Protein phosphorylation is one of several representative post translational modifications. Cyclic AMP-dependent protein kinase (PKA) plays the crucial and varying role of signal transduction. On the other hand, ras protein plays an important role in cell proliferation and growth. Although a previous report showed that H-ras protein was phosphorylated by PKA, the stiochiometry was not determined, so we investigated the stoichiometry of phosphorylation of the protein by PKA. H-ras cDNA inserted into a pGEX-2T expressing vector produced high levels of recombinant H-ras (rH-ras) in a fusion protein with glutathione S-transferase. rH-ras was obtained after cleavage by thrombin. Phosphorylation of ras protein by the catalytic subunit of PKA was performed, and the radioactivity was counted after SDS-PAGE and autoradiography. The results indicate that less than 0.1 mol of phosphate was incorporated per mol of H-ras protein, and suggest that H-ras protein could not be a physiologically meaningful substrate for PKA.
Arimura, S., Nakata, H., Tomiyama, K., & Watanabe, Y. (1997). Phosphorylation of H-ras proteins by protein kinase A. Cellular Signalling, 9(1), 37–40. https://doi.org/10.1016/S0898-6568(96)00090-3