Generation and characterization of a novel, permanently active S100P mutant

Biochim Biophys Acta. 2009 Jun;1793(6):1078-85. doi: 10.1016/j.bbamcr.2008.11.012. Epub 2008 Dec 8.

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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Calcium / metabolism*
  • Calcium-Binding Proteins* / genetics
  • Calcium-Binding Proteins* / metabolism
  • Cell Surface Extensions / metabolism
  • Cytoskeletal Proteins / metabolism
  • Epithelial Cells / cytology
  • Epithelial Cells / metabolism
  • Humans
  • Models, Molecular
  • Molecular Sequence Data
  • Mutagenesis, Site-Directed
  • Mutation*
  • Neoplasm Proteins* / genetics
  • Neoplasm Proteins* / metabolism
  • Protein Binding
  • Protein Structure, Tertiary
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Sequence Alignment

Substances

  • Calcium-Binding Proteins
  • Cytoskeletal Proteins
  • Neoplasm Proteins
  • Recombinant Fusion Proteins
  • S100P protein, human
  • ezrin
  • Calcium