Grant Philip, Pant Harish C
Laboratory of Neurochemistry, National Institute of Neurological Disorders and Stroke, National Institutes of Health, LNC, NINDS, Bldg. 36, Rm. 4D04, Bethesda, Maryland 20892, USA.
J Neurobiol. 2004 Mar;58(4):514-28. doi: 10.1002/neu.10305.
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.
在之前对鱿鱼星状神经节神经元磷酸化的研究中，我们证明了一种特定的多聚体磷酸化复合物可表征每个细胞区室。尽管通过考马斯亮蓝染色测定的细胞体提取物（巨纤维叶，GFL）的内源性蛋白质谱与巨轴突轴浆的相似，但在本研究中我们表明蛋白质磷酸化谱在质量上是不同的。虽然许多轴浆蛋白被磷酸化，包括大多数细胞骨架蛋白，但几乎所有核周体中的磷酸化都局限于低分子量化合物（<6 kDa）。由于外源底物组蛋白和酪蛋白的磷酸化在两个区室的提取物中同样活跃，未能检测到细胞体中的内源性蛋白质磷酸化归因于存在更活跃的磷酸酶。为了进一步探究磷酸酶在这些神经元中的作用，我们研究了在丝氨酸/苏氨酸和蛋白酪氨酸磷酸酶（PTP）抑制剂存在下的磷酸化情况。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，而细胞体磷酸化对蛋白酪氨酸磷酸酶抑制剂（如钒酸盐）更敏感。钒酸盐对PTPs的抑制刺激了GFL蛋白（包括细胞骨架蛋白）的内源性磷酸化。钒酸盐在细胞体和轴突全匀浆以及Triton X - 100无游离可溶性提取物中同样刺激蛋白酪氨酸激酶活性，但只有GFL的Triton X可溶性部分（膜结合蛋白）在钒酸盐存在下表现出显著激活，表明该部分中的PTP活性高于轴突中的。这些数据与以下假设一致，即轴突和细胞体中的神经元蛋白质磷酸化受与区室特异性多聚体复合物相关的不同磷酸酶调节。
