Phosphorylation of VeA by the MAP kinase AnFus3 [MpkB] and influence of AnFus3 on the interactions of the velvet complex.

Abstract

<p>(A) <i>In vitro</i> phosphorylation of VeA by AnFus3 in a radioactive kinase assay. Left panels; autoradiograph of dried SDS gel run for phosphorylation reactions (30 µl of total 45 µl reaction volume), Coomassie stain of the proteins from phosphorylation reaction (10 µl of total 45 µl reaction). VeA protein in AnFus3 reaction tube is shown with red rectangle. Right panels; ponceau staining of the immunoprecipitated (immobilized to the GFP trap sepharose) AnFus3::sGFP and only sGFP protein. Immunodetection of the fusion protein and free sGFP by the α-gfp. (B) Confirmation of specific VeA phosphorylation by a non-radioactive method. All recombinant proteins (10 µg each) were treated with both AnFus3 and GFP. AnFus3 treated samples were additionaly incubated with the lambda protein phosphatase (λ-PP). Proteins were immunodetected by α-Penta His, α-GST. After AnFus3 treatment, VeA showed a 3–5 kDA molecular weight shift (red arrow) that disappeared after L-PP treatment. Only VeA treated with MAPK was recognized by P-ser/thr specific antibody. (C) Protein levels of VeA in the wild type and <i>mpkB</i> mutant background. VeA::cTAP signals were normalized to the internal actin levels. VeA protein levels did not change in the absence of MpkB. (D) Reduced velvet complex formation in the <i>mpkB</i> mutant. The VeA-associated proteins from the cultures of the wild type and <i>mpkB</i>Δ strains grown in the darkness sexually at 30°C for 20 hours. Three independent experiments were performed and the associated proteins were identified. The ratio of the peptides from VelB and LaeA to the VeA protein drastically reduced in the MAPK mutant, whereas alpha importin KapA interaction slightly increased. Black bars represent the standard error. *LaeA was only found in one of the three purifications in <i>mpkB</i>Δ strain, thus no error bar is assigned.</p

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