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).