FIGURE 4

In vitro phosphorylation of Akt by CK2. Incubations were performed for 20 min in the presence of radioactive phosphorylation mixture (see Materials and Methods), with the indicated protein kinases. In all, 0.2 g of active or unactive Akt1 was used. Under the conditions used, the CK2 (0.1 g) or CK2 _{2 2} (0.02 g) amounts were equally active towards casein substrate (not shown). (a) An autoradiography is shown of phospho-proteins resolved on SDS/PAGE. Autophosphorylation of CK2 subunit is also visible. Stoichiometry of Akt1 phosphorylation was about 0.2 mol phosphate/mol protein. (b) Autoradiography of the high-voltage paper electrophoresis for the identification of the phospho-amino acid. Samples from lanes 4, 5, and 7 of panel a were hydrolyzed in 6 N HCl, and subjected to high-voltage paper electrophoresis, as described in Material and Methods, for the identification of the phospho-amino acid. An autoradiography of the paper is shown. The position of orthophosphate (Pi) and phospho-amino acids (Sp, Yp, and Tp), determined by cold standard migration, is indicated by the arrows