S6 PHOSPHORYLATION RESULTS FROM PROTHORACICOTROPIC HORMONE STIMULATION OF INSECT PROTHORACIC GLANDS - A ROLE FOR S6 KINASE

The insect prothoracic glands are the source of steroidal molting horm one precursors and the glands are stimulated by a brain neuropeptide, prothoracicotropic hormone (PTTH). Previous work from this laboratory revealed that PTTH acts via a cascade including Ca2+/calmodulin activa tion of adenylate cyclase, protein kinase A, and the subsequent phosph orylation of a 34 kDa protein (p34) hypothesized, but not proven, to b e the S6 protein of the 40S ribosomal subunit. The immunosuppressive m acrolide, rapamycin, is a potent inhibitor of cell proliferation, a si gnal transduction blocker, and also prevents ribosomal S6 phosphorylat ion in mammalian systems. We demonstrate here that rapamycin inhibited PTTH-stimulated ecdysteroidogenesis in vitro by the prothoracic gland s of the tobacco hornworm, Manduca sexta, with half-maximal inhibition at a concentration of about 5 nM. At concentrations above 5 nM, there was a 75% inhibition of ecdysteroid biosynthesis. Similar results wer e observed with the calcium ionophore (A23187), a known stimulator of ecdysteroidogenesis. Most importantly, the inhibition of ecdysteroid b iosynthesis was accompanied by the specific inhibition of the phosphor ylation of p34, indicating that p34 indeed is ribosomal protein S6. In vivo assays revealed that injection of rapamycin into day 6 fifth ins tar larvae resulted in a decreased hemolymph ecdysteroid titer and a d ose-dependent delay in molting and metamorphosis. When S6 kin ase (S6 K) activity was examined using rapamycin-treated prothoracic glands as the enzyme source and a synthetic peptide (S6-21) ora 40S ribosomal s ubunit traction from Manduca tissues as substrate, the date revealed t hat rapamycin inhibited S6K activity. The composite data suggest that rapamycin inhibits a signal transduction element leading to p34 phosph orylation that is necessary for PTTH-stimulated ecdysteroidogenesis in this insect endocrine gland, and lend further support to the concept that p34 is S6. (C) 1994 Wiley-Liss, Inc.