The molecular mechanism of regulated secretion: Insights from Xenopus oocytes.

摘要

Regulated exocytosis is the process by which the contents of secretory organelles are discharged into the extracellular medium. The goal of this thesis was to characterize the molecular machinery that regulates the exocytotic release of cortical granules in Xenopus oocytes. The objectives of this study were: (1) to identify the mechanism by which activators of protein kinase C (PKC) trigger cortical granule exocytosis, (2) to determine the substrates for the action of PKC, and (3) to investigate the mechanism governing the altered sensitivity of cortical granules to a calcium stimulus in progesterone-matured oocytes.;1. Phorbol esters or diacylglycerol trigger cortical granule exocytosis in Xenopus oocytes. We sought to identify the isoform(s) of PKC that mediate this calcium independent regulated secretory event. The selective PKC inhibitors, Go6976 and Go6983, excluded PKCdelta, epsilon and mu as secretory triggers. Sub-cellular fractionation and immunoblot data revealed that these oocytes expressed all five members of the novel PKC family, but it was only PKCeta that co-localized with cortical granules. Finally, expression of wild type or constitutively active forms of PKCdelta and eta strongly supported the conclusion that PKCeta initiates cortical granule exocytosis in these cells.;2. To establish the identity of protein(s) targeted for phosphorylation by PKCeta, I studied the incorporation of 32 P into oocyte proteins in response to phorbol esters. My data demonstrated that phorbol esters selectively enhanced the phosphorylation of a 35kDa protein in the cytosolic fraction of oocytes, but the role of this protein in secretion is unknown.;3. To investigate the molecular basis of the altered sensitivity of the secretory machinery to a calcium stimulus in mature oocytes, I found that this change required new protein synthesis, but did not require RNA synthesis. Calcium overlay experiments and immunoblot data indicated that there was no significant change in the amount or subcellular distribution of calmodulin, Troponin-C, synaptotagmin, munc-13, B/K, or Doc2 after progesterone. Pharmacological data argued against calmodulin, calpain, calcium-dependent PKCs, or calcineurin as being calcium sensors in these cells. Thus, the molecular basis of this transition remains to be clarified.