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First published online 25 September 2007
doi: 10.1242/jcs.003251

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DAP kinase mediates the phosphorylation of tropomyosin-1 downstream of the ERK pathway, which regulates the formation of stress fibers in response to oxidative stress

Le Centre de recherche en cancérologie de l'Université Laval, 9 rue McMahon, Québec G1R 2J6, Canada

View larger version (26K): [in this window] [in a new window]   Fig. 1. Phosphorylation of tropomyosin-1 downstream of ERK and its inhibition by ML-7. (A) Exponentially growing COS cells, HEK293 cells and HUVECs were extracted and processed for immunodetection of tropomyosin and actin using specific antibodies. (B) Exponentially growing HEK293 cells were transfected with a plasmid expressing wild-type (wt) tropomyosin-1. The next day, cells were incubated in phosphate-free culture medium in the presence of H3[32P]O4 for 90 minutes. Cells were pre-treated for 60 minutes with vehicle (DMSO 0.25%), PD098059 (50 µM; P) or UO0126 (50 µM; U), and treated or not with H2O2 for 30 minutes (250 µM) in the presence of the phosphatase inhibitor NaF (1 mM). The proteins were extracted and were subject to immunoprecipitation with a monoclonal antibody against tropomyosin (TM). Immunoprecipitated proteins were separated by SDS-PAGE. Dried gels were analyzed with PhosphorImager. Lower panel: extracts were made before and after transfection and were processed for immunodetection of tropomyosin. P-TM, phospho-tropomyosin. (C) HUVECs plated in a gelatin-coated Petri dish were electroporated with a plasmid expressing FLAG-tagged tropomyosin-1. The next day, cells were pre-treated or not with PD098059 (50 µM for 60 minutes), UO0126 (50 µM for 60 minutes), treated or not with H2O2 (250 µM for 30 minutes) and afterwards were extracted in IEF buffer. Proteins were separated by 2D electrophoresis (pH 4.5-5.5), transferred onto a nitrocellulose membrane and subject to immunodetection using a monoclonal against the FLAG epitope. (D) HUVECs plated in gelatin-coated Petri dishes were electroporated with a plasmid expressing HA-tagged tropomyosin-1. The next day, cells were pre-treated or not with ML-7 (25 µM for 60 minutes), treated or not with H2O2 (250 µM for 30 minutes) and were processed as in C, except that immunodetection was performed using a monoclonal antibody against HA.

View larger version (25K): [in this window] [in a new window]   Fig. 2. ERK does not directly phosphorylate tropomyosin-1. (A,B) Exponentially growing HUVECs were pre-treated with vehicle only (DMSO, 0.25%) or with the MEK inhibitor UO126 (50 µM, 60 minutes; U) and were treated or not with H2O2 (250 µM, 10 minutes). Then cells were extracted and processed for ERK immunoprecipitation. Immunoprecipitated proteins were used for an in vitro kinase assay using [-32P]ATP and (A) myelin basic protein (MBP) or (B) rh-tropomyosin-1 (rh-TM-1) as substrates, as described in Materials and Methods. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. Incorporation of 32PO4 into the corresponding bands was visualized using PhosphorImager. Western blots of the immunoprecipitated ERK (lower panels) and loading control of the total MBP (middle panel in A) and total rh-TM-1 (middle panel in B) are shown.

View larger version (25K): [in this window] [in a new window]   Fig. 3. DAP kinase (DAPK) is activated by H2O2 and is sensitive to ML-7. (A) Direct in vitro kinase assay was performed using increasing amounts of a purified constitutively active form of MLCK that was incubated with purified MLC (upper panel) or purified rh-tropomyosin-1 (third panel) as substrates. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Incorporation of 32PO4 into the corresponding bands was visualized using PhosphorImager. Loading controls of the total MLC (second panel) and total rh-tropomyosin-1 (lower panel) are shown. (B) HEK293 cells were transfected with a plasmid expressing FLAG-tagged wild-type DAPK. The next day, cells were treated or not with H2O2 (250 µM). The proteins were extracted and were subject to immunoprecipitation with a monoclonal antibody against FLAG. Immunoprecipitated proteins were used for an in vitro DAPK assay using purified myosin light chain (MLC) as a substrate. A western blot for phospho-MLC (Ser19) is shown in the upper panel and loading controls of the total MLC (middle panel) and FLAG-DAPK are shown in the lower panel. Quantification of the phospho-Ser19 within MLC is shown. (C) HEK293 cells were transfected with a plasmid expressing FLAG-tagged wild-type DAPK. The next day, cells were treated or not with H2O2 for 10 minutes (250 µM) and processed as in B except that an increasing concentration of ML-7 (0-10 µM) was added in the kinase reaction mixture.

View larger version (43K): [in this window] [in a new window]   Fig. 4. H2O2-activated DAP kinase (DAPK) directly phosphorylates tropomyosin-1. (A) HEK293 cells were transfected with either a plasmid expressing GFP, FLAG-tagged wild-type (wt) DAPK or the dominant-negative FLAG-tagged DAPK (DAP K42A). The next day, cells were treated or not with H2O2 for 10 minutes (250 µM). The proteins were extracted and were subject to immunoprecipitation with a monoclonal antibody against FLAG. Immunoprecipitated proteins were used for an in vitro DAPK assay using recombinant tropomyosin-1 (rTM) as a substrate and transferred to a nitrocellulose membrane. Incorporation of 32PO4 into the corresponding bands was visualized using PhosphorImager. A western blot of DAPK expression is shown in the middle panel, and tropomyosin-1 loading is shown in the bottom panel. (B) A direct in vitro kinase assay was performed using increasing amounts of a purified constitutively active form of DAPK that was incubated with purified MLC (upper panel) or purified rh-tropomyosin-1 (third panel) as substrates. Proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Incorporation of 32PO4 into the corresponding bands was visualized using PhosphorImager. Loading controls of the total MLC (second panel) and total rh-tropomyosin-1 (lower panel) are shown.

View larger version (47K): [in this window] [in a new window]   Fig. 5. H2O2-induced DAP kinase (DAPK) activation is downstream of ERK. (A) HEK293 cells were transfected with plasmids expressing the dominant-negative FLAG-tagged DAPK (DAPK K42A). The next day, cells were incubated in phosphate-free culture medium in the presence of H3[32P]O4 for 90 minutes. Cells were pre-treated with vehicle only (DMSO, 0.25%) or with the MEK inhibitor PD098059 (50 µM, 60 minutes; P) and treated or not with H2O2 for 10 minutes (250 µM). The proteins were extracted and were subject to immunoprecipitation with a monoclonal antibody against FLAG. Immunoprecipitated proteins (IP) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. Incorporation of 32PO4 into the corresponding bands was visualized using Phosphor Imager. A loading control of the immunoprecipitated FLAG-DAPK is shown (second panel). Total extracts (TE) were kept before the immunoprecipitation to monitor ERK activation (third panel) by immunodetection of phospho-ERK. Total ERK is shown in the lower panel. (B) HEK293 cells were transfected with plasmids expressing the dominant-negative FLAG-tagged DAPK K42A or a double-mutant DAPK K42A S735A. The next day, cells were incubated in phosphate-free culture medium in the presence of H3[32P]O4 for 90 minutes. Cells were processed as in A. Incorporation of 32PO4 into the corresponding FLAG-DAPK bands was visualized using PhosphorImager. A loading control of the immunoprecipitated FLAG-DAPK is shown (lower panel). (C) HEK293 cells were transfected with plasmids expressing the dominant-negative FLAG-tagged DAPK K42A. The next day, cells pre-treated with vehicle only (DMSO, 0.25%) or with the ERK inhibitor UO126 (50 µM, 60 minutes; U) were treated or not with H2O2 (250 µM) for 10 minutes. The proteins were extracted and were subject to immunoprecipitation with a monoclonal antibody against FLAG. Immunoprecipitated proteins (IP) were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was processed for immunodetection of ERK (upper panel) and DAPK (second panel). Total extracts (TE) were kept before the immunoprecipitation and processed as in A. (D) Exponentially growing HUVECs were pre-treated for 60 minutes with vehicle only (0.25% DMSO) or with the MEK inhibitor UO126 (50 µM; U) and were treated or not with H2O2 (250 µM, 10 minutes). Next, cells were extracted and processed for endogenous DAPK immunoprecipitation. Immunoprecipitated proteins were used for an in vitro kinase assay using purified MLC as a substrate, as described in Materials and Methods. Proteins were separated by SDS-PAGE and transferred onto a nitrocellulose membrane. The membrane was processed for immunodetection of phospho-Ser19 within MLC (upper panel), the total MLC (second panel), the immunoprecipitated DAPK (third panel) or phospho-ERK (lower panel) using specific antibodies.

View larger version (36K): [in this window] [in a new window]   Fig. 6. DAP kinase (DAPK) regulates H2O2-induced phosphorylation of tropomyosin-1. (A) HUVECs plated in gelatin-coated Petri dishes were electroporated with or without a siRNA for DAP kinase. The next day, cells were incubated in phosphate-free culture medium in the presence of H3[32P]O4 for 90 minutes and were treated or not with H2O2 (250 µM, 30 minutes) in the presence of the phosphatase inhibitor NaF (1 mM). The proteins were extracted and were subject to immunoprecipitation with a monoclonal antibody against tropomyosin. Immunoprecipitated proteins were separated by SDS-PAGE and transferred onto nitrocellulose membrane. The membrane was analyzed with PhosphorImager (upper panel). Western blots of DAPK (middle panel) and the immunoprecipitated tropomyosin (lower panels) are shown. (B) HEK293 cells were transfected with plasmids expressing wild-type tropomyosin-1 together with a plasmid expressing GFP or FLAG-tagged wild-type (wt) DAPK or the dominant-negative FLAG-tagged DAPK (DAPK K42A). The next day, cells were incubated in phosphate-free culture medium in the presence of H3[32P]O4 for 90 minutes. Cells were treated or not with H2O2 (250 µM) for 30 minutes in the presence of the phosphatase inhibitor NaF (1 mM). The proteins were extracted and were subject to immunoprecipitation with a monoclonal antibody against tropomyosin. Immunoprecipitated proteins were separated by SDS-PAGE and transferred to a nitrocellulose membrane. The membrane was analyzed with PhosphorImager (upper panel). Western blots of the immunoprecipitated tropomyosin (middle panels) and DAPK (lower panel) are shown.

View larger version (23K): [in this window] [in a new window]   Fig. 7. Tropomyosin is phosphorylated on Ser283 in response to oxidative stress. (A) Sequence comparison between human myosin light chain II (MLC II), a known substrate of DAP kinase, and human tropomyosin-1. DAP kinase phosphorylates MLC II on Ser19 (gray box). Sequence similarity is highlighted by the open boxes. (B) rh-tropomyosin 1 was phosphorylated in vitro by purified active DAP kinase. Following fractionation by SDS-PAGE, tropomyosin-1 was analyzed by LC-MS for identification of the phosphopeptides. The analysis revealed 17 peptides that belong to tropomyosin 1. Among those peptides, only peptide AISEELDHALNDMTSM was phosphorylated. This peptide has an observed m/z of 934.72+, whereas a m/z of 894.8 was expected, which is characteristic of the presence of a phosphate group. Analysis of this peptide revealed that the loss of one molecule of H2O associated with the loss of the phosphate group occurs on Ser15 (MpS on the graph). This serine corresponds to Ser283 within full-length tropomyosin 1. (C) HEK293 cells were transfected with plasmids expressing wild-type (wt) tropomyosin-1 (TM-1), tropomyosin-1 Ser283Ala or GFP. The next day, the cells were incubated in phosphate-free culture medium in the presence of H3[32P]O4 for 90 minutes. Cells were treated or not with H2O2 (250 µM) for 30 minutes in the presence of the phosphatase inhibitor NaF (1 mM). The proteins were extracted and were subject to immunoprecipitation with a monoclonal antibody against tropomyosin. Immunoprecipitated proteins were separated by SDS-PAGE. Dried gels were analyzed with PhosphorImager. In the lower panel, cells were extracted before and after immunoprecipitation, were separated by SDS-PAGE and then processed for western blotting using a monoclonal antibody against tropomyosin. A, TM-1 Ser283Ala.

View larger version (23K): [in this window] [in a new window]   Fig. 8. Phosphorylation of tropomyosin-1 enhances stress fiber formation in endothelial cells. HUVECs were electroporated with GFP-actin along with either wild-type tropomyosin-1 (A), the nonphosphorylatable mutant of tropomyosin-1 (S283A) (B) or the phosphorylation-mimicking mutant of tropomyosin-1 (S283E) (C). Live cells were visualized by fluorescence microscopy.

View larger version (89K): [in this window] [in a new window]   Fig. 9. DAP-kinase-induced phosphorylation of Ser283 within tropomyosin-1 promotes actin stress fiber formation. (A) Exponentially growing HUVECs were plated in gelatin-coated Petri dishes after being co-electroporated without (a-d) or with (e-p) a siRNA for DAP kinase together with or without (a-h) a plasmid expressing either tropomyosin-1 mutant S283A (i-l) or S283E (m-p). A plasmid expressing GFP was co-electroporated in each condition as a marker of positive cells. The next day, cells were treated (c,d,g,h,k,l,o,p) or not (a,b,e,f,i,j,m,n) with H2O2 (250 µM) for 30 minutes. Thereafter, cells were fixed and stained for F-actin using Alexa-488-phalloidin (b,c,f,g,j,k,n,o) and for GFP using a rabbit polyclonal antibody against GFP, revealed with anti-rabbit-IgG Alexa 568 (a,d,e,h,i,l,m,p), and then were examined by confocal microscopy. (B) A quantification from the above experiments was performed. GFP-positive cells expressing transcytoplasmic actin stress fibers or membrane blebs were counted and the ratio over the total number of counted GFP-positive cells was calculated from two separated experiments. Results are representative of means ± s.e.m. of at least three experiments. (C) Extracts of the cells from the above experiment were made and were processed for immunodetection of DAP kinase (DAPK; upper panel) and actin (lower panel) using specific antibodies. C, control cells; D, DAPK siRNA cells; A, DAPK siRNA + TM-1 S283A cells; E, DAPK siRNA + TM-1 S283E cells.

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