Proteomic analysis of cytokeratin 8 isoforms in cytosolic protein extracts of human colorectal mucosa reveals a complex pattern of partial degradation with hyperphosphoryation
Elliott, R. M., Polley , J. C., Bradburn, D. M., Commane, D., Arasaradnam, R. P., Mulholland, F., Mann, S., Belshaw, N., Mathers , J. C. and Johnson , I. T. (2012) Proteomic analysis of cytokeratin 8 isoforms in cytosolic protein extracts of human colorectal mucosa reveals a complex pattern of partial degradation with hyperphosphoryation. Journal of Proteome Research. ISSN 1535-3907 (In Press)
Keratin 8 (K8) is an intermediate filament (IF) protein widely expressed in mammalian epithelial tissues. Apart from mechanical functions, keratins are involved in regulation of key cellular processes, including proliferation, apoptosis and epithelial integrity. Building on previous work that showed increased expression of K8 isoforms in morphologically normal colorectal mucosa of patients with distant neoplastic lesions, we describe the characterisation of K8 isoforms in the soluble protein fraction of human colorectal epithelium. Using Western blot analysis of 2D gels, we applied antibodies specific for differing molecular regions, and for specific phosphorylated and acetylated forms of the protein. The full range of K8 immunoreactive features observed on the 2D blots of human colorectal mucosa were accounted for in terms of proteolytic fragmentation leading to the variations in molecular weight, and sequential increases in the numbers of phosphorylated amino acids, with consequent stepwise reductions in apparent pI. We found no significant acetylation of K8. Although the total extent of K8 phosphorylation exceeded that reported previously, the levels of phosphorylation at ser23, 73 or 431were relatively low. The truncated and phosphorylated forms of K8 detected in the present study probably represent the soluble cytoplasmic pool of K8 involved in cytoskeletal maintenance.