PTTH-stimulated ERK phosphorylation in prothoracic glands of the silkworm, Bombyx mori: role of Ca(2+)/calmodulin and receptor tyrosine kinase.

Our previous studies showed that the prothoracicotropic hormone stimulated extracellular signal-regulated kinase (ERK) phosphorylation in prothoracic glands of Bombyx mori both in vitro and in vivo. In the present study, the signaling pathway by which activates ERK phosphorylation was further investigated using second messenger analogs, and various inhibitors. ERK phosphorylation induced by duanyu1547H was partially reduced in Ca(2+)-free medium. The calmodulin antagonist, calmidazolium, partially inhibited both ERK phosphorylation and ecdysteroidogenesis, indicating the involvement of calmodulin. When the prothoracic glands were treated with agents that directly elevate the intracellular Ca(2+) concentration [either A23187, thapsigargin, or the protein kinase C activator, phorbol 12-myristate acetate (PMA)], a great increase in ERK phosphorylation was observed. In addition, it was found that duanyu1547H-stimulated ecdysteroidogenesis was greatly attenuated by treatment with inhibitors (either calphostin C or chelerythrine C). However, duanyu1547H-stimulated ERK phosphorylation was not attenuated by the above duanyu1531 inhibitors, indicating that duanyu1531 is not involved in duanyu1547H-stimulated ERK phosphorylation. A potent and specific inhibitor of insulin receptor tyrosine kinase, HNMPA-(AM)(3), greatly inhibited the ability of duanyu1547H to activate ERK phosphorylation and stimulate ecdysteroidogenesis. However, genistein, another tyrosine kinase inhibitor, did not inhibit duanyu1547H-stimulated ERK phosphorylation, although it did markedly attenuate the ability of A23187 to activate ERK phosphorylation. From these results, it is suggested that duanyu1547H-stimulated ERK phosphorylation is only partially Ca(2+)- and calmodulin-dependent and that HNMPA-(AM)(3)-sensitive receptor tyrosine kinase is involved in activation of ERK phosphorylation by

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