N-Terminal Domain of Annexin 2 Regulates Ca²⁺-Dependent Membrane Aggregation by the Core Domain:  A Site Directed Mutagenesis Study

Abstract

Annexin 2 binds and aggregates biological membranes in a Ca(2+)-dependent manner. This protein exists as a monomer (p36) or as a heterotetramer (p90) in which two p36 chains are associated with a dimer of p11, a member of the S100 protein family. Protein kinase C phosphorylates the protein at the level of the N-terminal tail on serines 11 and 25, thereby modifying its oligomeric structure and its properties of membrane aggregation. To analyze these effects, the properties of a series of mutants in which serines 11 and 25 were replaced by alanine and/or glutamic acid were investigated. The affinity for p11 light chain was decreased in the S11E mutants. Glutamic acid residues in positions 11 or 25 did not change membrane binding, either in the tetrameric or in the monomeric form. On the other hand, these mutations affected the aggregation properties of the two forms. For the tetramer, the aggregation efficiency was decreased but not the Ca(2+) sensitivity, whereas the latter was affected in the case of the monomer. The effects were stronger in the S11E mutants, and they were cumulative in the double mutant. They suggest a different conformation of the N-terminal domain in the mutants (and in the phosphorylated protein), a hypothesis which is supported by proteolysis experiments. This conformational change would affect aggregation by the monomer through a dimerization step.

Annexin A2 Is a Novel RNA-binding Protein

“…Expression and Purification of Recombinant ANXA2-The galactoseinducible Saccharomyces cerevisiae expression vector (pYeDP60) as well as the vector containing the cDNA for ANXA2 (pYeDP60-ANXA2) were kindly supplied by Jesus Ayala-Sanmartin (INSERM, Paris, France) and have been described previously (41). The cDNA of the S100A10 protein was PCR-amplified and inserted into the pYeDP60 vector, followed by the transformation of the vectors into the proteasedeficient S. cerevisiae strain FKY282 (kindly supplied by Francois Kepes, Genopole, Envy, France).…”

Novel Organization and Properties of Annexin 2-Membrane Complexes

“…AnxA2 produced in Saccharomyces cerevisiae and P11 produced in Escherichia coli were purified as described (17). (AnxA2-P11) 2 …”

“…Chromaffin granules were obtained as described (17) in buffer E (Hepes 10 mM, pH 7, saccharose 0.3 M). Aggregation experiments were performed as described (17).…”

“…Although the Ca 2ϩ -induced conformational change of AnxA2 in the presence of phospholipid membranes requires Ca 2ϩ close to the millimolar concentration, (AnxA2-P11) 2 needs only submicromolar concentration (8,9,16). The conformational change correlates with membrane aggregation and not with membrane binding (17). This suggested that the Ca 2ϩ -induced conformational change of annexin 2 is essential for its aggregative function but not for membrane binding.…”

The N-terminal Domain of Annexin 2 Serves as a Secondary Binding Site during Membrane Bridging

“…Protein Preparation and Labeling with Acrylodan and Pyrene Maleimide-AnxA2 produced in Saccharomyces cerevisiae was purified as previously described (35). Protein purity was estimated as Ͼ95% by gel electrophoresis.…”