Mapping of a regulatory important site for protein kinase C phosphorylation in the N-terminal domain of annexin II

Annexin II is a Ca2+-regulated membrane- and cytoskeleton-binding protein implicated in membrane transport events along the Ca2+-regulated secretory and the early endocytic pathway. Biochemical properties of this annexin and its intracellular distribution are regulated by complex formation with pll (S100A10), a member of the S100 protein family. The annexin II-pll interaction is mediated through the unique N-terminal domain of annexin II and is inhibited by protein kinase C phosphorylation of a serine residue in annexin II. To map this regulatory serine phosphorylation site we developed a baculovirus-mediated expression system for wild-type annexin II and for a series of annexin II mutants which contained substitutions in one or mole serine residues present in the N-terminal domain. The different mutant derivatives were purified and shown to display the same biochemical properties as recombinant wild-type annexin II and the authentic protein purified from porcine intestine. However, significant differences in phosphate incorporation were observed when the individual serine mutants were subjected to phosphorylation by protein kinase C. A comparison of the phosphorylation patterns obtained identified Ser-ll as the protein kinase C site responsible for regulating the annexin II-pll interaction. Ser-ll lies within the sequence mediating pll binding, i.e. amino-acid residues 1 to 14 of annexin II, and phosphorylation at this site most likely leads to a direct spatial interference with pll binding.