Fig. 6: GSK3β interaction with CDK5RAP2 inhibits its kinase activity.

A Lysates of BJ cells co-transfected with Myc-tagged CDK5RAP2 and FLAG-tagged GSK3β (upper panel) were subjected to immunoprecipitation (IP) using FLAG antibody (middle panel) or Myc antibody (lower panel) then analyzed for CDK5RAP2 and GSK3β co-immunoprecipitation by immunoblotting both IPs with Myc and FLAG antibodies. B. CDK5RAP2 directly interacts with GSK3β. GST-CDK5RAP2 bound to GSH-agarose beads was mixed with GSK3β as described in Materials and Methods. The resulting complexes were washed extensively then analyzed by SDS-PAGE and immunoblotting for GST and GSK3β. C Lysates of cells transfected with CDK5RAP2 siRNA #2 for 3 days were analyzed by SDS-PAGE and immunoblotting for CDK5RAP2, phosphoSer9-GSK3β, and GSK3β (left panel). Representative blots are from one of three independent experiments (n = 3) showing similar results. Ratios of levels of phosphoSer9-GSK3β vs GSK3β and standard deviation for the 3 independent sets of experiments (right panel) were calculated as described for CDK5RAP2 vs actin in Fig. 1, with values from cells transfected with control siRNA normalized to 1.0. *p = 0.01. D Lysates of cells transfected with CDK5RAP2 siRNA #2 for 3 days were subjected to IP using GSK3β antibody. The IPs were then examined for in vitro GSK3β kinase activity as described in Materials and Methods. Data represent means ± SD from three independent experiments (n = 3). *p = 0.0027. E Inhibition of GSK3β activity with TWS119 (5 μM in DMSO) in cells depleted of CDK5RAP2 restores β-catenin and WIP1 expression (left panel). Representative blots from one of three independent experiments (n = 3) showing similar results are shown. Ratios of levels of β-catenin and WIP1 vs actin and standard deviation for the 3 independent sets of experiments (right panel) were calculated as described for CDK5RAP2 vs actin in Fig. 1, with values from cells transfected with control siRNA and treated with DMSO normalized to 1.0. *p < 0.05.