Mass-spectrometric identification of proteins detected in forskolin-stimulated Xenopus laevis oocytes using antibody against phospho-(Ser/Thr) cAMP-dependent protein kinase substrate

In order to study the phosphorylated proteins in the resting Xenopus laevis oocytes, the proteins detected by Western blotting using phospho-(Ser/Thr) PKA substrate antibody (PKA substrate antibody) in forskolin-stimulated oocytes were purified and identified by mass spectrometry. Several proteins (ribosomal S6 protein, elongation factor-2 (EF-2), poly A binding protein, releasing factor 1) were identified, and the phosphorylation of EF-2 was further studied. Partially purified Xenopus EF-2 (xEF-2) was phosphorylated by PKA in vitro and this phosphorylation was detected by Western blotting using PKA substrate antibody. The phosphorylation of Thr-57 in xEF-2 (corresponding to Thr-56 of the mammalian enzyme) was detected in the partially purified xEF-2 from the resting oocytes, but this xEF-2 did not react with the PKA substrate antibody. These results suggest that Thr-57 in xEF-2 was phosphorylated, but xEF-2 does not seem to be phosphorylated by PKA in resting oocytes although PKA can phosphorylate xEF-2 in vitro and probably in forskolin-treated oocytes.