Generation and characterization of a novel, permanently active S100P mutant.
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.
Austermann
Judith
J
Center for Molecular Biology of Inflammation, Institute of Medical Biochemistry, Von-Esmarch-Str. 56, 48149 Münster, Germany.
Nazmi
Ali Reza
AR
Heil
Annika
A
Fritz
Günter
G
Kolinski
Michal
M
Filipek
Slawomir
S
Gerke
Volker
V
D000595:Amino Acid Sequence; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002135:Calcium-Binding Proteins / Q000235:genetics / Q000378:metabolism; D022081:Cell Surface Extensions / Q000378:metabolism; D003598:Cytoskeletal Proteins / Q000378:metabolism; D004847:Epithelial Cells / Q000166:cytology / Q000378:metabolism; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009363:Neoplasm Proteins / Q000235:genetics / Q000378:metabolism; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D016415:Sequence Alignment; D000099149:Ezrin
D002118:Calcium; D002135:Calcium-Binding Proteins; D003598:Cytoskeletal Proteins; D009363:Neoplasm Proteins; D011993:Recombinant Fusion Proteins; D000099149:Ezrin; C103003:S100P protein, human