BcMkk1 impedes BcRim15 phosphorylation mediated by BcSch9.
2018-09-13T18:14:52Z (GMT) by
<p>(A) The interactions of BcRim15 with BcMkk1 and BcSch9 were verified by the Y2H assays. Serial concentrations of yeast cells were drop-plated on SD-Leu-Trp-His plates. A pair of plasmids pGBKT7-53 and pGADT7 was used as a positive control. A pair of plasmids pGBKT7-Lam and pGADT7 was used as a negative control. (B) Domain architecture of the BcRim15 identified by Pfam (<a href="http://pfam.xfam.org/" target="_blank">http://pfam.xfam.org/</a>). (C) Phosphorylation of BcRim15<sup>PK</sup> (the protein kinase domain of BcRim15) in ΔBcMkk1 was dramatically higher than that in the wild type, and was reduced in the double mutant ΔBcMkk1-BcSch9. (D) Acid production and protease activity of each strain. (E) Both BcMkk1 and BcSch9 bind directly to BcRim15<sup>PK</sup>. Proteins associated with the GST beads or in the total proteins before GST pull-down (input) were probed by western blot analysis using the monoclonal mouse anti-His or anti-GST antibodies. (F) BcMkk1 inhibits the interaction of BcSch9 with BcRim15<sup>PK</sup>. BcRim15<sup>PK</sup> bound to Ni sepharose beads was incubated with 5 μg of BcSch9-GST and different amounts of BcMkk1-GST as indicated. Proteins associated with the beads or in the total proteins before His pull-down (input) were probed by western blot analysis using the monoclonal mouse anti-His or anti-GST antibodies. (G) Quantification of the binding data in (F). The relative binding activity of BcSch9 with BcRim15<sup>PK</sup> is calculated as the BcSch9 band intensity at a given concentration of BcMkk1 divided by the BcSch9 band intensity in the absence of BcMkk1. Values on the bars followed by the same letter are not significantly different at <i>P</i> = 0.05.</p>