Figure 5
PKA phosphorylates GPKOW at S27 and T316 in vitro. A. Recombinant GPKOW proteins were expressed in bacteria and purified on a HisTrap column followed by an ion exchange column. GPKOW wt (lane 1), GPKOW S27A (lane 2), GPKOW T316A (lane 3) and GPKOW S27A+T316A (lane 4) were all recognized by anti-GPKOW B01 after separation on SDS-PAGE and immunoblotting. One representative immunoblot is shown. B. Purified GPKOW wt (lanes 1 and 2), single- (lanes 3-6) or double-mutated GPKOW (lanes 7 and 8) were incubated with active (+) or heat inactivated (-) PKA Cα1 (7.4 ng) and γ-32P-ATP in a reaction buffer. The samples were analyzed by SDS-PAGE and autoradiography. The CBB staining in the lower panel shows the amount of the different proteins. C. Unsaturated images from Syngene G-box and Typhoon 9400 phosphoimager were quantified by Genetools (Syngene) and the statistical analysis was performed with paired students T-test in GraphPad Prism. The asterisk indicates a significant difference between the columns with a p-value < 0.01 (n = 3). D. 293T cells were harvested and lysed. The lysates were adjusted to equal protein concentration, precleared with magnetic beads before immunoprecipitation using anti-GPKOW (lane 1-2) and magnetic beads. Sample 2 was treated with alkaline phosphatase for 30 minutes before both samples were analyzed by SDS-PAGE and immunoblotting using anti-GPKOW. One representative experiment is shown (n = 3).