It is well known that phosphorylation of extracellular signal‐regulated kinase (ERK) is involved in prothoracicotropic hormone (PTTH)‐stimulated ecdysteroidogenesis in insect prothoracic glands (PGs). In the present study, we further investigated the downstream signalling pathways. Our results showed that PTTH stimulated p90 ribosomal S6 kinase (RSK) phosphorylation at Thr573 in Bombyx mori PGs both in vitro and in vivo. The in vitro PTTH stimulation was stage‐ and dose‐dependent. The absence of Ca2+ reduced PTTH‐stimulated RSK phosphorylation. Stimulation of RSK phosphorylation was also observed after treatment with either A23187 or thapsigargin. A phospholipase C (PLC) inhibitor, U73122, blocked PTTH‐stimulated RSK phosphorylation. These results indicate the involvement of Ca2+ and PLC. Treatment with diphenylene iodonium (DPI), a mitochondrial oxidative phosphorylation inhibitor, blocked PTTH‐regulated RSK phosphorylation, indicating its redox regulation. A mitogen‐activated protein kinase (MAPK)/ERK kinase (MEK) inhibitor, U0126, but not a phosphatidylinositol 3‐kinase (PI3K) inhibitor, LY294002, decreased PTTH‐stimulated RSK phosphorylation, indicating that ERK is an upstream signalling. A protein kinase C (PKC) inhibitor, chelerythrine C, inhibited PTTH‐stimulated RSK phosphorylation, and a PKC activator, phorbol 12‐myristate acetate (PMA) stimulated RSK phosphorylation, indicating the involvement of PKC. BI‐D1870, a specific RSK inhibitor, partly prevented PTTH‐stimulated RSK phosphorylation and significantly inhibited PTTH‐stimulated ecdysteroid secretion, indicating that PTTH‐stimulated RSK phosphorylation is involved in ecdysteroidogenesis. Taken together, these data indicate that PTTH activates RSK phosphorylation which plays important roles in PTTH‐stimulated ecdysteroidogenesis.

In the present study, the participation of protein kinase C (PKC) signalling in prothoracicotropic hormone (PTTH)-stimulated ecdysteroidogenesis in Bombyx prothoracic glands (PGs) is demonstrated and characterized. PTTH stimulated phosphorylation of a 37-kDa protein in Bombyx PGs both in vitro and in vivo, as recognized by a PKC substrate antibody. Treatment with either A23187 or thapsigargin also stimulated this 37-kDa protein phosphorylation. PTTH-stimulated phosphorylation of the 37-kDa protein was markedly attenuated in the absence of Ca 2+ . The phospholipase C (PLC) inhibitor, U73122, greatly inhibited PTTHstimulated phosphorylation of this protein, indicating the involvement of Ca 2+ and PLC. A mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor (U0126), a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002) and a chemical activator of adenosine 5 0 -monophosphate-activated protein kinase (AMPK) (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside) did not affect PTTHstimulated phosphorylation of the 37-kDa protein, implying that ERK and PI3K/AMPK are not the upstream signalling pathways for PKC-dependent protein phosphorylation. The mitochondrial oxidative phosphorylation inhibitors (the uncoupler carbonyl cyanide ptrifluoromethoxyphenylhydrazone and diphenylene iodonium) inhibited PTTH-stimulated phosphorylation of the 37-kDa protein, indicating its redox regulation.Treatment with PKC inhibitors (either calphostin C, chelerythrine C or rottlerin) reduced PTTH-stimulated phosphorylation of the 37-kDa protein. PTTH-stimulated ecdysteroidogenesis was also inhibited by treatment with rottlerin, thus further confirming participation of PKC-dependent phosphorylation in PTTH signalling. From these results, we demonstrated that redoxregulated PTTH-stimulated PKC signalling is involved in ecdysteroid secretion in Bombyx PGs.