Phosphorylation by Protein Kinase C of Annexin 2 in Chromaffin Cells Stimulated by Nicotine

Abstract

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...

Annexin 2 Promotes the Formation of Lipid Microdomains Required for Calcium-regulated Exocytosis of Dense-Core Vesicles

“…Indeed, it has been reported that phosphorylation by PKC triggers the fusion of purified secretory granules preaggregated by unphosphorylated p36 (Regnouf et al, 1995), suggesting that annexin 2 becomes fusogenic when phosphorylated by PKC. Because secretagogueevoked stimulation activates PKC to phosphorylate endogenous annexin 2 in chromaffin cells (Delouche et al, 1997), annexin 2 has been proposed to mediate membrane fusion once the granule is brought in proximity to the plasma membrane by SNARE proteins (Regnouf et al, 1995). Our amperometric data do not support this hypothesis.…”

Differential regulation of keratinocyte chemokinesis and chemotaxis through distinct nicotinic receptor subtypes

“…The signaling pathway involving both PKC-δ and RhoA has been implicated in mediating regulation of cell motility downstream of other types of cell surface receptors, such as the leukotriene D 4 , lysophosphatidic acid and bombesin receptors (Barry and Critchley, 1994;Massoumi et al, 2002). Both PKCs and Rho GTPases are known to mediate nicotinergic signaling in nonneuronal cells (Delouche et al, 1997;Gasman et al, 1999;Zidovetzki et al, 1999). Although the involvement of PKC-δ and RhoA in regulation of KC motility was independently reported (Cozzolino et al, 2003;Li et al, 2002), the newly discovered co-operation between PKC-δ and RhoA in mediating nicotinergic control of KC migration presents a novel paradigm of intracellular signaling from ACh-gated ion channels.…”

Annexin A2 Heterotetramer: Structure and Function

“…Several growth factors such as platelet derived growth factor receptor; hepatocyte growth factor and insulin growth factor induce tyrosine phosphorylation of annexin A2. Nicotine stimulation of adrenal cells causes PKC activation which induces serine (Ser-25) phosphorylation of annexin A2 and calcium- dependent exocytosis [74,75]. One of the other critical residues in annexin A2 that is phosphorylated in response to several stimuli described above is Tyr-23, located in the amino-terminal tail region of the protein.…”