Biochemical characterization of S100A2 in human keratinocytes: Subcellularlocalization, dimerization, and oxidative cross-linking

S100A2 is a calmodulin-like protein of unknown function, whose transcriptio n is positively regulated in response to ErbB and p53 signaling. Expression of S100A2 is markedly increased in the context of ErbB-driven reactive epi dermal hyperplasia, and decreased in the context of hypofunctional p53 muta tions in carcinoma cell lines and tumors. This bimodal pattern of regulatio n suggests an important function for S100A2 in keratinocyte differentiation and carcinogenesis. Taking the biochemical approach to the determination o f S100A2 function, we have characterized its physical state and subcellular localization in normal human keratinocytes. S100A2 in hypotonic lysates re mained soluble after centrifugation at 100 000 x g, indicating that it is n ot associated with cell membranes. Permeabilization experiments confirmed t he lack of membrane association and revealed a digitonin-insoluble nuclear fraction of S100A2, which was confirmed by immunofluorescence microscopy. P ulldown assays of epitope-tagged S100A2 and yeast two-hybrid screening reve aled that S100A2 displays a strong propensity to homodimerize. Naturally ex pressed S100A2 dimers in normal human keratinocytes readily underwent inter molecular disulfide cross-linking unless a strong denaturant was present du ring cell lysis. Treatment of intact normal human keratinocytes with hydrog en peroxide strongly promoted S100A2 cross-linking. These results demonstra te that native S100A2 is a homodimer that does not depend on disulfide cros s-linking for stability, but undergoes intermolecular cross-linking at cyst eine 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.