K-252A, A POTENT PROTEIN-KINASE INHIBITOR, BLOCKS NERVE GROWTH-FACTOR INDUCED NEURITE OUTGROWTH AND CHANGES IN THE PHOSPHORYLATION OF PROTEINS IN PC12H CELLS
K-252A, A POTENT PROTEIN-KINASE INHIBITOR, BLOCKS NERVE GROWTH-FACTOR INDUCED NEURITE OUTGROWTH AND CHANGES IN THE PHOSPHORYLATION OF PROTEINS IN PC12H CELLS
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DOI：
10.1083/jcb.107.4.1531
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发表时间：
1988-10-01
影响因子：
7.8
通讯作者：
HASHIMOTO, S
中科院分区：
文献类型：
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作者：
HASHIMOTO, S
神经生长因子(NGF)促进PC12嗜铬细胞瘤细胞向神经元分化。神经元分化最显著和最显著的特征之一是轴突生长。NGF导致细胞形成神经突起的机制尚不清楚。本研究表明，一种有效的蛋白激酶抑制剂K-252a可以阻断NGF诱导的轴突生长和NGF引起的蛋白磷酸化的变化。在用32P-正磷酸磷酸化完整细胞的实验中，PC12h细胞暴露于NGF(50 ng/ml)可导致酪氨酸羟化酶和35,000-D蛋白的磷酸化增加，而36,500-D蛋白的磷酸化减少。用K-252A(100 NM)预处理PC12h细胞可抑制NGF对这三种蛋白磷酸化的影响。在无细胞提取物与[γ-32P]ATP的磷酸化过程中，用NGF(50 ng/ml)处理PC12h细胞后，Nsp100的磷酸化水平降低。用K-252A(30 NM)处理细胞，几乎完全阻断了NGF对随后用NGF处理的细胞Nsp100磷酸化的影响。神经生长因子处理PC12h细胞的实验研究加入K-252A(100 NM)几乎完全阻断了NGF诱导的嵌合体的产生。早期的研究表明，NGF诱导的PC12细胞突起生长至少涉及两种成分：第一种是转录依赖的，第二种是转录不依赖的。为了确定K-252a作用的成分，进行了NGF诱导的轴突启动或再生实验。当K-252a存在于启动步骤中时，NGF仅诱导放线菌素D敏感的突起，表明K-252a干扰NGF的转录依赖作用。当已经被NGF激活的细胞被NGF处理时，出现了对放线菌素D抗性的突起，这些突起被K-252a阻断，这种抑制物也干扰了NGF的转录非依赖性作用。尽管K-252A抑制神经生长因子促进的轴突形成的确切机制尚不清楚，但最可能的解释是，转录依赖和非转录依赖成分都参与了某些特定蛋白激酶(S)的至少一个步骤的激活，而K-252A可以抑制该蛋白的激活。
Nerve growth factor (NGF) promotes neuronal differentiation of PC12 pheochromocytoma cells. One of the most prominent and distinguishing features of neuronal differentiations is neurite outgrowth. The mechanism by which NGF causes the cells to elaborate neurites is unknown. This study shows that K-252a, a potent protein kinase inhibitor, blocks NGF-induced neurite outgrowth and the changes in protein phosphorylation elicited by NGF. In the experiment with intact cells phosphorylated with 32 P-orthophosphoric acid, an exposure of PC12h cells to NGF (50 ng/ml) caused an increased in the phosphorylation of tyrosine hydroxylase and a 35,000-D protein and a decrease in a 36,500-D protein. Pretreatment of PC12h cells with K-252a (100 nM) inhibited the effects of NGF on the phosporylation of these three proteins. In the phosphorylation of cell-free extract with [.gamma.-32P] ATP, treatment of PC12h cells with NGF (50 ng/ml) caused a decrease in the phosphorylation of Nsp100. Pretreatment of the cells with K-252a (30 nM) almost completely blocked the NGF effect on the phosphorylation of Nsp100 elicited by subsequent treatment of the cells with NGF. Treatment of PC12h cells with NGF outgrowth of neurites. The addition of K-252a (100 nM) into the culture almost completely blocked the generation of neurities elicited by NGF. Earlier studies demonstrated that NGF-induced neurite outgrowth in PC12 cells involves at least two components: the first of these is transcription-dependent and the second is transcription-independent. To determine the component on which K-252a acts, experiments were carried out on NGF-induced priming or regeneration of neurites. When K-252a was present in the priming step, NGF induced only actinomycin D-sensitive neurites, showing that K-252a interferes with the transcription-dependent actions of NGF. When already primed cells were treated with NGF, actinomycin D-resistant neurites were formed and these were blocked by K-252a, that the inhibitor interferes with the transcription-indepent actions of NGF as well. Although the exact mechanism of inhibition of NGF-promoted neurite formation by K-252a is unknown, the most probale explanation is that both transcription-dependent and -independent components are involved in at least one step of the activation of some specific proteins kinase(s) that can be suppressed by K-252a.