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First published online April 24, 2006
doi: 10.1242/10.1242/jcs.02874

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Phosphorylation on Ser5 increases the F-actin-binding activity of L-plastin and promotes its targeting to sites of actin assembly in cells

Bassam Janji^1,*, Adeline Giganti^1,*, Veerle De Corte², Marie Catillon¹, Erik Bruyneel³, Delphine Lentz¹, Julie Plastino⁴, Jan Gettemans² and Evelyne Friederich^1,

¹ Laboratory for Molecular Biology, Genomics and Modelling, Public Research Centre for Health (CRP-Santé), 84 Val Fleuri, 1526 Luxembourg
² Department of Biochemistry, Faculty of Medicine and Health Sciences, Ghent University, Albert Baertsoenkaai 3, 9000 Ghent, Belgium and Flanders Interuniversity, Institute for Biotechnology (V.I.B.), 9052 Ghent, Belgium
³ Laboratory of Experimental Cancerology, Department of Radiotherapy and Nuclear Medicine, Ghent University Hospital (1P7), De Pintelaan 185, 9000 Ghent, Belgium
⁴ Laboratoire Physicochimie "Curie", UMR168 CNRS/Institut Curie, 11, rue Pierre et Marie Curie, 75231 Paris CEDEX 05, France

View larger version (27K): [in a new window]   Fig. 1. Characterisation of the L-plastin Ser5 phospho antibody and expression of wild-type L-plastin and phosphorylation variants in Vero cells. (A) Schematic representation of wild-type L-plastin protein (WT) and of phosphorylation variants, and a modular arrangement of L-plastin domains. The amino-acid sequences of the wild-type phosphorylation sites (Ser5 and Ser7) and of the sv-tagged phosphorylation variants (S/A, S/E) are indicated. (B) The anti-Ser5-P antibody reacts specifically with in vitro phosphorylated recombinant L-plastin. Equal amounts of recombinant wild-type L-plastin (WT) or or L-plastin Ser5Ala (S/A) were incubated with the catalytic domain of protein kinase A for various time points as described in Materials and Methods. L-plastin was analysed by immunoblotting with anti-Ser5-P (upper panel) or anti-L-plastin antibodies (lower panel). (C) The anti-Ser5-P antibody specifically reacts with phosphorylated L-plastin in Jurkat T lymphoid cells. Jurkat cells were stimulated with 1 mM 8-Bromo-cAMP or 0.1 mM forskolin for 45 minutes. Before stimulation, cells were treated (+) or not (-) with 50 µM of H-89 for 45 minutes. Equal amounts of cell lysates were analysed by immunoblotting with anti-Ser5-P (upper panel) or anti-L-plastin antibodies (lower panel). (D) Expression and Ser5 phosphorylation of wild-type L-plastin and phosphorylation variants in Vero cells. Vero cells were transfected with cDNA constructs encoding sv-tagged wild-type (WT), Ser5Ala (S/A) or Ser5Glu (S/E) L-plastin. Untransfected cells (UT) were used as a negative control. After 48 hours, equal amounts of cell extracts were analysed by immunoblotting. Transfected proteins were detected with anti-sv-tag (upper panel) or anti-Ser5-P antibodies (lower panel).

View larger version (78K): [in a new window]   Fig. 2. L-plastin phosphorylated on Ser5 localises to the actin cytoskeleton in Vero cells. Transfected Vero cells expressing wild-type L-plastin or S/A variant were processed for immunofluorescence staining. (A) Intracellular distribution of L-plastin phosphorylated on Ser5. (Upper row) Cells expressing wild-type L-plastin (WT) were stained with Rhodamine-phalloidin (left panel) and anti-Ser5-P antibody (middle panel). Alexa-Fluor-488-coupled anti-rabbit IgG antibody served as secondary antibody. Merged image of enlarged regions (boxes) of left and middle images is shown on the right. Red, F-actin; green, anti-Ser5-P antibody. (Lower row) Cells expressing wild-type L-plastin were double-stained with anti-sv-tag antibody (left panel) and anti-Ser5-P antibody (middle panel) with Alexa-Fluor-594-coupled anti-mouse and Alexa-Fluor-488-coupled anti-rabbit IgG antibody as secondary antibodies, respectively. Merged image of enlarged regions (boxes) of left and middle images is shown on the right. Red, total WT L-plastin; green, phosphorylated WT L-plastin. Bars, 15 µm. (B) The anti-Ser5-P antibody does not react with Ser5Ala L-plastin. Cells expressing the non-phosphorylatable Ser5Ala variant (S/A) were double-stained with anti-sv-tag antibody (left) and anti-Ser5-P antibody (right) as described above. Bar, 15 µm.

View larger version (69K): [in a new window]   Fig. 3. Intracellular distribution of L-plastin phosphorylation variants in Vero cells. (A,B) Transfected Vero cells expressing wild-type L-plastin (upper rows), L-plastin Ser5Ala (middle rows) or L-plastin Ser5Glu (lower rows) were processed for immunofluorescence double-staining. (A) Co-distribution of L-plastin variants with F-actin. Cells were stained with Rhodamine-phalloidin (left column) and anti-sv-tag antibody (middle column). Secondary antibody as in Fig. 2A. Merged images of enlarged regions (boxes) of left and middle images are shown on the right. Red, F-actin; green, total L-plastin. Bars, 15 µm. (B) Co-distribution of L-plastin variants with vinculin. Cells were double-stained with vinculin (left column) and sv-tag antibody (middle column). Secondary antibodies as in Fig. 2A. Merged images of enlarged regions (boxes) of left and middle images are shown on the right. Red, vinculin; green, total L-plastin. Bars, 15 µm.

View larger version (91K): [in a new window]   Fig. 4. Targeting of phosphorylated L-plastin to areas of fast actin assembly. Transfected Vero cells expressing Ser5Glu (S/E), Ser5Ala (S/A) or wild-type L-plastin were processed for immunofluorescence double- or triple-staining. (Upper row) Cells expressing Ser5Glu variant were double-labelled with anti-cortactin and anti-sv-tag antibodies. Alexa-Fluor-488-coupled anti-mouse IgG (L-plastin S/E, green) and Texas-Red-coupled anti-rabbit IgG antibody (cortactin, red) served as secondary antibodies. The two right panels show merges of enlarged regions (boxes) of anti-sv-tag- and anti-cortactin-stained images. Bar, 15 µm. (Middle row) Cells expressing wild-type L-plastin were triple-labelled with Alexa-Fluor-350-phalloidin, anti-cortactin and anti-Ser5-P antibodies. Alexa-Fluor-488-coupled anti-mouse IgG (cortactin, green) and Texas-Red-coupled anti-rabbit IgG antibody (Ser5-P WT L-plastin, red) served as secondary antibodies. The right panel shows a merge of cortactin and anti-Ser5-P staining. Bar, 3 µm. (Lower row) Cells expressing L-plastin Ser5Ala were labelled with anti-cortactin and anti-sv-tag antibody. Secondary antibodies were used as described above (middle row). The right panel shows a merge of cortactin and sv-tag images. Bar, 3 µm.

View larger version (152K): [in a new window]   Fig. 5. Targeting of L-plastin phosphorylation variants to surface-F-actin spikes in epithelial LLCPK1 cells. LLCPK1 cells expressing Ser5Glu (S/E, upper panels), Ser5Ala (S/A, middle panels) or WT L-plastin (WT, lower panels) were double-stained with Rhodamine-phalloidin (left panels) and anti-sv-tag antibody (right panels). Cy2-coupled anti-rabbit IgG served as secondary antibody. Bars, 15 µm.

View larger version (81K): [in a new window]   Fig. 6. Probing the avidity of L-plastin phosphorylation variants for cellular actin structures by detergent extraction. (A) Immunolocalisation of L-plastin variants after detergent extraction. Transfected Vero cells expressing L-plastin wild-type (upper panels), Ser5Ala (middle panels) or Ser5Glu (lower panels) were extracted with Triton X-100 prior to fixation. Cells were stained with anti-sv-tag antibody (right row) and Rhodamine-phalloidin (left row) as described in Fig. 3A. Bars, 15 µm. (B) Quantification of extraction experiments. Cells exhibiting a sv-positive staining pattern were counted on untreated (no extraction) and detergent-extracted (extraction) coverslips, as described in Materials and Methods. (C) Immunolocalisation of phosphorylated wild-type L-plastin after detergent extraction. Transfected Vero cells expressing wild-type L-plastin were extracted as described in A and stained with Rhodamine-phalloidin (left panel) and anti-Ser5-P antibody (middle panel). Right panel shows a merge of enlarged regions (boxes) of left and middle images. Green, Ser5-P WT L-plastin; red, F-actin. Bar, 15 µm.

View larger version (37K): [in a new window]   Fig. 7. Phosphorylated wild-type L-plastin and S/E variant bind with a higher stoichiometry to F-actin than non-phosphorylated wild-type protein. (A) Binding of phosphorylated and non-phosphorylated wild-type L-plastin to F-actin. Wild-type L-plastin (L-plastin WT) was incubated in the absence or presence of the catalytic domain of PKA for 120 minutes at 30°C. G-actin (6 µM) was copolymerised with various concentrations of phosphorylated [L-plastin WT (P)] or non-phosphorylated wild-type L-plastin (L-plastin WT) and centrifuged at high speed. Supernatants and pellets were analysed by SDS-PAGE. Coomassie-staining patterns of pellets (top panel) and supernatants (bottom panel) of triplicate samples are shown. The molar ratios of L-plastin to actin are indicated. (B) Quantification of binding of phosphorylated or non-phosphorylated wild-type L-plastin and L-plastin S/A variant to F-actin. Amounts of phosphorylated (black) or non-phosphorylated (blue) wild-type L-plastin and L-plastin S/A variant (red) in pellets and supernatants were quantified by densitometry of Coomassie-stained protein bands. L-plastin in the pellets is plotted as a function of increasing L-plastin concentrations. Each point is the mean of three experiments ± s.d. (C) Immunoblotting analysis of phosphorylated wild-type L-plastin, co-sedimented with F-actin. A fraction of protein pellets shown in A (upper panel) was analysed by immunoblotting using anti-Ser5-P antibody (lower panel). After stripping the membrane, total L-plastin was detected with anti-L-plastin antibody (upper panel). Triplicate samples are shown for each actin to plastin ratio. (D) Binding of WT L-plastin and S/E variant to F-actin. G-actin (12 µM) was copolymerised with various concentrations of WT L-plastin or S/E variant and centrifuged at high speed. Coomassie-staining patterns of supernatants (S) and pellets (P) are shown. The molar ratios of L-plastin to actin are indicated. (E) Binding curves of WT L-plastin or S/E variant to F-actin. Amounts of F-actin-bound WT L-plastin (rhombus) and S/E variant (square) in the pellet are plotted as a function of increasing L-plastin concentrations. Each point is the mean of four experiments ± s.d.

View larger version (46K): [in a new window]   Fig. 8. L-plastin Ser5Glu has highly increased bundling activity when compared to non-phosphorylated wild type protein. (A) Co-sedimentation of WT L-plastin or S/E variant with F-actin bundles. Co-sedimentation of 12 µM G-actin with WT L-plastin or S/E variant as described in Fig. 7, with the exception that mixtures were centrifuged at low speed to sediment F-actin bundles. Coomassie-staining patterns of pellets (upper panels) and supernatants (lower panels) are shown. Lane 1, actin alone (input). The molar ratios of L-plastin to actin are indicated. (B) Quantification of F-actin bundle formation. Actin in the pellet (% of total) corresponding to bundled F-actin, was plotted as a function of increasing concentrations of WTL-plastin () or L-plastin Ser5Glu (). Each point is the mean of four experiments ± s.d. (C) Electron microscopy reveals increased bundle formation in the presence of S/E variant. Actin was copolymerised with L-plastin WT (left) or L-plastin S/E (right) at a molar ratio of 1:4 as described in A. Actin filaments were negatively stained with 1% uranyl acetate and analysed by transmission electron microscopy. Electron microscopy images are shown. (D) Effect of Ca2+ on bundling activities of L-plastin WT or S/E variant. Low-speed co-sedimentation of 12 µM G-actin with L-plastin WT () or S/E variant () was performed as described in A, at a 1:4 molar ratio, in the presence of various concentrations of Ca2+. pCa was varied by the addition of various volumes of 1 mM CaCl2. pCa=8 (0.024 mM); pCa=7 (0.195 mM); pCa=6 (0.709 mM); pCa=5 (0.971 mM). Note that pCa=-log [Ca2+]. F-actin in the pellet (% of total) was plotted as a function of increasing concentrations of free Ca2+. Each point is the mean of three experiments ± s.d.

View larger version (29K): [in a new window]   Fig. 9. Ser-Ala substitution in L-plastin is sufficient to abolish L-plastin-dependent collagen invasion. (A) Expression of wild-type L-plastin and phosphorylation variants in HEK293T cells. HEK293T cells were transfected with cDNA constructs encoding wild type (WT), Ser-Ala (S/A), Ser5Ala-Ser7Ala (S5-7/A) or Ser5Glu L-plastin (S/E). Untransfected cells were used as a negative control (UT). After 48 hours, equal amounts of cell extracts (20 µg) were analysed by immunoblotting with anti-sv-tag antibody. (B) Collagen-invasion-capacity of HEK293T cells expressing WT L-plastin or phosphorylation variants. Transfected cells expressing wild-type L-plastin or L-plastin phosphorylation variants were tested for their capacity to invade a collagen-type-I gel as described in Materials and Methods. DHD-FIB rat colon myofibroblasts were used as positive control for invasion and untransfected (UT) HEK293T cells as negative control. Results are representative of three independent experiments (mean ± s.d.). (C) Fast aggregation assay of HEK293T cells transfected with WT L-plastin and phosphorylation variants. Plotted curves of relative volume distribution (y-axis) as a function of particle diameter (x-axis) are shown for mock-transfected (MT) HEK293T cells, HEK293T cells transfected with L-plastin (WT), L-plastin Ser5Ala (S5/A), L-plastin Ser5Ala-Ser7Ala (S5-7/A) or L-plastin Ser5Glu (S/E) after 30 minutes. The arrow indicates the peak position of HEK293T cells after 0 minutes of aggregation (not shown). (D) Wound healing assay. 2D-migration of cells expressing wild-type L-plastin is similar to cells expressing mutant L-plastin. MT, mock-transfected HEK293T cells. The migration distance in µm (y-axis) as a function of time in hours (x-axis) is shown. Data are representative for two independent experiments. (E) PKA but not PKC inhibitors block L-plastin-induced invasion of HEK293T cells. Transfected cells expressing wild-type L-plastin were tested for their capacity to invade a collagen-type-I gel as described in B with the exception that PKC inhibitors (GF109 or Gö796) or PKA (KT5720) inhibitors (all at 10 µM) were present during the assay. Control, untransfected cells. -inhibitor, untreated cells expressing wild-type L-plastin. Results are representative of three independent experiments (mean ± s.d.). (F) PKA activation increases phosphorylation of L-plastin wild-type in HEK293T cells. Transfected HEK293T cells were incubated for 45 minutes in the absence (-) or in the presence (+) of PKA inhibitor H-89 and stimulated for an additional 45 minutes with 1 mM 8-Bromo-cAMP. Equal amounts of cell lysates were analysed by immunoblotting using anti-Ser5-P antibody (upper panel) or anti-sv-tag antibody (lower panel).

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