MULTIPLE PHOSPHORYLATION OF RIBOSOMAL-PROTEIN S6 AND SPECIFIC PROTEIN-SYNTHESIS ARE REQUIRED FOR PROTHORACICOTROPIC HORMONE-STIMULATED ECDYSTEROID BIOSYNTHESIS IN THE PROTHORACIC GLANDS OF MANDUCA-SEXTA

Prothoracicotropic hormone (PTTH)-stimulated protein phosphorylation leads to ecdysteroidogenesis (molting hormone biosynthesis) in the prothoracic glands of the tobacco hornworm, Manduca sexta. The phosphorylation of 34 and 50 kDa peptides (p34 and p50) paralleled the increase in ecdysteroidogenesis, and the dephosphorylation of p34 and p50 preceded a decrease in ecdysteroidogenesis. Inhibition by rapamycin of p34, but not p50, phosphorylation prevented PTTH-stimulated ecdysteroidogenesis in a dose-dependent manner, suggesting that p34 phosphorylation is requisite for PTTH-stimulated ecdysteroidogenesis. Two proteins whose synthesis was rapidly stimulated by PTTH were p50 and p70. The time-course of PTTH-stimulated synthesis of p50 paralleled that of p34 phosphorylation and that of ecdysteroidogenesis, Rapamycin inhibited PTTH-stimnlated synthesis of p50 and p70, suggesting that specific protein synthesis is also required for PTTH-stimulated ecdysteroidogenesis, confirming the results of Rybczynski and Gilbert [(1994) Insect Biochem, Molec, Biol, 24, 175-189], and that p34 phosphorylation may regulate the downstream synthesis of p50 and p70, possible key regulatory proteins leading to ecdysteroidogenesis. Results from two-dimensional (2D)-PAGE analysis of the ribosomal proteins purified from prothoracic glands, demonstrated that p34 is indeed ribosomal S6, and is phosphorylated at up to five sites (P-1-5) upon PTTH stimulation. The multiple phosphorylation of S6 was inhibited completely by rapamycin as shown in 2D gel maps, further confirming that p34 is ribosomal protein S6. Temporal analysis of PTTH-stimulated S6 phosphorylation by 2D-PAGE revealed that phosphorylation of S6 at the P-1 site was temporally correlated with the initiation of ecdysteroidogenesis, and that multiple phosphorylation at all five sites (P-1-5) was correlated with the maximal synthesis of ecdysteroids. Dephosphorylation of S6 was accompanied by a decrease in ecdysteroidogenesis. These data demonstrate that p34 is ribosomal protein S6 and that both the phosphorylation of S6 and specific protein synthesis are required for PTTH-stimulated ecdysteroidogenesis in the prothoracic gland.