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First published online 6 March 2007
doi: 10.1242/jcs.03408

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Phosphorylation of adducin by protein kinase C promotes cell motility

¹ Department of Life Science and the Graduate Institute of Biomedical Sciences, National Chung Hsing University, Taichung 40227, Taiwan
² Department of Obstetrics and Gynecology, Taichung Veterans General Hospital, Taichung 40705, Taiwan

View larger version (47K): [in this window] [in a new window]   Fig. 1. Overexpression of PKC in MDCK cells induces the lamellipodia. (A) The expression of endogenous PKC and GFP-PKC was analyzed by immunoblotting with anti-PKC (top panel). The activity of GFP-PKC or its kd mutant was analyzed by an in vitro kinase assay using myelin basic protein (MBP) as an exogenous substrate (bottom panel). (B) Total PKC (including endogenous PKC and GFP-PKC) was immunoprecipitated by polyclonal anti-PKC and its activity was analyzed by an in vitro kinase assay. The phosphorylation of MBP was measured and expressed as fold increase relative to the level of the control MDCK cells. The value (mean ± s.d.) was from three experiments. *P<0.05. (C) The activity of GFP-PKC was measured in the presence of PMA (150 nM) or rottlerin (5 µM). The phosphorylation of MBP was measured and expressed as fold increase relative to the level in the absence of PMA and rottlerin. The value (mean ± s.d.) was from three experiments. *P<0.05. (D) MDCK cells stably expressing GFP, GFP-PKC or its kd mutant were allowed to grow as cell colonies and the micrographs were taken under a phase-contrast microscope. Bar, 50 µm. (E) MDCK cells were plated on collagen-coated glass coverslips for 24 hours and stained for actin filaments with TRITC-conjugated phalloidin. The fluorescence of GFP and the actin filaments was visualized under a confocal laser-scanning microscope. Arrowheads indicate the distribution of GFP-PKC and the cortical actin filaments at the edge of membrane protrusions. Bars, 20 µm. (F) An equal amount of the whole cell lysates from MDCK cells stably expressing FLAG-PKC or their neomycin-resistant control cells (Neo) was analyzed by immunoblotting with anti-FLAG or anti-PKC. (G) MDCK cells as described in F were plated on collagen-coated glass coverslips for 24 hours and stained for FLAG-PKC with anti-FLAG. Bar, 20 µm.

View larger version (54K): [in this window] [in a new window]   Fig. 2. The catalytic activity of PKC is required for its ability to promote cell spreading and migration. (A) Cell spreading assay. An equal number (105) of MDCK cells or those stably expressing GFP, GFP-PKC (wt), or its kd mutant was suspended in serum-free medium and plated onto collagen-coated dishes. At various times after plating, spread cells in several random fields were counted under a phase-contrast microscope. Each data point is the average of three experiments and expressed as the percentage of spread cells in all counted cells (n=200). Representative micrographs were taken from the cells that had been plated for 60 minutes. (B) Trans-well cell migration assay. An equal number (2x104) of the cells were suspended in serum-free medium and subjected to the assay, as described in Materials and Methods. 6 hours later, the migrated cells were fixed, stained and counted using a light microscope. Values (means ± s.d.) are from three independent experiments and expressed as fold increase relative to the level of the control MDCK cells. *P<0.05. (C) Wound healing assay. Cells grown as a monolayer on glass were wounded by manual scratching with a pipette tip. Time-lapse micrographs were taken every 5 minutes for 12 hours to record the healing process. The representative micrographs at 0 hour, 6 hours and 12 hours are shown. The average migratory speed of ten cells at the front was measured by Meta Imaging SeriesTM software version 4.5.

View larger version (27K): [in this window] [in a new window]   Fig. 3. siRNA-mediated knockdown of PKC in MDCK cells suppresses their migration. (A) Doxycycline-inducible expression of the PKC siRNA was established in MDCK cells. Parental MDCK cells, a control siRNA clone and two PKC-specific siRNA clones were grown in the medium supplemented with (+) or without (-) 2 µg/ml doxycycline for 72 hours before lysis. The expression and activity of PKC was analyzed. The expression levels of PKC, PKC and tubulin were analyzed and served as the control. (B) Cells were grown in medium with (+) or without (-) 2 µg/ml doxycycline for 72 hours and then subjected to a Trans-well cell migration assay. Values (means ± s.d.) are from three independent experiments, relative to the level of the control MDCK cells, which was defined as 100%. *P<0.05 compared with their counterparts in the medium without doxycycline.

View larger version (37K): [in this window] [in a new window]   Fig. 4. PKC phosphorylates -adducin at its Ser726 both in vivo and in vitro. (A) GFP, GFP-PKC or its kd mutant was transiently expressed in COS cells. 48 hours later, whole cell lysates were analyzed by immunoblotting with anti-phospho-adducin (Ser726), anti-adducin or anti-GFP. Ser726 phosphorylation of -adducin was measured and expressed as fold increase relative to the level in the cells expressing GFP. Values (means ± s.d.) are from three independent experiments. (B) Whole cell lysates from MDCK cells stably expressing GFP, GFP-PKC or its kd mutant were analyzed by immunoblotting with anti-phospho-adducin (Ser726) or anti-adducin. The Ser726 phosphorylation of adducin was measured and expressed as fold increase relative to the level in the control GFP cells. Values (means ± s.d.) are from three independent experiments. (C) MDCK cells stably expressing GFP-PKC were serum starved for 16 hours and treated with (+) or without (-) 100 nM PMA and 10 µM rottlerin for 15 minutes. The Ser726 phosphorylation of -adducin was analyzed. (D) MDCK cells stably expressing GFP-PKC were plated on collagen-coated glass coverslips for 24 hours and then stained for adducin and its Ser726 phosphorylated form. The fluorescent images were scanned by a confocal microscope on the z-axis 2 µm above the substratum. Bars, 2 µm. (E) GFP--adducin (GFP-add) or its mutants (S716A, S726A and S716A/S726A) were transiently overexpressed in HEK293 cells and immunoprecipitated by anti-GFP. The bound GFP--adducin proteins were eluted from the beads with citric acid and neutralized in Tris buffer. The purity and yield of GFP--adducin proteins were analyzed by SDS-polyacrylamide gel electrophoresis and visualized by Coomassie Blue stain. (F) GFP-PKC was transiently expressed in HEK293 cells and immobilized on protein A beads with anti-GFP. The washed immunocomplexes were analyzed by immunoblotting with anti-GFP or subjected to an in vitro kinase assay in the presence of [-32P]ATP using purified GFP--adducin proteins as the substrates. The 32P-incorporated proteins were fractionated by SDS-polyacrylamide gel electrophoresis and visualized by autoradiography. The phosphorylation of GFP--adducin proteins was measured and expressed as a percentage relative to the phosphorylation of GFP--adducin (wt). Values (means ± s.d.) are from three independent experiments. IVK, in vitro kinase assay.

View larger version (24K): [in this window] [in a new window]   Fig. 5. PKC does not phosphorylate the Ser726 of -adducin. (A) GFP, GFP-PKC and GFP-PKC were transiently overexpressed in COS cells. Whole cell lysates were analyzed by immunoblotting with anti-phospho-adducin (Ser726), anti-adducin or anti-GFP. The Ser726 phosphorylation of adducin was measured and expressed as fold increase relative to the level in control GFP cells. (B) Endogenous PKC was immunoprecipitated by anti-PKC. The washed immunocomplexes were subjected to in vitro kinase assays using purified GFP--adducin (GFP-add) or its mutants as the substrate. The phosphorylation of GFP--adducin (wt) is defined as 100%. Values (means ± s.d.) are from three independent experiments. IVK, in vitro kinase assay.

View larger version (33K): [in this window] [in a new window]   Fig. 6. PKC-mediated phosphorylation of adducin promotes cell spreading and migration. (A) GFP, GFP--adducin or GFP--adducin (or its S726A mutant) was expressed alone or co-expressed with FLAG-PKC in MDCK cells. An equal amount of the whole cell lysates from those cells was analyzed by immunoblotting with anti-adducin, anti-phospho-adducin or anti-FLAG. (B) MDCK cells stably co-expressing FLAG-PKC with GFP--adducin were fixed and stained with anti-FLAG. The fluorescent images of FLAG-PKC and GFP--adducin were scanned by a confocal microscope on the z-axis 2 µm above the substratum. Bar, 5 µm. The enlarged images show the colocalization of GFP--adducin and FLAG-PKC at the plasma membrane. (C) MDCK cells were suspended in serum-free medium and plated on collagen-coated dishes. 40 minutes later, spread cells were counted and expressed as the percentage in total counted cells (n=200). Values (means ± s.d.) are from three independent experiments. (D) The cells were suspended in serum-free medium and subjected to a Trans-well cell migration assay. 6 hours later, the migrated cells were fixed, stained and counted using a light microscope. Values (means ± s.d.) are from three independent experiments and expressed as fold increase relative to the level of the cells expressing GFP alone. *P<0.05 (compared with the cells expressing GFP alone). **P<0.05 (compared with the cells co-expressing FLAG-PKC and GFP).