GRK6 binds to and is regulated by the chaperone HSP90 in human myeloma cells and is required for phospho-STAT3 pathway signaling. (A) 3xFlag-tagged GRK6 protein complexes were affinity purified from 108 KMS11 myeloma cells using M2 antibody and separated by SDS-PAGE. A single GRK6 copurified protein (*) was detected by Coomassie staining, migrating at 90 kDa, and was subsequently identified by nanoLC-ESI-MS/MS as HSP90A/B. Additional immunoprecipitation (IP) Coomassie staining bands reflect M2 antibody fragments and “sticky” lysate proteins retained nonspecifically by sepharose. The identity of isolated 3xFLAG-GRK6 was confirmed by nanoLC-ESI-MS/MS. (B-C) HSP90 potently and rapidly (< 24 hours) regulates GRK6 protein levels in KMS11 (B) and OPM1 (C) myeloma cells. Both myeloma tumor lines were treated with the HSP90 inhibitor, geldanamycin, 1μM, for the specified times, and were then assayed for ensuing effects on GRK6 protein expression by immunoblot. (D) To assess the effect and kinetics of GRK6 inhibition on the activity of myeloma survival pathways, FLAG-GRK6–expressing OPM1 cells were infected with control lentivirus expressing nontargeting shRNA, or with lentivirus expressing GRK6 shRNA, and the phospho-activation status of key intracellular signaling mediators AKT, MAPK p42 and p44 (ERK1/2), JNK, and STAT3 was determined by immunoassay sequentially from 24 to 96 hours after viral shRNA infection. MCL1 phosphorylation and levels were examined in parallel.
