Fig. 4

HSP90 increased PKM2 phosphorylation at Thr-328. a HA-tagged PKM2 was co-transfected with empty vector or Flag-tagged HSP90 in HEK293 cells. Representative IP experiments were performed to examine PKM2 Ser/Thr phosphorylation. Total cell lysates were subjected to immunoblotting analysis using specific antibodies against HA and Flag. Forced expression of Flag-tagged HSP90 in HEK293 cells increased the phosphorylation of HA-tagged PKM2. b Huh7 cells were transfected with empty vector or Flag-tagged HSP90. Representative IP experiments were performed to examine PKM2 Ser/Thr phosphorylation. Total cell lysates were subjected to immunoblotting analysis using specific antibodies against HSP90 and GAPDH. Overexpression of HSP90 increased PKM2 Ser/Thr phosphorylation in Huh7 cells. c Hep3B cells were transfected with negative control (NC) shRNA or HSP90 shRNA. Representative IP experiments were performed to examine PKM2 Ser/Thr phosphorylation. Total cell lysates were subjected to immunoblotting analysis using specific antibodies against HSP90 and GAPDH. Knockdown of HSP90 decreased PKM2 Ser/Thr phosphorylation in Hep3B cells. d Phosphor-specific protein enrichment and MS identified two putative phosphorylation sites in PKM2 regulated by HSP90. S405 and T326 residues were highlighted by red. e Huh7 cells were transfected with empty vector or Flag HSP90, along with PKM2-S405A mutant (Serine 405 to alanine mutation) or HA-T328A mutant (Threonine 328 to alanine mutation). T328A mutant, instead of S405A, abrogated the increased phosphorylation of PKM2 induced by HSP90 overexpression. f Huh7 cells were co-transfected with empty vector or Flag-HSP90, and, HA-tagged wild type (WT) PKM2 or HA-tagged T328A mutant PKM2. The protein half-life of HA-tagged WT or mutated PKM2 was analyzed following treatment with cycloheximide (CHX). HSP90 overexpression increased the half-life of the wild type PKM2 while failed to increase the half time of T328A PKM2 mutant