Landreth G E, Williams L K
Department of Neurology, Medical University of South Carolina, Charleston 29425.
Neurochem Res. 1987 Oct;12(10):943-50. doi: 10.1007/BF00966317.
Nerve growth factor (NGF) rapidly stimulates the phosphorylation of a 250 kDa cytoskeletally-associated protein (pp250) by a protein kinase which is also associated with structural elements of the cell. We have solubilized these proteins and demonstrated that NGF-stimulated phosphorylation can be observed in cell free extracts of cytoskeletons from NGF-treated PC12 cells. The pp250 substrate and the 250-kinase were solubilized from PC12 cytoskeletons by treatment with 2 M urea. Phosphorylation of pp250 was maximally stimulated following treatment of the cells for 5 min with NGF. This effect was transient, diminishing with longer exposure of the cells to hormone. The 250-kinase preferred Mn2+ over Mg2+ and was inhibited by both Na+ and K+. The phosphorylation of pp250 was not affected by Ca2+. Upon fractionation of the urea-soluble cytoskeletal proteins by gel filtration, the 250-kinase eluted in two peaks; one peak of enzyme activity coeluting with the pp250 substrate, and a second peak of enzyme activity eluting with an apparent Mr of approximately 60 kDa. Treatment of the PC12 cells with the phorbol ester TPA also stimulated the phosphorylation of pp250, although this effect was not as great as that produced by NGF. This cell free system should be a valuable tool in the investigation of the mechanisms of NGF action.
神经生长因子（NGF）可通过一种与细胞结构成分相关的蛋白激酶迅速刺激一种250 kDa的细胞骨架相关蛋白（pp250）的磷酸化。我们已将这些蛋白溶解，并证明在经NGF处理的PC12细胞的细胞骨架无细胞提取物中可观察到NGF刺激的磷酸化。通过用2 M尿素处理，从PC12细胞骨架中溶解出pp250底物和250激酶。用NGF处理细胞5分钟后，pp250的磷酸化受到最大程度的刺激。这种效应是短暂的，随着细胞与激素接触时间延长而减弱。250激酶对Mn2+的偏好超过Mg2+，并受到Na+和K+的抑制。pp250的磷酸化不受Ca2+影响。通过凝胶过滤对尿素可溶性细胞骨架蛋白进行分级分离时，250激酶在两个峰中洗脱；一个酶活性峰与pp250底物共洗脱，另一个酶活性峰以约60 kDa的表观分子量洗脱。用佛波酯TPA处理PC12细胞也刺激了pp250的磷酸化，尽管这种效应不如NGF产生的效应大。这个无细胞系统应该是研究NGF作用机制的一个有价值的工具。
