASP, GFP-VASP.S322E, or vector control as indicated. Cells were lysed and analyzed by Western blot for phosphorylation at Ser-157 using a phosphospecific antibody. VASP expression was controlled by re-probing with anti-GFP.

FIGURE 3.

PKD1-mediated phosphorylations of VASP occur in vivo. A, NMuMG cells (0.5 × 10⁶ cells, 6-cm dish) were transfected with vector control, HA-tagged constitutively active PKD1 (PKD1.CA) and FLAG-tagged VASP, VASP.S157A, VASP.S322A, or VASP.S157A.S322A as indicated. VASP was immunoprecipitated (anti-FLAG) and analyzed for PKD1-mediated phosphorylation using the pMOTIF antibody. Shown are short and long exposures. Samples were re-probed with anti-FLAG for equal expression of wild type or mutant VASP. Control Western blots were performed for PKD1.CA expression (anti-PKD1). B, HeLa cells (0.65 × 10⁶ cells, 6-cm dish) were transfected as indicated. VASP was immunoprecipitated (anti-FLAG) and analyzed for phosphorylation at Ser-157 or Ser-322 using anti-pS157-VASP or anti-pS322-VASP antibodies. Samples were re-probed with anti-FLAG for equal loading of wild type or mutant VASP. Control Western blots were performed for PKD1.CA expression (anti-PKD1).

FIGURE 4.

PKD1-mediated phosphorylations of VASP occur in response to active RhoA. A, HeLa cells (0.35 × 10⁶ cells, 6-cm dish) were lentivirally infected with scrambled control or PKD-shRNA as indicated. The next day, cells were additionally transfected with combinations of VASP, vector control (pEBG), or active RhoA.CA (pEBG-RhoA.CA) as indicated. VASP was immunoprecipitated (anti-FLAG) and analyzed for phosphorylation at Ser-157 or Ser-322 using indicated phosphospecific antibodies. Samples were re-probed with anti-FLAG for VASP. Control Western blots were performed for PKD1 knockdown (anti-PKD1/2) and for RhoA.CA expression (anti-GST). B, HeLa cells (0.35 × 10⁶ cells, 6-cm dish) were transfected with vector control (pEBG) or active RhoA.CA (pEBG-RhoA.CA) and treated with PKD inhibitor (CID755673, 20 μm) as indicated. Endogenous VASP was immunoprecipitated (anti-VASP) and analyzed for phosphorylation at Ser-157 or Ser-322 using indicated phosphospecific antibodies. Samples were re-probed with anti-VASP. Control Western blots were performed for RhoA.CA expression (anti-GST).

FIGURE 5.

PKD activity is necessary for localization of VASP to the cell periphery. A and B, HeLa cells were treated with the PKD inhibitor CID755673 or left untreated. After 16 h, samples were fixed and analyzed for localization of endogenous VASP using immunofluorescence. Shown is a representative cell under each condition. In untreated cells, arrows indicate localization of VASP at the leading edge/cell periphery (bar is 10 μm).

FIGURE 6.

Ser-157 and Ser-322 phosphorylation is necessary for localization of VASP to the cell periphery. A–R, HeLa cells were transfected with GFP-tagged VASP, VASP.S157E, VASP.S322E, VASP.S157E.S322E, VASP.S157E.S322A, or VASP.S322A as indicated. After 24 h cells were fixed, and F-actin was stained with phalloidin. Localization of GFP or GFP-tagged proteins was determined using immunofluorescence analysis (bar is 10 μm). Insets are 2.5-fold enhanced and shown in G–I and P–R. VASP is stained in gray (green in insets), phalloidin in deep red. G3, H3 I3, P3, Q3, R3 show an overlay. In A–O, changes in cellular localization were observed in all cells analyzed (typically n = 100). All data were confirmed in at least three independent experiments.

FIGURE 7.

VASP phosphorylation is necessary for actin polymerization. A, Hek293T cells (0.5 × 10⁶ cells/well, 6-well plate) were transfected with vector control, HA-tagged constitutively active PKD1 (PKD1.CA) and VASP or VASP mutants as indicated, as well as SRE promoter luciferase and Renilla luciferase reporters. Induced luciferase activity was measured. Error bars shown represent standard deviations. * indicates statistical significance (p < 0.05) as compared with vector control. p values were acquired with the Student's t test using Graph Pad software. Protein expression was controlled by Western blotting with anti-FLAG (VASP), anti-PKD1 (PKD1), or anti-tubulin (loading control) antibodies. B, Hek293T cells (0.5 × 10⁶ cells/well, 6-well plate) were transfected with vector control, GFP-tagged VASP or VASP.S157E.S322E mutant as indicated, as well as SRE promoter luciferase and Renilla luciferase reporters. Induced luciferase activity was measured. Error bars shown represent standard deviations. * indicates statistical significance (p < 0.05) as compared with vector control; ** indicates statistical significance (p < 0.05) as compared with wild type VASP. p values were acquired with the Student's t test using Graph Pad software. Protein expression and loading was controlled by Western blotting with anti-GFP (VASP) or anti-β-actin (loading control) antibodies.

FIGURE 8.

β-Actin recovery and mobility are different in cells expressing VASP or the VASP. S157E.S322E mutant. A and B, HeLa cells were co-transfected with GFP-β-actin and mCherry-VASP or mCherry-VASP.S157E.S322E. FRAP analysis was performed to measure actin recovery and mobility. At least 10 cells were analyzed per transfection condition. * indicates statistical significance (p < 0.05).

FIGURE 9.

PKD1-mediated phosphorylation of VASP induces leading edge formation and elongated filopodia. A–F, HeLa cells were co-transfected with GFP-actin and Cherry-VASP or the Cherry-VASP.S157E.S322E mutant as indicated. The next day cells were reseeded into ibidi μ-Slides in phenol-red free media, and after 12 h live cell imaging was performed as described under “Experimental Procedures.” A1, A2, C1, and C2 show single channel pictures for Cherry-VASP, Cherry-VASP.S157E.S322E or GFP-actin. The bar represents 10 μm. The insets in A and C show typical localization of VASP (at the focal contacts) or VASP.S157E.S322E (red, VASP, green, actin, yellow, overlay). The bar shows 10 μm. B and D show kymographic analysis (t = 1200 s, length of line in B = 17.3 μm, in C = 18.2 μm) of several areas at sites of VASP or VASP mutant location. E shows single pictures of an area from C showing rapid formation, protrusion, and retraction of filopodia at sites of Cherry-VASP.S157E.S322E localization. Pictures shown are at a difference of 20 s. Cherry-VASP.S157E.S322E is in red pseudocolor (at t = 0), GFP-actin is in gray in all pictures. F and G show quantifications of distances from nuclei to the leading edge (F) or length of filopodia formed (G). Analysis was performed using Image J. p values were acquired with the student's t test using Graph Pad software. In F, n represents the number of cells (50) analyzed for nucleus to leading edge distance, combined from three independent experiments. In G, n represents the numbers of filopodia analyzed for their length from 10 different cells, combined from three independent experiments.

FIGURE 10.

PKD1-mediated VASP phosphorylations induce membrane ruffling and negatively affect cell migration. A–H, HuMEC cells were co-transfected with GFP-tagged active PKD1 (GFP-PKD1.CA) and VASP, or the VASP.S157A.S322A mutant as indicated. 16 h after transfection, cells were fixed, and F-actin was stained with phalloidin. Localization of proteins was determined using immunofluorescence analysis (bar is 10 μm). Insets are 2.5-fold enhanced. Changes in cellular localization were observed in all cells (typically n = 50) analyzed. All data were confirmed in at least three independent experiments. I and J, HeLa cells were transfected with vector control, VASP or VASP phosphorylation mimicking mutant as indicated (I), or with vector or PKD1.CA in combination with VASP or the VASP.S157A.S322A mutant (J). Real-time cell migration was monitored with an impedance-based assay system (ECIS) over a time period of 5 h. K, schematic of the here proposed PKD1-mediated signaling events downstream active RhoA that lead to VASP localization to the leading edge of cells, to filopodia formation and cause F-actin filament extension. If persistent such signaling results in membrane ruffling and eventually to decreased cell migration.