Biochemical characterization of S100A2 in human keratinocytes: subcellular localization, dimerization, and oxidative cross-linking.
S100A2 is a calmodulin-like protein of unknown function, whose transcription is positively regulated in response to ErbB and p53 signaling. Expression of S100A2 is markedly increased in the context of ErbB-driven reactive epidermal hyperplasia, and decreased in the context of hypofunctional p53 mutations in carcinoma cell lines and tumors. This bimodal pattern of regulation suggests an important function for S100A2 in keratinocyte differentiation and carcinogenesis. Taking the biochemical approach to the determination of S100A2 function, we have characterized its physical state and subcellular localization in normal human keratinocytes. S100A2 in hypotonic lysates remained soluble after centrifugation at 100 000 x g, indicating that it is not associated with cell membranes. Permeabilization experiments confirmed the lack of membrane association and revealed a digitonin-insoluble nuclear fraction of S100A2, which was confirmed by immunofluorescence microscopy. Pulldown assays of epitope-tagged S100A2 and yeast two-hybrid screening revealed that S100A2 displays a strong propensity to homodimerize. Naturally expressed S100A2 dimers in normal human keratinocytes readily underwent intermolecular disulfide cross-linking unless a strong denaturant was present during cell lysis. Treatment of intact normal human keratinocytes with hydrogen peroxide strongly promoted S100A2 cross-linking. These results demonstrate that native S100A2 is a homodimer that does not depend on disulfide cross-linking for stability, but undergoes intermolecular cross-linking at cysteine residues in response to oxidative stress. Based on these findings, we propose that S100A2 may protect normal keratinocytes against carcinogens by participating in the cellular proof-reading response to oxidative stress.
Deshpande
R
R
Departments of Dermatology and Radiation Oncology (Cancer Biology), University of Michigan Medical School, Ann Arbor, Michigan, USA.
Woods
T L
TL
Fu
J
J
Zhang
T
T
Stoll
S W
SW
Elder
J T
JT
D000911:Antibodies, Monoclonal / Q000276:immunology; D000918:Antibody Specificity; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification; D002413:Cations, Divalent / Q000494:pharmacology; D002630:Chemotactic Factors / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D019281:Dimerization; D004220:Disulfides / Q000737:chemistry; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology; D015603:Keratinocytes / Q000737:chemistry*; D008970:Molecular Weight; D010084:Oxidation-Reduction; D009418:S100 Proteins / Q000737:chemistry* / Q000276:immunology / Q000302:isolation & purification*; D013347:Subcellular Fractions / Q000737:chemistry
D000911:Antibodies, Monoclonal; D002135:Calcium-Binding Proteins; D002413:Cations, Divalent; D002630:Chemotactic Factors; D004220:Disulfides; D009418:S100 Proteins; C098997:S100A2 protein, human; D006861:Hydrogen Peroxide