"In vitro" phosphorylation of annexin 2 heterotetramer by protein kinase C. Comparative properties of the unphosphorylated and phosphorylated annexin 2 on the aggregation and fusion of chromaffin granule membranes.
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 microM 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.
Regnouf
F
F
Centre National de la Recherche Scientifique, Institut de Biologie Physico-Chimique, Paris, France.
Sagot
I
I
Delouche
B
B
Devilliers
G
G
Cartaud
J
J
Henry
J P
JP
Pradel
L A
LA
D000818:Animals; D017306:Annexin A2 / Q000737:chemistry* / Q000378:metabolism / Q000494:pharmacology; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002837:Chromaffin Granules / Q000187:drug effects / Q000378:metabolism / Q000648:ultrastructure; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008561:Membrane Fusion / Q000187:drug effects; D008854:Microscopy, Electron; D010766:Phosphorylation; D011485:Protein Binding; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism*; D051381:Rats
D017306:Annexin A2; D011493:Protein Kinase C