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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