Protein phosphatase-2A associates with and dephosphorylates keratin 8 after hyposmotic stress in a site- and cell-specific manner.
Keratins 8 and 18 (K8 and K18) are regulated by site-specific phosphorylation in response to multiple stresses. We examined the effect and regulation of hyposmotic stress on keratin phosphorylation. K8 phospho-Ser431 (Ser431-P) becomes dephosphorylated in HT29 cells, but hyperphosphorylated on other K8 but not K18 sites in HRT18 and Caco2 cells and in normal human colonic ex vivo cultures. Hyposmosis-induced dephosphorylation involves K8 but not K18, K19 or K20, occurs preferentially in mitotically active cells, and peaks by 6-8 hours then returns to baseline by 12-16 hours. By contrast, hyperosmosis causes K8 Ser431 hyperphosphorylation in all tested cell lines. Hyposmosis-induced dephosphorylation of K8 Ser431-P is inhibited by okadaic acid but not by tautomycin or cyclosporine. The PP2A catalytic subunit co-immunoprecipitated with K8 and K18 after hyposmotic stress in HT29 cells, but not in HRT18 or Caco2 cells where K8 Ser431 becomes hyperphosphorylated. K8 Ser431-P dephosphorylation after hyposmosis was independent of PP2A levels but correlated with increased PP2A activity towards K8 Ser431-P. Therefore, hyposmotic stress alters K8 phosphorylation in a cell-dependent manner, and renders K8 Ser431-P a physiologic substrate for PP2A in HT29 cells as a result of PP2A activation and the physical association with K8 and K18. The divergent hyposmosis versus hyperosmosis K8 Ser431 phosphorylation changes in HT29 cells suggest that there are unique signaling responses to osmotic stress.
Department of Medicine, Palo Alto VA Medical Center, 3801 Miranda Avenue, Mail Code 154J, Palo Alto, CA 94304, USA. guozhongtao@stanford.edu
Guo-Zhong
GZ
Tao
Diana M
DM
Toivola
Qin
Q
Zhou
Pavel
P
Strnad
Baohui
B
Xu
Sara A
SA
Michie
M Bishr
MB
Omary
D018938:Caco-2 Cells; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D016650:Fluorescein-5-isothiocyanate; D019084:Fluorescent Antibody Technique, Indirect; D005456:Fluorescent Dyes; D019073:HT29 Cells; D006801:Humans; D007038:Hypotonic Solutions; D007633:Keratins / Q000737:chemistry* / Q000378:metabolism*; D018613:Microscopy, Confocal; D008954:Models, Biological; D019319:Okadaic Acid / Q000494:pharmacology; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D054648:Protein Phosphatase 2; D012694:Serine / Q000378:metabolism*; D013312:Stress, Physiological / Q000378:metabolism*; D013379:Substrate Specificity / Q000187:drug effects; D014966:Xanthenes
D004791:Enzyme Inhibitors; D005456:Fluorescent Dyes; D007038:Hypotonic Solutions; D014966:Xanthenes; D019319:Okadaic Acid; D012694:Serine; D007633:Keratins; C034657:Texas red; D010749:Phosphoprotein Phosphatases; D054648:Protein Phosphatase 2; D016650:Fluorescein-5-isothiocyanate