Modulation of pp60c‐srctyrosine kinase activity during secretion in stimulated bovine adrenal chromaffin cells

摘要

AbstractHigh 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‐srclocalizes 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‐srcin exocytosis, cultured bovine chromaffin cells were analyzed for changes in c‐srctyrosine 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‐srcimmunoprecipitated 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‐srcrevealed that approximately 50 less c‐srcprotein 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‐srcactivity, 50 of the maximal reduction in pp60c‐srcactivity occurred within 2–4 min while 50 maximal release of 3H‐norephinephrine occurred within 1–3 min. Taken together, these results suggest that pp60c‐srcmay play some role (di