Study on role of protein phosphorylation in sialagogue-stimulated parotid gland.

本实验观察了大鼠腮腺组织块在含硅透镜纸上无血清199培养液上培养时，促涎素刺激后蛋白磷酸化水平、鸟氨酸脱羧酶（ODC）和原癌基因mRNA水平的变化。1）异丙肾上腺素（IPR）对表观分子量分别为17 K、20 K、20 K的四种蛋白质的丝氨酸/苏氨酸残基的磷酸化有促进作用，31 K和32 K及卡巴胆碱（CC）刺激31 K和32 K蛋白磷酸化。这些蛋白磷酸化先于ODC活性的增加。IPR依赖的ODC诱导和蛋白磷酸化被莫能菌素选择性抑制，但不被多粘菌素B，而CC依赖的ODC诱导和蛋白磷酸化被多粘菌素B抑制，但不被莫能菌素抑制。莫能菌素和多粘菌素B都不能抑制IPR或CC刺激的淀粉酶分泌。2）北方/斑点和Western印迹分析显示， ...更多信息 甲氧胺）使ODC mRNA和ODC蛋白的稳态水平分别在刺激后2 h和6 h达到最大值。这些增加大致与ODC活性成比例，表明唾液腺刺激因子依赖性酶诱导在转录水平上受到调节。9个原癌基因中有4个的mRNA在刺激后30分钟（c-fos）或60分钟（c-jun，c-myc和c-src）时表现出依赖于涎蛋白的增加。3）酪氨酸激酶抑制剂如除莠霉素A、染料木黄酮和2，5-二羟基肉桂酸甲酯抑制了促涎素刺激的ODC诱导，但增强了促涎素诱导的淀粉酶分泌。酪氨酸磷酸酶抑制剂Na_3VO_4可增加ODC活性，除莠霉素A可抑制ODC活性的增加。这些结果表明，蛋白质的丝氨酸/苏氨酸和酪氨酸磷酸化参与了促涎素刺激的ODC诱导，而蛋白质酪氨酸磷酸化对淀粉酶分泌有抑制作用。少

Changes in protein phosphorylation and mRNA levels of ornithine decarboxylase (ODC) and proto-oncogenes in sialagogue-stimulated parotid gland were investigated in rat parotid explants cultured on siliconized lens paper floating on serum-free 199 medium.The results were summarized as follows;1) Isoproterenol (IPR) enhanced the phosphorylation of serine/threonine residues of four proteins with apparent molecular weights of 17K, 20K, 31K and 32K and carbachol (CC) stimulated the phosphorylation of31K and 32K proteins. These protein phosphorylations preceded the increase of ODC activity. IPR-dependent ODC induction and phosphorylation of the proteins were selectively suppressed by monensin but not by polymyxin B, whereas CC-dependent ODC induction and protein phosphorylation were inhibited by polymyxin B but not by monensin. Neither monensin nor polymyxin B suppressed IPR- or CC-stimulated amylase secretion.2) Northern/dot and Western blot analyses revealed that the sialagogues (IPR, CC, … More methoxamine) elevated the steady-state levels of ODC mRNA and ODC protein to maxima at 2 h and 6 h, respectively, after stimulation. The increases were roughly proportional to those in ODC activity, suggesting that sialagogue-dependent enzyme induction is regulated at the transcriptional level. The mRNAs of four of nine proto-oncogenes examined showed sialagogue-dependent increases to maxima at 30 min (c-fos) or 60 min (c-jun, c-myc and c-src) after stimulation. These increases were all transient, with the levels returning to the unstimulated levels within 60 min.3) Tyrosine kinase inhibitors such as herbimycin A, genistein and methyl 2,5-dihydroxycinnamate inhibited a sialagogue-stimulated ODC induction but enhanced a sialagogue-induced amylase secretion. A tyrosine phosphatase inhibitor, Na_3 VO_4, increased ODC activity and this increase was suppressed by herbimycin A.These results suggest that serine/threonine and tyrosine phosphorylations of proteins participate in a sialagogue-stimulated ODC induction, and that protein tyrosine phosphorylation shows an inhibitory effect on amylase secretion. Less