Phosphorylation of glycogen synthase kinase-3β in relation to diapause processing in the silkworm, Bombyx mori

Abstract

Glycogen synthase kinase-3 (GSK-3) is a multifunctional protein kinase that plays important roles in regulating both glycogen synthesis and protein synthesis. In the present study, we investigated GSK-3β phosphorylation of silkworm eggs by immunoblotting with a conserved phospho-specific antibody to GSK-3β. Results showed that the temporal changes in GSK-3β phosphorylation were closely related to changes in glycogen levels previously reported by other researchers. In diapause eggs, an abrupt decrease in phosphorylation of GSK-3β was found with the onset of diapause, and phosphorylation level of GSK-3β reached a minimum level within 1 week after oviposition. However, when diapause eggs were incubated at 25 °C for 15 days and then transferred to 5 °C, a great increase in GSK-3β phosphorylation was observed 5 days after transfer to 5 °C and high levels were maintained throughout the chilling period. In both non-diapause eggs and eggs whose diapause initiation was prevented by HCl, levels of GSK-3β phosphorylation appeared to remain relatively high for several days and then greatly decreased 2 or 3 days before hatching. Moreover, GSK-3β phosphorylation dramatically increased when dechorionated eggs were incubated in medium. The addition of the mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK) kinase (MEK) inhibitor, U0126, did not inhibit GSK-3β phosphorylation in dechorionated eggs, although U0126 dose-dependently inhibited ERK phosphorylation. This result showed that ERK phosphorylation is not involved in upstream signaling for GSK-3β phosphorylation and that there may be two distinct signaling pathways involved in diapause processing in Bombyx mori eggs.