N-terminal domain of annexin 2 regulates Ca2+-dependent membrane aggregation by the core domain: A site directed mutagenesis study

Annexin 2 binds and aggregates biological membranes in a Ca2+-dependent man ner. 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 S 100 protein family. Protein kinase C phosphorylates the protein at the leve l of the N-terminal tail on serines 11 and 25, thereby modifying its oligom eric structure and its properties of membrane aggregation. To analyze these effects, the properties of a series of mutants in which serines ii and 25 were replaced by alanine and/or glutamic acid were investigated. The affini ty for pll light chain was decreased in the S11E mutants. Glutamic acid res idues 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 aff ected the aggregation properties of the two forms. For the tetramer, the ag gregation efficiency was decreased but not the Ca2+ sensitivity, whereas th e 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 s uggest a different conformation of the N-terminal domain in the mutants (an d in the phosphorylated protein), a hypothesis which is supported by proteo lysis experiments. This conformational change would affect aggregation by t he monomer through a dimerization step.