An elevation of cytosolic protein phosphorylation modulates trimeric G-protein regulation of secretory vesicle formation from the trans-Golgi network.

Abstract

The role of protein phosphorylation in the formation of secretory vesicles from the trans-Golgi network (TGN) and in the regulation of this process by TGN-associated trimeric G-proteins was investigated, using a previously established and a novel cell-free system derived from the neuroendocrine cell line PC12. In the absence of exogenous activators of trimeric G-proteins, okadaic acid, an inhibitor of protein serine/threonine phosphatase types 1, 2A, and PPX, had no significant effect on secretory vesicle formation as reconstituted in a postnuclear supernatant. However, okadaic acid antagonized the inhibition of secretory vesicle formation which occurred upon activation of trimeric G-proteins by either aluminum fluoride or guanosine 5'-3-O-(thio)-triphosphate (GTP gamma S). Microcystin-LR, a protein phosphatase inhibitor structurally distinct from okadaic acid, also antagonized the trimeric G-protein-mediated inhibition of secretory vesicle formation but, in contrast to okadaic acid, alone was sufficient to stimulate this process. The antagonistic effect of the phosphatase inhibitors was abolished by a broad spectrum protein kinase inhibitor, staurosporine, which alone, however, did not affect vesicle formation. The effect of okadaic acid was promoted by activators of protein kinase C (phorbol myristate acetate) and protein kinase A (cyclic AMP). To investigate the subcellular localization of the phosphoprotein that is involved in the antagonistic effect of protein phosphatase inhibitors, a novel cell-free system was established which reconstitutes the formation of secretory vesicles from TGN membranes supplemented with cytosol. Using this cell-free system, the relevant phosphoprotein was found to reside in the cytosol. In conclusion, our results suggest that serine/threonine protein phosphorylation is not required for secretory vesicle formation from the TGN but modulates, via a cytosolic phosphoprotein, the regulation of this process by TGN-associated trimeric G-proteins.