title: Two functional S100A4 monomers are necessary for regulating nonmuscle myosin-IIA and HCT116 cell invasion.
abstract: S100A4, a member of the Ca(2+)-activated S100 protein family, regulates the motility and invasiveness of cancer cells. Moreover, high S100A4 expression levels correlate with poor patient survival in several cancers. Although biochemical, biophysical, and structural data indicate that S100A4 is a noncovalent dimer, it is unknown if two functional S100A4 monomers are required for the productive recognition of protein targets and the promotion of cell invasion. To address this question, we created covalently linked S100A4 dimers using a glycine rich flexible linker. The single-chain S100A4 (sc-S100A4) proteins exhibited wild-type affinities for calcium and nonmuscle myosin-IIA, retained the ability to regulate nonmuscle myosin-IIA assembly, and promoted tumor cell invasion when expressed in S100A4-deficient colon carcinoma cells. Mutation of the two calcium-binding EF-hands in one monomer, while leaving the other monomer intact, caused a 30-60-fold reduction in binding affinity for nonmuscle myosin-IIA concomitant with a weakened ability to regulate the monomer-polymer equilibrium of nonmuscle myosin-IIA. Moreover, sc-S100A4 proteins with one monomer deficient in calcium responsiveness did not support S100A4-mediated colon carcinoma cell invasion. Cross-linking and titration data indicate that the S100A4 dimer binds a single myosin-IIA target peptide. These data are consistent with a model in which a single peptide forms interactions in the vicinity of the canonical target binding cleft of each monomer in such a manner that both target binding sites are required for the efficient interaction with myosin-IIA.
authors: Department of Biochemistry, Albert Einstein College of Medicine, Bronx, New York 10461, United States.
authors: Reniqua P
authors: RP
authors: House
authors: Maria
authors: M
authors: Pozzuto
authors: Purvi
authors: P
authors: Patel
authors: Natalya G
authors: NG
authors: Dulyaninova
authors: Zhong-Hua
authors: ZH
authors: Li
authors: Wendy D
authors: WD
authors: Zencheck
authors: Michele I
authors: MI
authors: Vitolo
authors: David J
authors: DJ
authors: Weber
authors: Anne R
authors: AR
authors: Bresnick
mesh_terms: D000595:Amino Acid Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D002850:Chromatography, Gel; D002942:Circular Dichroism; D019281:Dimerization; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009361:Neoplasm Invasiveness; D024507:Nonmuscle Myosin Type IIA / Q000378:metabolism*; D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins / Q000737:chemistry / Q000378:metabolism / Q000502:physiology*
chemical_list: D000071999:S100 Calcium-Binding Protein A4; D009418:S100 Proteins; C077591:S100A4 protein, human; D024507:Nonmuscle Myosin Type IIA