Carbachol activates ERK2 in isolated gastric parietal cells via multiple signaling pathways.
Carbachol activates ERK2 in isolated gastric parietal cells via multiple signaling pathways.
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Carbachol 通过多种信号通路激活分离的胃壁细胞中的 ERK2。
DOI：
10.1152/ajpgi.1999.276.6.g1484
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发表时间：
1999
期刊：
影响因子：
--
通讯作者：
Todisco,A
中科院分区：
文献类型：
--
作者：
Takeuchi,Y;Pausawasdi,N;Todisco,A
我们之前报道过，乙醇和表皮生长因子（EGF）都是分离的犬胃壁细胞细胞外信号调节蛋白激酶（ERKs）的有效诱导剂，这些激酶的诱导导致胃酸分泌的急性抑制和慢性刺激作用。在这项研究中，我们研究了这些作用的分子机制。carbachol （100 μM）和EGF （10 nM）均可诱导Ras活化。通过用表达血凝素（HA）标记的ERK2 （HA-ERK2）和显性表达的突变（无活性）Ras基因的载体转染顶壁细胞，研究了Ras在ERK2诱导中的作用。凝胶内激酶法测定HA-ERK2活性。显性负Ras降低了60%的碳醇诱导HA-ERK2活性，并完全抑制了EGF的刺激作用。由于Ras激活需要多蛋白复合物的组装，我们研究了碳醇和EGF对Shc酪氨酸磷酸化的影响及其与Grb2和鸟嘌呤核苷酸交换因子Sos的关联。用抗磷酸酪氨酸抗体对抗shc免疫沉淀物进行的Western blot分析表明，碳二酚和EGF均诱导了52-kDashcisoform的酪氨酸磷酸化。用抗Shc抗体对Grb2免疫沉淀物进行印迹，证实了Grb2与Shc的相关性。用抗Grb2抗体探测抗Sos免疫沉淀，发现Sos与Grb2组成性结合。为了研究Sos在ERK2激活中的功能作用，我们用HA-ERK2载体和一个显性表达的突变（失活）sosgene转染了壁细胞。显性负Sos不影响HA-ERK2的碳醇刺激，但抑制EGF的刺激作用达60%。然后我们研究了βγ-亚基在碳醇诱导HA-ERK2中的作用。将HA-ERK2载体与表达β-肾上腺素能受体激酶1羧基末端的载体一起转染顶壁细胞，已知其可阻断βγ-亚基介导的信号传导。在该载体的存在下，HA-ERK2的碳醇诱导被抑制了40%。综上所述，这些数据表明，在胃壁细胞中，carbachol通过Ras-和βγ依赖的机制激活ERKs，这些机制需要鸟嘌呤核苷酸交换因子而不是Sos。
We previously reported that both carbachol and epidermal growth factor (EGF) are potent inducers of the extracellular signal-regulated protein kinases (ERKs) in isolated gastric canine parietal cells and that induction of these kinases leads to acute inhibitory and chronic stimulatory effects on gastric acid secretion. In this study we investigated the molecular mechanisms responsible for these effects. Both carbachol (100 μM) and EGF (10 nM) induced Ras activation. The role of Ras in ERK2 induction was examined by transfecting parietal cells with a vector expressing hemoagglutinin (HA)-tagged ERK2 (HA-ERK2) together with a dominantly expressed mutant (inactive)rasgene. HA-ERK2 activity was quantitated by in-gel kinase assays. Dominant negative Ras reduced carbachol induction of HA-ERK2 activity by 60% and completely inhibited the stimulatory effect of EGF. Since Ras activation requires the assembly of a multiprotein complex, we examined the effect of carbachol and EGF on tyrosyl phosphorylation of Shc and its association with Grb2 and the guanine nucleotide exchange factor Sos. Western blot analysis of anti-Shc immunoprecipitates with an anti-phosphotyrosine antibody demonstrated that both carbachol and EGF induced tyrosyl phosphorylation of a major 52-kDashcisoform. Grb2 association with Shc was demonstrated by blotting Grb2 immunoprecipitates with an anti-Shc antibody. Probing of anti-Sos immunoprecipitates with an anti-Grb2 antibody revealed that Sos was constitutively bound to Grb2. To examine the functional role of Sos in ERK2 activation, we transfected parietal cells with the HA-ERK2 vector together with a dominantly expressed mutant (inactive)sosgene. Dominant negative Sos did not affect carbachol stimulation of HA-ERK2 but inhibited the stimulatory effect of EGF by 60%. We then investigated the role of βγ-subunits in carbachol induction of HA-ERK2. Parietal cells were transfected with the HA-ERK2 vector together with a vector expressing the carboxy terminus of the β-adrenergic receptor kinase 1, known to block signaling mediated by βγ-subunits. In the presence of this vector, carbachol induction of HA-ERK2 was inhibited by 40%. Together these data suggest that, in the gastric parietal cells, carbachol activates the ERKs through Ras- and βγ-dependent mechanisms that require guanine nucleotide exchange factors other than Sos.
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影响因子：
4.1
作者：
HER, JH;LAKHANI, S;WEBER, MJ
通讯作者：
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DOI：
--
发表时间：
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期刊：
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DOI：
10.1152/ajpgi.1996.271.4.g640
发表时间：
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期刊：
The American journal of physiology
影响因子：
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作者：
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影响因子：
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作者：
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通讯作者：
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影响因子：
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