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 prot ein implicated in membrane transport events along the Ca2+-regulated s ecretory and the early endocytic pathway. Biochemical properties of th is annexin and its intracellular distribution are regulated by complex formation with pll (S100A10), a member of the S100 protein family. Th e annexin II-pll interaction is mediated through the unique N-terminal domain of annexin II and is inhibited by protein kinase C phosphoryla tion of a serine residue in annexin II. To map this regulatory serine phosphorylation site we developed a baculovirus-mediated expression sy stem for wild-type annexin II and for a series of annexin II mutants w hich 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 w ild-type annexin II and the authentic protein purified from porcine in testine. However, significant differences in phosphate incorporation w ere observed when the individual serine mutants were subjected to phos phorylation by protein kinase C. A comparison of the phosphorylation p atterns obtained identified Ser-ll as the protein kinase C site respon sible for regulating the annexin II-pll interaction. Ser-ll lies withi n 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.