Loss of PP2A binding to striatin causes Mst3 hyperphosphorylation. (A) FLAG-Mst3 (FL-Mst3) immunoprecipitates were prepared, and then aliquots were incubated without ATP, with ATP, or with ATP plus the kinase inhibitor, staurosporine. FLAG-Mst3 was visualized by immunoblotting. (B) Mst3 hyperphosphorylation caused by incubation of HEK293 cells with 100 nM okadaic acid for the times indicated retards the migration of Mst3 in SDS-PAGE. U, untreated. Vehicle Control, DMSO. (C) Three days after transfection of HEK293 cells with HA-tagged wild-type striatin and striatin mutants deficient in PP2A binding, HA-striatin immune complexes (HA-IP) and lysates were prepared and immunoblotted. (D) Relative hyperphosphorylation of Mst3 in striatin complexes and lysates of transfected cells was quantitated by measuring the ratios of the upper and lower bands of Mst3 using a chemilumimager and normalizing to wild-type HA-striatin. The error bars represent the standard error of at least four independent experiments. *, p ≤ 0.05; **, p ≤ 0.01 relative to wild-type. The p values for R88S/K89E and 84A/94A/105A mutant effects in lysates were 0.19 and 0.06, respectively. (E) Three days after transfection of HEK293 cells with HA-tagged R100S/R101E striatin, HA-striatin immune complexes were prepared in the absence of phosphatase inhibitors, denatured, and divided into three equal portions. Samples were incubated without PP2A or with purified PP2A plus either DMSO (vehicle control) or 100 nM okadaic acid. After incubation, the Mst3 protein bands were detected by immunoblotting. All lanes are from the same gel and exposure but the first two lanes were originally separated by a blank lane.
Gordon et al. BMC Biochemistry 2011 12:54 doi:10.1186/1471-2091-12-54