(A) HEK293 cells transfected with either wild-type V5-tau or V5-tau harboring the double mutation of S262A/S356A were cotransfected with myc-CHIP, PAR-1, or both. V5 coimmunoprecipitation showed that CHIP bound to and greatly enhanced the polyubiquitination of wild-type tau; however, PAR-1 phosphorylation of tau prevented this interaction. Mutation of the S262 and S356 sites to alanine residues attenuated CHIP binding; however, ubiquitination activity was entirely abrogated. Inputs confirmed the hyperphosphorylation of wild-type tau at the S262/S356 residues in the presence of PAR-1. (B) HeLa cells expressing wild-type V5-tau were cotransfected with constitutively active GSK3β, PAR-1, or empty vector and then treated with EC102 or vehicle. In the absence of EC102, constutively active GSK3β promoted the phosphorylation of tau at S396/S404 relative to empty vector and PAR-1 promoted the phosphorylation of tau at S262/S356 relative to empty vector. In the presence of EC102, phosphorylated S396/S404 was dramatically reduced compared with treatment with vehicle, while phosphorylated S262/S356 was largely unaffected by drug treatment. (C) HeLa cells expressing wild-type V5-tau were cotransfected with constitutively active GSK3β or empty vector. Tau was then coimmunoprecipitated with the V5 antibody, and Hsp90 binding was assessed. Hsp90 bound more avidly to tau phosphorylated by constutively active GSK3β compared with cells transfected with vector alone.