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First published online March 22, 2006
doi: 10.1242/10.1242/jcs.02861

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Protein phosphatase-2A associates with and dephosphorylates keratin 8 after hyposmotic stress in a site- and cell-specific manner

¹ Department of Medicine, Palo Alto VA Medical Center, 3801 Miranda Avenue, Mail Code 154J, Palo Alto, CA 94304, USA
² Stanford University Digestive Disease Center, 300 Pasteur Drive, Stanford, CA 94305, USA
³ Department of Pathology, Stanford University School of Medicine, Stanford, CA 94305, USA

View larger version (32K): [in a new window]   Fig. 1. Effect of osmotic milieu changes on keratin phosphorylation: (A) HT29 cells were cultured at 37°C in isosmotic (lanes 1,6; 12 hours), hyposmotic (lanes 2-4; 1,6 or 12 hours, respectively) and hyperosmotic (lanes 7-9; 1, 6 or 12 hours, respectively) conditions. As a positive control for keratin hyperphosphorylation, cells were cultured at 42°C for 12 hours (lanes 5, 10). Total cell lysates were then prepared, transferred to PVDF membranes, followed by blotting with antibodies to the indicated epitopes (pS431, Ser431-P; pS73, Ser73-P; pS33, Ser33-P; pS52, Ser52-P). Asterisks highlight a unique K8 phosphorylated species (owing to K8 Ser73 phosphorylation) that has a slightly delayed migration in SDS-PAGE gels (Liao et al., 1997). Blotting with antibodies to total K8 or K18 is included to ensure equal protein loading. (B) HT29 cells were plated followed by exposure to isosmotic (I; lanes 1, 3, 5, 7) or hyposmotic (H; lanes 2, 4, 6, 8) conditions for 6 hours during log-phase (40-50% confluency) or subconfluent (70-80%) growth in the presence or absence of serum. Total cell lysates were subjected to immunoblot analysis using the indicated antibodies.

View larger version (28K): [in a new window]   Fig. 2. Time course of K8 and K18 phosphorylation changes after hyposmotic exposure. HT29 cells were cultured in isosmotic (I) or hyposmotic (H) conditions (37°C) for the indicated time periods. Total cell lysates were prepared by mixing the cells with hot Laemmli sample buffer, followed by SDS-PAGE then immunoblotting using anti-phospho-keratin or anti-total-keratin antibodies as indicated.

View larger version (45K): [in a new window]   Fig. 3. Analysis of K8 phosphorylation and dephosphorylation by two-dimensional gel electrophoresis. HT29 cells were cultured in isosmotic or hyposmotic conditions for 6 hours, solubilized by 1% Emp in PBS (4°C, 60 minutes), followed by immunoprecipitation of K8 and K18. Precipitates were analyzed in duplicate by isoelectric focusing (IEF) in the first dimension followed by SDS-PAGE in the second dimension. One gel was then stained with Coomassie Blue and the second was transferred to a PVDF membrane for immunoblot analysis using anti-K8 Ser431-P antibodies. After blotting analysis, the PVDF membrane was stained with Coomassie Blue (not shown) in order to assign the indicated K8 charged isoforms.

View larger version (53K): [in a new window]   Fig. 4. Effect of hyposmosis on K19 and K20 phosphorylation. (A) Total cell extracts were prepared from HT29 cells (Total) or high salt extraction (HSE) was used to generate a highly enriched keratin fraction followed by analysis with SDS-PAGE and Coomassie Blue staining. (B) HT29 cells were incubated in isotonic or hypotonic medium for 6 hours, harvested, then subjected to SDS-PAGE followed by immunoblotting using antibodies to K19 or K20, or phospho-K19 or phosho-K20 (pK19 and pK20). Lanes 3 included homogenates isolated from OA-treated HT29 cells, as a positive control for the phospho-antibody reactivity. (C) Total cell lysates from iso- or hypo-treated HT29 cells (as in panel B) were analyzed by isoelectric focusing (IEF) in the first dimension followed by SDS-PAGE in second dimension then immunoblotting using anti-K19 or anti-K20 antibodies. Spot 1 is the non-phosphorylated isoform; others are phosphorylated isoforms for K19 and K20.

View larger version (23K): [in a new window]   Fig. 5. Effect of hyposmotic stress on K8 and K18 phosphorylation in human colonic cell lines and ex-vivo-cultured colonic biopsies. (A) Three human colonic cell lines (HT29, HRT18 and Caco2) were cultured in isosmotic (I) or hyposmotic (H) medium at 37°C for 6 hours. Total cell lysates were blotted with antibodies to the indicated antigens. (B) Total tissue lysates were generated from normal human colon biopsies immediately upon isolation (none), or after culturing in isosmotic (Iso) or hyposmotic (Hypo) conditions (37°C, 6 hours). Lysates were then blotted with antibodies to total K8 or to K8 Ser431-P.

View larger version (73K): [in a new window]   Fig. 6. Immunofluorescence analysis of keratins and phospho-keratins under isosmotic and hyposmotic conditions. HT29 (A) and Caco2 (B) cells were cultured in isosmotic (Iso) or hyposmotic (Hypo) conditions (6 hours, 37°C), then fixed and triple (K8/K18, K8 Ser431-P and nuclei in HT29 cells) or double (K8/K18, K8 Ser431-P in Caco2 cells) stained. The panels on the right are enlargements of the outlined `boxed' areas. Bars, 25 µm.

View larger version (18K): [in a new window]   Fig. 7. Effect of protein phosphatase inhibition on K8 Ser73 and Ser431 phosphorylation. (A) HT29 cells, cultured under isosmotic conditions, were treated with tautomycin, cyclosporine-A or carrier control (DMSO) for 1 hour (37°C). Total cell lysates were then prepared and blotted with antibodies to total K8 or K8 Ser431-P. (B) HRT18 and HT29 cells were cultured in isosmotic or hyposmotic conditions for 5 hours, then DMSO (vehicle control; lanes 1, 4, 7, 10) or OA (100, 625 nM) were added to the culture media (1 hour). Total cell lysates were then prepared followed by immunoblotting.

View larger version (25K): [in a new window]   Fig. 8. Association of PP2A with K8 and K18 in HT29 cells after hyposmotic stress. HRT18 and HT29 cells were cultured in isosmotic (I) or hyposmotic (H) conditions (6 hours, 37°C), then solubilized with 1% NP40 in PBS (4°C). (A) The lysates were then analyzed by blotting using antibodies to K8, K18, PP1 or PP2A. (B) Alternatively, the NP40 lysates were used to immunoprecipitate K8 and K18 followed by blotting using the indicated antibody. Asterisk indicates the antibody heavy chain band.

View larger version (19K): [in a new window]   Fig. 9. Schematic of K8 phosphorylation changes in response to osmotic alterations. K8 (and all other IF proteins) consist of a central, relatively conserved, coiled-coil -helical `rod' domain that is flanked by non--helical relatively non-conserved `head' and `tail' domains (Herrmann and Aebi, 2004). The head and tail domains contain most of the known posttranslational modification of IF proteins (Coulombe and Omary, 2002; Omary et al., 2004). Hyperosmotic stress results in human K8 hyperphosphorylation (upward-pointing double arrows) at Ser73 and Ser431. Hyposmotic stress (downward-pointing double arrows) also induces K8 hyperphosphorylation at Ser73 and Ser431 except in the case of HT29 cells where it induces K8 dephosphorylation via PP2A.