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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.

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