Fig. 4. Requirements of the Thr⁴⁴⁰ and Ser⁴³⁵ phosphosites for Ric-8A-stimulated Gα_q steady-state GTP hydrolysis activity.

(A to F) Purified Gα_q (50 nM) was incubated with the indicated concentrations of purified Ric-8A proteins and [ γ−³²P]GTP. The linear rate of GTP hydrolysis was determined by measuring the production of free ³²Pi. (A) Full-length WT Ric-8A proteins purified from insect cells (Hi5) and treated with or without alkaline phosphatase or purified from E. coli and treated with or without CK2 were tested for their ability to stimulate the steady-state GTPase activity of Gα_q. (B) WT Ric-8A-ACT protein purified from E. coli and treated with or without CK2 was tested for its ability to stimulate the steady-state GTPase activity of Gα_q. (C to F) WT Ric-8A purified from insect cells (Hi5) was used as a positive standard of phosphorylated Ric-8A activity for comparison to the activities of (C) Ric-8A–T440A purified from insect cells and Ric-8A–T440A purified from E. coli and treated with or without CK2, (D) Ric-8A–S435A purified from insect cells and Ric-8A-S435A purified from E. coli and treated with or without CK2, (E) Ric-8A-T440D purified from insect cells and Ric-8A-T440D purified from E coli and treated with or without CK2, and (F) Ric-8A-S435D purified from insect cells and Ric-8A-S435D purified from E. coli and treated with or without CK2. Data were plotted on semilog graphs and fitted to one-phase exponential association functions using GraphPad Prism. Experiments were performed in triplicate. Error bars indicate the SEM and were sometimes smaller than the size of the plotted symbols.