First published online November 3, 2003
doi: 10.1242/10.1242/jcs.00794
Uncoordinated regulation of stress fibers and focal adhesions by DAP kinase
Jean-Cheng Kuo1,
Jia-Ren Lin1,
James M. Staddon2,
Hiroshi Hosoya3 and
Ruey-Hwa Chen1,*
1 Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan
2 Eisai London Research Laboratories, University College London, London, UK
3 Department of Biological Science, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan
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Fig. 1. DAP-kinase phosphorylates MLC in vitro and in vivo. (A) DAP-kinase interacts with MLC. GSTMLC fusion protein or GST alone immobilized on glutathione-Sepharose beads was incubated with DAP kinase purified from recombinant baculovirus. The precipitates and the purified DAP kinase (5% of the input amount) were subjected to SDS-PAGE and immunoblotted with anti-DAP-kinase antibody (top). The same membrane was stained by amido black to show equal input of the GST and GST-MLC proteins (bottom). (B) DAP kinase phosphorylates a  20 kDa protein. The Triton-soluble (S) and -insoluble (I) fractions of the 293 cell lysate were separated, heated to inactivate cellular kinases and then used as substrates in the kinase reactions catalysed by Flag/DAP-kinase immunoprecipitated from lysates of transfected cells. The anti-Flag immunoprecipitates derived from untransfected cells (DAPK–) and purified MLC were included as negative and positive controls, respectively. (C) Determination of MLC phosphorylation sites by DAP kinase. Wild-type and mutant MLC fused with GST were phosphorylated in vitro by Flag/DAP-kinase or its kinase-dead mutant (K42A) immunoprecipitated from transfected cells. Wild-type MLC (MLC) was added to each kinase reaction as an internal control. The kinase reactions were subjected to SDS-PAGE and transferred to PVDF membrane. The membrane was stained by amido black to reveal the equal input of MLC, GST-MLC and DAP kinase in the kinase reactions, and then subjected to autoradiography to detect the phosphorylated proteins. (D) Phosphoamino-acid analysis of MLC phosphorylated by Flag-DAP kinase. The positions of phosphorylated serine (pS), threonine (pT) and tyrosine (pY) are indicated. (E) DAP kinase enhances MLC phosphorylation in vivo. NIH3T3 cells infected with recombinant retrovirus as indicated or 293 cells transfected with various constructs were serum-starved for 6 hours. Cell lysates were then subjected to immunoblot with antibodies specific to DAP kinase, diphosphorylated MLC (MLC-pp), monophosphorylated MLC (MLC-p) or MLC as indicated.
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Fig. 2. DAP kinase promotes the assembly or stabilization of stress fibers. (A) NIH3T3 cells were infected with recombinant retrovirus carrying vector alone or various forms of DAP kinase as indicated. Infected cells were selected by puromycin and then subjected to immunoblot analysis to detect the expression of DAP kinase proteins. (B) Cells as in (A) were serum-starved for 6 hours, treated with or without 25 mM BDM for 10 minutes or 100 nM cytochalasin D for 30 minutes, and then stained with rhodamine-phalloidin. (C) Quantitation of cells with stress fibers in experiments as described in (B). Only cells that were not exposed to BDM and cytochalasin D were analysed. The values shown are means ± s.d. from at least three independent experiments and more than 300 individual cells were counted for each experiment. (D) NIH3T3 cells transiently transfected with DAP kinase were serum starved and then double stained with anti-DAP-kinase and rhodamine-phalloidin. A cell that overexpresses DAP kinase is indicated by strong DAP kinase staining pattern. (E) NIH3T3 cells were cotransfected with DAP kinase, MLCS19A mutant (or a control vector) and GFP at a ratio of 5:5:1. Cells were serum starved and stained with rhodamine-phalloidin. (F) Quantitation of GFP-positive cells with stress fibers in experiments described in (E). Scale bars, 10 µm.
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Fig. 4. DAP kinase and MLCK display distinct roles in the assembly of stress fibers. (A) DAP kinase is insensitive to ML-7. ML-7 was added at indicated concentrations to kinase reactions containing MLC, Flag-DAP kinase and Ca2+/calmodulin. MLC phosphorylation (bottom) and DAP kinase autophosphorylation (top) were detected by autoradiography. (B) Kinase reactions as in (A) but without ML-7 were performed for various times. The extent of phosphorylation was quantified and expressed as percentage, assigning the maximum phosphorylation of MLC to 100%. (C) Virus-infected NIH3T3 cells (Fig. 2B) were serum starved and treated with 5 µM of ML-7 for 30 minutes. Cells were then fixed and stained with rhodaminephalloidin. (D) Subcellular localization of endogenous DAP kinase. NIH3T3 cells cultured in serum-containing medium were double stained with anti-DAP-kinase for endogenous DAP kinase (a) and with rhodamine-phalloidin for F-actin (b). The merged image is shown in (c). Scale bars, 10 µm.
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Fig. 5. DAP kinase is involved in serum-induced stress-fiber formation. (A) NIH3T3 cells were infected with recombinant retrovirus carrying vector alone or DAPK42A, as indicated. Serum-starved cells were re-stimulated with serum for 20 minutes and then stained with rhodamine-phalloidin. Scale bar, 10 µm. The percentages of cells with stress fibers seen in serum-starved and stimulated conditions are indicated on the bottom. (B) NIH3T3 cells were transiently transfected with various combinations of constructs as indicated. Myc-ROCK and Myc-CAT were immunoprecipitated from cell lysates and then used to phosphorylate recombinant MLC in vitro in the presence or absence of 2 µM Y27632. The kinase reactions were resolved by SDS-PAGE and MLC phosphorylation was detected by autoradiography (top). The same kinase reactions were subjected to immunoblot analysis to detect the precipitated ROCK or CAT (middle). The expression of DAPK42A was detected by immunoblot (bottom). (C) Endogenous MLCK was immunoprecipitated from lysates of 293T cells transfected with various constructs, as indicated. The immunoprecipitates were used to phosphorylate recombinant MLC in vitro, in the presence or absence of 2 µM ML-7 (top). The same kinase reactions were subjected to immunoblot analysis to detect equal input of MLCK (middle). The expression of various DAP-kinase proteins was detected by immunoblot (bottom).
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Fig. 6. DAP kinase is required for serum-induced stress fiber formation and is activated by serum. (A) Reduction of endogenous DAP kinase expression by siRNA. Cell lysates from untreated (–), control-siRNA- or DAPK-siRNA-treated cells were immnuoblotted with antibodies as indicated. (B) siRNA-mediated knockdown of DAP kinase inhibits stress-fiber formation in response to serum. Cells as in (A) were stimulated with serum for 20 minutes and then stained with rhodamine-phalloidin. Cells receiving siRNA were visualized by their FITC fluorescence. Scale bar, 10 µm. (C) NIH3T3 cells were transfected with Flag-DAP kinase and stimulated with or without serum for various times. The Flag-DAP kinase was immunoprecipitated from cell lysates and subjected to in vitro kinase reactions using MLC as a substrate. The kinase reactions were resolved by SDS-PAGE, and MLC phosphorylation was detected by autoradiography (top). The same kinase reactions were subjected to immunoblot analysis to detect the precipitated Flag-DAP kinase (bottom). The result shown is a representative experiment from three independent experiments. (D) Quantification of DAP kinase activities in response to serum stimulation. Experiments were performed as in (C), and the amounts of phosphorylated MLC were normalized by those of DAP kinase input in the kinase reactions. Data are expressed as fold inductions relative to cells that were not stimulated by serum. Each value represents means ± s.d. from three independent experiments.
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Fig. 7. DAP kinase does not stimulate the formation of focal adhesions. (A,B) NIH3T3 cells co-transfected with various DAP kinase constructs and GFP at a ratio of 10:1 were serum starved for 6 hours and then stained with anti-paxillin (A) or anti-vinculin (B). The transfected cells are visualized by GFP fluorescence. (C) Quantitation of cells with focal adhesions in experiments as described in (A). The values shown are means ± s.d. from at least three independent experiments, and more than 300 individual cells were counted for each experiment. (D) NIH3T3 cells infected with recombinant retrovirus carrying DAP kinase or DAPK CaM were serum starved and then double stained with rhodamine-phalloidin (red) and anti-paxillin or anti-vinculin (green). Alternatively, NIH3T3 cells were treated with 200 ng ml–1 LPA for 15 minutes and then subjected to the same immunostaining. Images shown are the superimpositions of red and green fluorescence. (E) NIH3T3 cells transfected with DAP kinase were serum starved for 6 hours and then triple stained with rhodamine-phalloidin (a), anti-paxillin followed by FITC-conjugated anti-mouse secondary antibody (b) and anti-DAP-kinase followed by AMCA-conjugated anti-rabbit secondary antibody (c). The overlay of three images is shown in (d). Scale bars, 10 µm.
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Fig. 8. IRM analysis shows the inability of DAP kinase to induce focal adhesion assembly. NIH3T3 cells cotransfected with various constructs as indicated together with GFP were serum starved and then subjected to IRM analysis. Transfected cells are visualized by GFP fluorescence and marked with white arrows in the IRM images. In cells transfected with control vector, DAP kinase or DAPKCaM, matched phase-contrast images are shown on the left to indicate the positions of the cells. Scale bars, 10 µm.
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Fig. 9. DAP kinase promotes focal adhesion disassembly under serum-stimulated conditions. (A) NIH3T3 cells co-transfected with various DAP kinase constructs and GFP were cultured in serum-containing medium. Two days after transfection, cells were fixed and stained with anti-paxillin. The percentages of GFP-positive cells that display a weaker paxillin-staining pattern than the neighboring GFP-negative cells were quantified and are indicated on the right. The values shown are means ± s.d. from at least three independent experiments. (B) Cells as in (A) were directly analysed by IRM without fixation. (A,B) Transfected cells are visualized by GFP fluorescence and marked with arrows; (A) the phase-contrast image is also included. Scale bars, 10 µm.
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Fig. 10. DAP kinase does not affect stress fibers under serum-stimulated conditions. (A) NIH3T3 cells cotransfected with DAP kinase and GFP were cultured in serum-containing medium. Two days after transfection, cells were stained with rhodamine-phalloidin. Scale bars, 10 µm. (B) NIH3T3 cells transfected with DAP kinase or control vector together with GFP were cultured and stained as in (A). The percentages of GFP-positive cells containing stress fibers were quantified and plotted.
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© The Company of Biologists Ltd 2003
