Figure 9

Mutation of either threonine 178 or threonine 182 to alanine in the activation loop of Mst3 prevents the hyperphosphorylation-induced gel shift induced by okadaic acid (OA) treatment. Three days after transfection with empty vector or FLAG epitope-tagged wild-type (WT) or mutant Mst3 isoform b-expressing plasmids, HEK293 cells were treated with either DMSO or OA (100 nM) for four hours and then lysed. Lysates were denatured as described in Methods and FLAG-Mst3 was immunoprecipitated with anti-FLAG antibody. Cell lysates (Lysates) and FLAG-Mst3 immunoprecipitates (FLAG IP) were separated by SDS-PAGE and immunoblotted with anti-FLAG antibody (FL-Mst3) and phospho-specific antibody recognizing the threonine 178 autophosphorylation site of Mst3 (pMst3). The small arrows denote the position of IgG heavy chain in the immunoprecipitates. In anti-FLAG immunoblots of lysates from DMSO-treated cells (top panel), a background band is seen at the expected position of hyperphosphorylated Mst3 in all lanes. The fact that this band is present in the vector control lysate lane and absent in anti-FLAG Mst3 immunoprecipitates prepared from these lysates (bottom panel) shows that this band is not Mst3. Okadaic acid induces the presence of the upper band of FLAG-tagged Mst3 in wild-type (WT) and T172A Mst3-expressing cells (clearly darker than the vector control background band), but not in T178A and T182A Mst3-expressing cells (second panel from the top).