Gu S-H, Chen C-H, Lin P-L
Department of Biology, National Museum of Natural Science, Taichung, Taiwan.
Chung Hwa University of Medical Technology, Tainan, Taiwan.
Insect Mol Biol. 2022 Feb;31(1):115-126. doi: 10.1111/imb.12744. Epub 2021 Nov 16.
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 . The phospholipase C (PLC) inhibitor, U73122, greatly inhibited PTTH-stimulated phosphorylation of this protein, indicating the involvement of Ca 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'-monophosphate-activated protein kinase (AMPK) (5-aminoimidazole-4-carboxamide-1-β-d-ribofuranoside) did not affect PTTH-stimulated 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 p-trifluoromethoxyphenylhydrazone 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 redox-regulated PTTH-stimulated PKC signalling is involved in ecdysteroid secretion in Bombyx PGs.
在本研究中，证明并表征了蛋白激酶C（PKC）信号通路参与家蚕前胸腺（PGs）中促前胸腺激素（PTTH）刺激的蜕皮激素合成过程。PKC底物抗体识别结果显示，PTTH在体外和体内均刺激家蚕PGs中一种37 kDa蛋白的磷酸化。用A23187或毒胡萝卜素处理也能刺激这种37 kDa蛋白的磷酸化。在无Ca的情况下，PTTH刺激的37 kDa蛋白磷酸化明显减弱。磷脂酶C（PLC）抑制剂U73122极大地抑制了PTTH刺激的该蛋白磷酸化，表明Ca和PLC参与其中。丝裂原活化蛋白激酶/细胞外信号调节激酶（ERK）激酶（MEK）抑制剂（U0126）、磷酸肌醇3激酶（PI3K）抑制剂（LY294002）以及5'-单磷酸腺苷激活蛋白激酶（AMPK）的化学激活剂（5-氨基咪唑-4-甲酰胺-1-β-D-呋喃核糖苷）均不影响PTTH刺激的37 kDa蛋白磷酸化，这意味着ERK和PI3K/AMPK不是PKC依赖性蛋白磷酸化的上游信号通路。线粒体氧化磷酸化抑制剂（解偶联剂羰基氰化物对三氟甲氧基苯腙和二苯碘鎓）抑制了PTTH刺激的37 kDa蛋白磷酸化，表明其氧化还原调节作用。用PKC抑制剂（钙泊三醇C、白屈菜红碱C或罗勒素）处理可降低PTTH刺激的37 kDa蛋白磷酸化。用罗勒素处理也抑制了PTTH刺激的蜕皮激素合成，从而进一步证实PKC依赖性磷酸化参与PTTH信号传导。从这些结果来看，我们证明了氧化还原调节的PTTH刺激的PKC信号通路参与家蚕PGs中的蜕皮激素分泌。
