Heterotetrameric annexin 2 phosphorylated "in vitro"by rat brain protein kinase C is purified and obtained devoid of unphosphorylated protein; it contains 2 mol of phosphate/mol of heterotetramer. The aggregative and binding properties of the phosphorylated annexin 2 toward purified chromaffin granules are compared with those of the unphosphorylated annexin 2. Annexin 2 binds to chromaffin granules with high affinity. Phosphorylation of annexin 2 decreases the affinity of this binding without affecting the maximum binding capacity. The binding curves are strongly cooperative. It is suggested that a surface oligomerization of the proteins may take place upon binding. Besides, phosphorylation of annexin 2 is followed by a dissociation of the light chains from the heavy chains in the heterotetramer. Whereas annexin 2 induces the aggregation of chromaffin granules at M calcium concentration, the phosphorylated annexin 2 does not induce aggregation at any concentration of calcium either at pH 6 or 7. The phosphorylation of annexin 2 by protein kinase C, MgATP, and 12-O-tetradecanoylphorbol-13-acetate on chromaffin granules induces a fusion of chromaffin granules membranes observed in electron microscopy. The fusion requires the activation of protein kinase C by 12-O-tetradecanoylphorbol-13-acetate. Given these results and since annexin 2 is phosphorylated by protein kinase C under stimulation of chromaffin cells, it is suggested that phosphorylated annexin 2 may be implicated in the fusion step during exocytosis of chromaffin granules.Annexin 2 is a calcium-phospholipid-binding protein of the annexin family which has been characterized in the adrenal medulla as chromobindin 8. The heterotetrameric molecule formed of two heavy chains of 36 kDa and two light chains of 11 kDa possesses the unique property among the other annexins to aggregate chromaffin granules at micromolar calcium concentration (1).It has been shown by immunoelectron microscopy (2) that in chromaffin cells annexin 2 was closely associated with the inner face of the plasma membranes. In cultured chromaffin cells, thin strands were found cross-linking the chromaffin vesicles to the plasma membrane after stimulation with acetylcholine. Similar thin strands were also observed between aggregated chromaffin vesicles when they were mixed with annexin 2 in the presence of calcium. These data strongly suggested that conformational changes were induced in annexin 2 to cross-link the vesicles and the plasma membrane after stimulation of cultured chromaffin cells. When primary cultured chromaffin cells were stimulated by nicotine, annexin 2 was phosphorylated by protein kinase C. The phosphorylation of the protein was concomitant with the catecholamine release.1 In streptolysin-permeabilized cells, annexin 2 phosphorylated "in vitro" by brain protein kinase C was able to reconstitute secretion of catecholamines in cells depleted of protein kinase C activity (3). Taken together, all these results suggest that annexin 2 could be involved in the exocytotic process....
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