Topographic regulation of phosphorylation in giant neurons of the squid, Loligo pealei: role of phosphatases.
Topographic regulation of phosphorylation in giant neurons of the squid, Loligo pealei: role of phosphatases.
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乌贼巨型神经元磷酸化的地形调控：磷酸酶的作用。
DOI：
10.1002/neu.10305
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Pant,HarishC
中科院分区：
文献类型：
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作者：
Grant,Philip;Pant,HarishC
在以前对鱿鱼星状神经节神经元磷酸化的研究中，我们证明了每个细胞隔室都有一个特定的多聚体磷酸化复合体。虽然Coomassie染色测定的细胞体提取物(巨纤维叶，GFL)的内源蛋白质谱与巨轴突轴浆的蛋白质谱相似，但在本研究中，我们发现蛋白质磷酸化谱在性质上是不同的。虽然许多轴浆蛋白是磷酸化的，包括大多数细胞骨架蛋白，但实际上核周膜的所有磷酸化都局限于低分子化合物(<；6 kDa)。由于外源底物组蛋白和酪蛋白的磷酸化在两个隔室的提取物中同样活跃，因此未能检测到细胞体中内源蛋白的磷酸化是由于存在更活跃的磷酸酶。为了进一步探索磷酸酶在这些神经元中的作用，我们研究了丝氨酸/苏氨酸和蛋白酪氨酸磷酸酶(PTP)抑制剂存在下的磷酸化。我们发现轴突细胞骨架蛋白的磷酸化受冈田酸敏感的丝氨酸/苏氨酸磷酸酶调节，而细胞体的磷酸化对蛋白酪氨酸磷酸酶的抑制剂更敏感，如钒酸。钒酸盐抑制PTPs可刺激包括细胞骨架蛋白在内的GFL蛋白的内源性磷酸化。胞体、轴突全匀浆和Triton X-100游离提取物对蛋白酪氨酸激酶活性的刺激作用相同，但只有GFL的Triton X可溶部分(膜结合蛋白)在钒的存在下表现出显著的激活，表明该部分的PTP活性高于轴突。这些数据与假设是一致的，即轴突和细胞体中的神经元蛋白磷酸化是由不同的磷酸酶调节的，这些磷酸酶与隔室特异性多聚体复合体相关。©2004威利期刊，Inc.神经生物学杂志58：514-528,2004
In previous studies of phosphorylation in squid stellate ganglion neurons, we demonstrated that a specific multimeric phosphorylation complex characterized each cellular compartment. Although the endogenous protein profile of cell body extracts (giant fiber lobe, GFL), as determined by Coomassie staining, was similar to that of axoplasm from the giant axon, in this study we show that the protein phosphorylation profiles are qualitatively different. Whereas many axoplasm proteins were phosphorylated, including most cytoskeletal proteins, virtually all phosphorylation in perikarya was confined to low molecular weight compounds (<6 kDa). Because phosphorylation of exogenous substrates, histone and casein, was equally active in extracts from both compartments, failure to detect endogenous protein phosphorylation in cell bodies was attributed to the presence of more active phosphatases. To further explore the role of phosphatases in these neurons, we studied phosphorylation in the presence of serine/threonine and protein tyrosine phosphatase (PTP) inhibitors. We found that phosphorylation of axonal cytoskeletal proteins was modulated by okadaic acid‐sensitive ser/thr phosphatases, whereas cell body phosphorylation was more sensitive to an inhibitor of protein tyrosine phosphatases, such as vanadate. Inhibition of PTPs by vanadate stimulated endogenous phosphorylation of GFL proteins, including cytoskeletal proteins. Protein tyrosine kinase activity was equally stimulated by vanadate in cell body and axonal whole homogenates and Triton X‐100 free soluble extracts, but only the Triton X soluble fraction (membrane bound proteins) of the GFL exhibited significant activation in the presence of vanadate, suggesting higher PTP activities in this fraction than in the axon. The data are consistent with the hypothesis that neuronal protein phosphorylation in axons and cell bodies is modulated by different phosphatases associated with compartment‐specific multimeric complexes. © 2004 Wiley Periodicals, Inc. J Neurobiol 58: 514–528, 2004