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 l eads to ecdysteroidogenesis (molting hormone biosynthesis) in the prot horacic glands of the tobacco hornworm, Manduca sexta. The phosphoryla tion of 34 and 50 kDa peptides (p34 and p50) paralleled the increase i n ecdysteroidogenesis, and the dephosphorylation of p34 and p50 preced ed a decrease in ecdysteroidogenesis. Inhibition by rapamycin of p34, but not p50, phosphorylation prevented PTTH-stimulated ecdysteroidogen esis in a dose-dependent manner, suggesting that p34 phosphorylation i s requisite for PTTH-stimulated ecdysteroidogenesis. Two proteins whos e synthesis was rapidly stimulated by PTTH were p50 and p70. The time- course of PTTH-stimulated synthesis of p50 paralleled that of p34 phos phorylation 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, co nfirming 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 prote ins leading to ecdysteroidogenesis. Results from two-dimensional (2D)- PAGE analysis of the ribosomal proteins purified from prothoracic glan ds, demonstrated that p34 is indeed ribosomal S6, and is phosphorylate d at up to five sites (P-1-5) upon PTTH stimulation. The multiple phos phorylation of S6 was inhibited completely by rapamycin as shown in 2D gel maps, further confirming that p34 is ribosomal protein S6. Tempor al analysis of PTTH-stimulated S6 phosphorylation by 2D-PAGE revealed that phosphorylation of S6 at the P-1 site was temporally correlated w ith the initiation of ecdysteroidogenesis, and that multiple phosphory lation at all five sites (P-1-5) was correlated with the maximal synth esis of ecdysteroids. Dephosphorylation of S6 was accompanied by a dec rease in ecdysteroidogenesis. These data demonstrate that p34 is ribos omal protein S6 and that both the phosphorylation of S6 and specific p rotein synthesis are required for PTTH-stimulated ecdysteroidogenesis in the prothoracic gland.