Figure 5.

GRK2-stimulated HDAC activity is relevant for pseudopodia formation in response to chemotactic cues. (A) Expression of GRK2 mutants unable to phosphorylate HDAC6 inhibits pseudopodia formation. Parental, wt5, A1 or K1 HeLa cells were serum starved for 16 h and subjected to transwell migration assays as detailed in Materials and methods and in the absence or presence of serum in the bottom chamber. Levels of pseudopodia protein recovered on the underside of porous filters were analysed using the Bradford method. Data are mean±s.e.m. of three independent experiments. (B, C) Accumulation of both HDAC6 and GRK2 phosphorylated at S670 at pseudopodia correlates with local deacetylation of tubulin. Cells as in (A) were allowed to migrate in the absence or presence of a serum gradient, and 2 h later purified pseudopodia were collected and the levels of α-Tubulin acetylation (B) or of GRK2, its phosphorylation at S670 and HDAC6 (C) were determined by immunoblot. Data in (B) are mean±s.e.m. from 3 to 4 experiments. Representative blots are shown. ^*P<0.05, ^**P<0.01, compared with control, untreated HeLa cells; ^†P<0.05, ^††P<0.01 compared with serum-stimulated HeLa cells, unpaired two-tailed t-test. Figure source data can be found in Supplementary data.