Stress, apoptosis, and mitosis induce phosphorylation of human keratin 8 at Ser-73 in tissues and cultured cells

Simple epithelia express keratins 8 (K8) and 18 (K18) as their major intermediate filament proteins, We previously showed that several types of cell stress such as heat and virus infection result in a distinct hyperphosphorylated form of K8 (termed HK8). To better characterize K8/18 phosphorylation, we generated monoclonal antibodies by immunizing mice with hyperphosphorylated keratins that were purified from colonic cultured human HT29 cells pretreated with okadaic acid. One antibody specifically recognized HK8, and the epitope was identified as (71)LLpSPL which corresponds to K8 phosphorylation at Ser-73, Generation of HK8 occurs in mitotic HT29 cells, basal crypt mitotic cells in normal mouse intestine, and in regenerating mouse hepatocytes after partial hepatectomy, Prominent levels of HK8 were also generated in HT29 cells that were induced to undergo apoptosis using anisomycin or etoposide. In addition, mouse hepatotoxicity that is induced by chronic feeding with griseofulvin resulted in HK8 formation in the liver. Our results demonstrate that a ''reverse immunological'' approach, coupled with enhancing in vivo phosphorylation using phosphatase inhibitors, can result in the identification of physiologic phosphorylation states, As such, K8 Ser-73 phosphorylation generates a distinct HK8 species under a variety of in vivo conditions including mitosis, apoptosis, and cell stress. The low steady state levels of HK8 during mitosis, in contrast to stress and apoptosis, suggest that accumulation of HK8 may represent a physiologic stress marker for simple epithelia.