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.
Sugimoto
Isamu
I
Division of Pathological Biochemistry, Department of Biomedical Sciences, School of Life Sciences, Faculty of Medicine, Tottori University, Nishi-cho, Yonago.
Li
Zhijun
Z
Sakamoto
Yoko
Y
Ito
Susumu
S
Hashimoto
Eikichi
E
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000276:immunology / Q000378:metabolism*; D005260:Female; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D009865:Oocytes / Q000187:drug effects* / Q000378:metabolism*; D020652:Peptide Elongation Factor 2 / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D010766:Phosphorylation; D012694:Serine / Q000378:metabolism; D013379:Substrate Specificity / Q000276:immunology; D013912:Threonine / Q000378:metabolism; D029867:Xenopus Proteins / Q000737:chemistry*; D014982:Xenopus laevis
D000906:Antibodies; D020652:Peptide Elongation Factor 2; D029867:Xenopus Proteins; D005576:Colforsin; D013912:Threonine; D012694:Serine; D017868:Cyclic AMP-Dependent Protein Kinases