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

Guo-Zhong Tao,Baohui Xu,Sara A Michie,Diana M Toivola,M Bishr Omary,Qin Zhou,Pavel Strnad

doi:10.1242/jcs.02861

Abstract

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.

Highlights

Intermediate filaments (IF) are a major component of the three cytoskeletal networks that include the actin microfilament and tubulin microtubule networks (Ku et al, 1999; Chang and Goldman, 2004)
We show that hyposmosis results in sitespecific dephosphorylation of K8 in HT29 cells by association with the catalytic subunit of phosphatase 2A (PP2A), whereas hyperosmosis induces K8 hyperphosphorylation in the same cells
Phosphorylation changes of HT29 cell K8 Ser431 in response to extracellular osmotic alterations We examined the effect of hyper- and hyposmotic changes on K8 and K18 phosphorylation using site-specific antiphosphokeratin antibodies

Summary

Introduction

Intermediate filaments (IF) are a major component of the three cytoskeletal networks that include the actin microfilament and tubulin microtubule networks (Ku et al, 1999; Chang and Goldman, 2004). Compared with the limited number of actins and tubulins, IF proteins make up a large family of tissue- and differentiation-specific proteins that are encoded by more than 60 unique genes (Hesse et al, 2001; Herrmann and Aebi, 2004). Mutations of IF genes are associated with a wide spectrum of diseases that reflect their tissue-selective expression (Fuchs and Cleveland, 1998; Omary et al, 2004). The major IF proteins consist of keratins (K) which include type I (K9-K20, relatively acidic) and type II (K1-K8, neutral or basic) IF (Coulombe and Omary, 2002). All epithelial cells express at least one type I and one type II keratin that associate as obligate heteropolymers in a noncovalent fashion

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