Nerve growth factor- and epidermal growth factor-stimulated phosphorylation of a PC12 cytoskeletally associated protein in situ.
Nerve growth factor- and epidermal growth factor-stimulated phosphorylation of a PC12 cytoskeletally associated protein in situ.
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DOI：
10.1083/jcb.100.3.677
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发表时间：
1985-03
影响因子：
7.8
通讯作者：
Rieser, G D
中科院分区：
文献类型：
--
作者：
Landreth, G E;Rieser, G D
神经生长因子 (NGF) 和表皮生长因子 (EGF) 在 PC12 细胞中产生稳定的改变，这些改变持续存在于去污剂不溶性细胞骨架中，导致 250,000 mol-wt 细胞骨架相关蛋白原位磷酸化。用 NGF 或 EGF 处理 PC12 细胞，然后用去污剂裂解细胞，并用 γ-32P-ATP 孵育所得细胞骨架，从而可以检测激素刺激的能量依赖性事件，从而导致细胞骨架相关蛋白的磷酸化增强，这是受体占据的直接结果。这些事件只有在生理温度下处理完整细胞时才会引发。 NGF 和 EGF 刺激事件发生得很快；然而，它们只是激素作用的短暂影响。研究发现，NGF 和 EGF 通过独立的机制发挥作用，刺激 250,000 mol-wt 蛋白质的原位磷酸化，因为甲基转移酶抑制剂可阻断 NGF（而非 EGF）的作用。 250,000 mol-wt 蛋白质在丝氨酸和苏氨酸残基上被磷酸化，以响应 NGF 和 EGF，尽管比例略有不同。数据表明，250,000 mol-wt 蛋白质的激素刺激标记可能是蛋白激酶直接激活、激素作用导致激酶相对于其底物重新分布或这些事件同时发生的结果。
Nerve growth factor (NGF) and epidermal growth factor (EGF) produce stable alterations in PC12 cells that persist in the detergent- insoluble cytoskeleton, resulting in the phosphorylation of a 250,000- mol-wt cytoskeletally associated protein in situ. Treatment of PC12 cells with NGF or EGF, followed by detergent lysis of the cells and incubation of the resulting cytoskeletons with gamma-32P-ATP, permitted detection of hormonally stimulated, energy-dependent events, which result in the enhanced phosphorylation of a cytoskeletally associated protein as an immediate consequence of receptor occupancy. These events were elicited only upon treatment of intact cells at physiological temperatures. The NGF- and EGF-stimulated events occurred rapidly; however, they were a transient effect of hormone action. NGF and EGF were found to act through independent mechanisms to stimulate the in situ phosphorylation of the 250,000-mol-wt protein, as the effects of NGF, but not EGF, were blocked by methyltransferase inhibitors. The 250,000-mol-wt protein was phosphorylated on serine and threonine residues in response to both NGF and EGF although in somewhat different proportions. The data suggest that the hormone-stimulated labeling of the 250,000-mol-wt protein may be the result of either the direct activation of a protein kinase, the redistribution of the kinase relative to its substrates as a consequence of hormone action, or the coincident occurrence of these events.