Lipocortin 1 co-associates with cytokeratins 8 and 18 in A549 cells via the N-terminal domain

Abstract

An affinity chromatography strategy was used to search for proteins in A549 cells which interact with the N-terminus of lipocortin 1 (annexin 1). Using the biologically active fragment Lc13-25 as the affinity ligand, two proteins of molecular weight (m.w.) 52 and 48 kDa were extracted. Affinity blots of these proteins bound iodinated Lc13-25. Partial tryptic digests of these proteins were analysed by matrix assisted laser desorption mass spectrometry and found to display fragmentation patterns with a strong similarity to those of cytokeratin 8 and 18 respectively. Subsequent blotting with a panel of specific cytokeratin antibodies strongly supported the idea that the two proteins were cytokeratin 8 and cytokeratin 18. Cytokeratin 8 was isolated from A549 cells in intermediate filament (IF) preparations which were also found to contain lipocortin 1 as a potential intermediate filament associated protein (IFAP). This association persisted throughout cycles of IF assembly and disassembly. Dual-labelling immuno-histochemistry in A549 cells showed strong co-localization of lipocortin and cytokeratin 8. The implications of this finding are discussed in the light of the biological activity and possible function of lipocortin 1.