Generation and characterization of a novel, permanently active S100P mutant.

ABSTRACT

S100 proteins function as Ca2+ signal transducers by regulating cellular targets in their Ca2+ bound conformation. S100P is a member of the S100 protein family that can activate the membrane and F-actin binding protein ezrin in a Ca2+ dependent manner at least in vitro. Here we generated a novel tool to elucidate directly the S100P-ezrin interaction in vivo. This was achieved by constructing a S100P derivative that contained mutations in the two EF hand loops predicted to lock the protein in a permanently active state. The resulting S100P mutant, termed here S100P pa, could be purified as a soluble protein and showed biochemical properties displayed by wild-type S100P only in the presence of Ca2+. Importantly, S100P pa bound to the N-terminal domain of ezrin in the absence of Ca2+ showing an affinity only slightly reduced as compared to that of Ca2+-bound WT S100P. In line with this permanent complex formation, S100P pa colocalized with ezrin to plasma membrane protrusions of epithelial cells even in the absence of intracellular Ca2+ transients. Thus, S100P pa is a novel type of S100 protein mutant locked in a permanently active state that shows an unregulated complex formation with its cellular target ezrin.