Oddie K M, Litz J S, Balserak J C, Payne D M, Creutz C E, Parsons S J
Department of Microbiology, University of Virginia Cancer Center, University of Virginia School of Medicine, Charlottesville 22908.
J Neurosci Res. 1989 Sep;24(1):38-48. doi: 10.1002/jnr.490240107.
High levels of the proto-oncogene product, pp60c-src, have been found in developing and adult neural tissues as well as in certain fully mature cells of the hematopoietic lineage, e.g., platelets and myelomonocytes. Adrenal medullary chromaffin cells exhibit characteristics of both types of cells, i.e., they are derived from the neural crest and carry out exocytosis in response to specific stimuli. Earlier studies have shown that pp60c-src localizes not only to the plasma membrane of chromaffin cells but also to the membranes of chromaffin granules, the secretory vesicles of these cells that store catecholamines and other secretory products. To investigate the possible involvement of pp60c-src in exocytosis, cultured bovine chromaffin cells were analyzed for changes in c-src tyrosine kinase activity in response to stimulation by several secretagogues. Results of in-vitro immune complex kinase assays showed that pp60c-src, derived from cells that had been stimulated for various lengths of time, exhibited decreased auto- and transphosphorylating activities as compared to pp60c-src immunoprecipitated from control cells. The greatest reduction in activity was observed 10 min post-stimulation, while normal levels were regained 2-6 hr after secretagogue treatment. Western immunoblot analysis of the immunoprecipitated pp60c-src revealed that approximately 50% less c-src protein was present in immune complexes prepared 10 min after stimulation as compared to those prepared from mock-stimulated controls, resulting in a specific autophosphorylating activity that was 42-47% of control and little or no reduction in the transphosphorylating specific activity. In experiments in which the rate of secretion of [3H]-norepinephrine from cells preloaded with this compound was compared to the rate of modulation of pp60c-src activity, 50% of the maximal reduction in pp60c-src activity occurred within 2-4 min while 50% maximal release of [3H]-norepinephrine occurred within 1-3 min. Taken together, these results suggest that pp60c-src may play some role (direct or indirect) in the exocytotic process.
在发育中的和成年的神经组织以及造血谱系的某些完全成熟的细胞（如血小板和骨髓单核细胞）中，已发现原癌基因产物pp60c-src的高水平表达。肾上腺髓质嗜铬细胞表现出这两种细胞类型的特征，即它们起源于神经嵴，并在特定刺激下进行胞吐作用。早期研究表明，pp60c-src不仅定位于嗜铬细胞的质膜，还定位于嗜铬颗粒的膜，嗜铬颗粒是这些细胞储存儿茶酚胺和其他分泌产物的分泌囊泡。为了研究pp60c-src在胞吐作用中可能的参与情况，分析了培养的牛嗜铬细胞在几种促分泌剂刺激下c-src酪氨酸激酶活性的变化。体外免疫复合物激酶分析结果表明，与从对照细胞免疫沉淀的pp60c-src相比，来自不同刺激时间的细胞的pp60c-src的自身磷酸化和转磷酸化活性降低。刺激后10分钟观察到活性的最大降低，而促分泌剂处理后2 - 6小时恢复到正常水平。对免疫沉淀的pp60c-src进行的蛋白质免疫印迹分析表明，与模拟刺激对照制备的免疫复合物相比，刺激后10分钟制备的免疫复合物中c-src蛋白含量减少约50%，导致特定的自身磷酸化活性为对照的42 - 47%，而转磷酸化比活性几乎没有降低。在将预先加载该化合物的细胞中[3H] - 去甲肾上腺素的分泌速率与pp60c-src活性调节速率进行比较的实验中，pp60c-src活性最大降低的50%在2 - 4分钟内出现，而[3H] - 去甲肾上腺素最大释放的50%在1 - 3分钟内出现。综上所述，这些结果表明pp60c-src可能在胞吐过程中发挥某种作用（直接或间接）。
