Abstract. Annexin II is a Ca2+-dependent membranebinding protein present in a wide variety of cells and tissues. Within cells, annexin II is found either as a 36-kD monomer (p36) or as a heterotetrameric complex (p90) coupled with the S-100-related protein, pll. Annexin II has been suggested to be involved in exocytosis as it can restore the secretory responsiveness of permeabilized chromaffin cells. By quantitative confocal immunofluorescence, immunoreplica analysis and immunoprecipitation, we show here the translocation of p36 from the cytosol to a subplasmalemmal Triton X-100 insoluble fraction in chromaffin cells following nicotinic stimulation. A synthetic peptide corresponding to the NH2-terminal domain of p36 which contains the phosphorylation sites was microinjected into individual chromaffin cells and catecholamine secretion was monitored by amperometry. This peptide blocked cornpletely the nicotine-induced recruitment of p36 to the cell periphery and strongly inhibited exocytosis evoked by either nicotine or high K ÷. The light chain of annexin II, pll, was selectively expressed by adrenergic chromaffin cells, and was only present in the subplasmalemmal Triton X-100 insoluble protein fraction of both resting and stimulated cells, pll can modify the Ca 2÷-and/or the phospholipid-binding properties of p36. We found that less Ca 2+ was required to stimulate the translocation of p36 and to trigger exocytosis in adrenergic chromaffin cells. Our findings suggest that the translocation of p36 to the subplasmalemmal region is an essential event in regulated exocytosis and support the idea that the presence of pll in adrenergic cells may confer a higher Ca 2+ affinity to the exocytotic pathway in these cells.
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....
Annexin 2 phosphorylated in vitro by protein kinase C has been shown to restore partially catecholamine secretion in streptolysin 0-permeabilized chromaffin cells depleted of their protein kinase C activity. This result suggested a phosphorylation of annexin 2 in stimulated cells. Nicotine stimulation induced an increase of 32p incorporation in annexin 2 heavy chain concomitant with catecholamine release. This incorporation results from phosphorylation by protein kinase C because (a) serin.e was the only phosphorylated residue, (b) 32P incorporation was inhibited by the protein kinase inhibitors H7, GF 1 09203X, and staurosporine, and (c) activators of this enzyme, 1 2-O-tetradecanoylphorbol 13-acetate and 1,2-dioctanoylglycerate, increased the incorporation of radioactivity. The phosphorylated heavy chain had an electrophoretic mobility lower than that of the unmodified one, thus allowing determination of the fraction of phosphorylated protein. In the resting state, a significant fraction of annexin 2 heavy chain was phosphorylated, and nicotine stimulation resulted in an activation of both phosphorylation and dephosphorylation. Phosphorylation was largely increased in the presence of okadaic acid, indicating the involvement of type 1 and 2A phosphatases. Key Words: Annexin-Protein kinase C-Phosphorylation-Chromaffin cells-Secretion. J. Neurochem. 68, 1720-1727 (1997).Adrenal chromaffin cells, which secrete catecholamines on stimulation by acetylcholine, have served as a good model for the study of the molecular mechanisms involved in regulated exocytosis. Primary cultures of chromaffln cells have allowed the study of stimulation by acetylcholine of catecholamine secretion and the characterization of factors essential for secretion (Fenwick et al., 1978;Kilpatrick et al., 1980). Along this line, Michener et al. (1986) and Creutz et a!. (1987) demonstrated the phosphorylation of two phospholipid and calcium binding proteins, chromobindins 8 and 9, when chromaffin cells were preincubated with 32P0 4 3 and stimulated by nicotine. These chromobindins were identified as annexins 1 and 2. Annexin 2 differs from the other annexins in being a heterotetramer composed of two 36-kDa heavy chains and two I l-kDa light chains or a monomer with one 36-kDa heavy chain. Only the heterotetramer is able to bind chromaffin granules, to promote their aggregation at a physiological calcium concentration of 5 ,uM, and, at pH 6, to induce the fusion of granules after addition of arachidonic acid (Drust and Creutz, 1988). The heavy chain of annexin 2 is phosphorylated by various protein kinases, and it is the major substrate for protein kinase C and for the Rous sarcoma virus enzyme pp60~"°. In human cells treated with epiderma! growth factor and platelet-derived growth factor, annexin 2 is phosphorylated on a tyrosine as well as on a serine (Isacke et a!., 1986). The serine residue was identified as Ser25 within the amino-terminal region (Glenney and Tack, 1985). Alternatively, in vitro phosphorylations of annexin 2 with ca...
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