Protease-activated kinase II mediates multiple phosphorylation of ribosomal protein S6 in reticulocytes.

Ribosomal protein S6 was phosphorylated in vitro by the cAMP-independent protein kinase, protease-activated kinase II. The enzyme incorporated up to four phosphates into S6 as shown by analysis of the phosphorylated derivatives by two-dimensional gel electrophoresis. When tryptic digests of S6 phosphorylated by the enzyme were examined by two-dimensional peptide mapping, five phosphopeptides were observed. In contrast, only two phosphopeptides were identified with the cAMP-dependent protein kinase. One phosphopeptide was common to both peptide maps; the others were unique for each enzyme. The physiological significance of the phosphorylation by protease-activated kinase II was verified with phosphopeptide maps of S6 phosphorylated in intact cells. Highly phosphorylated forms of S6 were obtained in rabbit reticulocytes incubated in slightly acidified medium (pH 7.2 to 6.8). The phosphopeptide pattern contained the same five phosphopeptides observed with protease-activated kinase II in vitro indicating the pH-dependent phosphorylation of S6 was mediated by protease-activated kinase II.