Signaling involved in PTTH-stimulated 4E-BP phosphorylation in prothoracic gland cells of Bombyx mori.

Our previous studies showed that adenosine 5'-monophosphate-activated protein kinase (AMPK)/the target of rapamycin (TOR) signaling is involved in prothoracicotropic hormone ecdysteroidogenesis in Bombyx mori prothoracic glands (PGs). In the present study, we further investigated the signaling involved in phosphorylation of 4E-BP. We found that 4E-BP phosphorylation stimulated by was partially reduced in Ca2+-free medium, indicating the involvement of Ca2+. In addition, we found that a potent and specific inhibitor of phospholipase C (PLC), U73122, greatly inhibited 4E-BP phosphorylation. However, duanyu1547H-stimulated 4E-BP phosphorylation was not attenuated by a protein kinase C inhibitor (chelerythrine C). These results indicate that PLC, but not is involved in duanyu1547H-stimulated 4E-BP phosphorylation. When PGs were treated with agents that directly elevate the intracellular Ca2+ concentration (either A23187 or thapsigargin), a great increase in 4E-BP phosphorylation was observed. A23187-stimulated phosphorylation of 4E-BP was blocked by a chemical activator of AMPK (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside, AICAR) and a phosphoinositide 3-kinase (PI3K) inhibitor (LY294002), but not by U0126, indicating involvement of AMPK and PI3K. Determination of AMPK phosphorylation showed that treatment with either A23187 or thapsigargin inhibited AMPK phosphorylation. Moreover, duanyu1547H appeared to inhibit AMPK phosphorylation in a Ca2+-dependent manner. Altogether, these results indicate interconnections among Ca2+ signaling, AMPK, and 4E-BP phosphorylation in PGs of B. mori.

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