Keratin 8 Phosphorylation by p38 Kinase Regulates Cellular Keratin Filament Reorganization: MODULATION BY A KERATIN 1-LIKE DISEASE-CAUSING MUTATION

Nam-On Ku,M Bishr Omary,Salman Azhar

doi:10.1074/jbc.m107623200

Abstract

Keratin 8 (K8) serine 73 occurs within a relatively conserved type II keratin motif ((68)NQSLLSPL) and becomes phosphorylated in cultured cells and organs during mitosis, cell stress, and apoptosis. Here we show that Ser-73 is exclusively phosphorylated in vitro by p38 mitogen-activated protein kinase. In cells, Ser-73 phosphorylation occurs in association with p38 kinase activation and is inhibited by SB203580 but not by PD98059. Transfection of K8 Ser-73 --> Ala or K8 Ser-73 --> Asp with K18 generates normal-appearing filaments. In contrast, exposure to okadaic acid results in keratin filament destabilization in cells expressing wild-type or Ser-73 --> Asp K8, whereas Ser-73 --> Ala K8-expressing cells maintain relatively stable filaments. p38 kinase associates with K8/18 immunoprecipitates and binds selectively with K8 using an in vitro overlay assay. Given that K1 Leu-160 --> Pro ((157)NQSLLQPL --> (157)NQSPLQPL) leads to epidermolytic hyperkeratosis, we tested and showed that the analogous K8 Leu-71 --> Pro leads to K8 hyperphosphorylation by p38 kinase in vitro and in transfected cells, likely due to Ser-70 neo-phosphorylation, in association with significant keratin filament collapse upon cell exposure to okadaic acid. Hence, K8 Ser-73 is a physiologic phosphorylation site for p38 kinase, and its phosphorylation plays an important role in keratin filament reorganization. The Ser-73 --> Ala-associated filament reorganization defect is rescued by a Ser-73 --> Asp mutation. Also, disease-causing keratin mutations can modulate keratin phosphorylation and organization, which may affect disease pathogenesis.

Highlights

Keratin 8 (K8) serine 73 occurs within a relatively conserved type II keratin motif (68NQSLLSPL) and becomes phosphorylated in cultured cells and organs during mitosis, cell stress, and apoptosis
This was aided by an antibody termed LJ4, which was generated by immunizing mice with keratins that were purified from okadaic acidtreated HT-29 cells
The HK8 species are present in very small amounts in exponentially growing HT-29 cells as determined by immunoprecipitation with monoclonal antibody (mAb) L2A1, which recognizes the entire keratin pool [31], but become markedly enriched after immunoprecipitation with mAb LJ4

Summary

Introduction

Keratin 8 (K8) serine 73 occurs within a relatively conserved type II keratin motif (68NQSLLSPL) and becomes phosphorylated in cultured cells and organs during mitosis, cell stress, and apoptosis. Transgenic mouse studies showed that K18 Ser-52 phosphorylation facilitates a protective role against hepatotoxic injury [28], a finding that has provided direct evidence for a number of correlative data that document increased keratin phosphorylation in association with a variety of stresses in cultured cells and in intact animals [29]. Ser-23 is a highly conserved site among all type II keratins, which suggests a common keratin function for this modification, whereas Ser-431 is a basally phosphorylated site that increases its phosphorylation specific activity during mitosis and upon exposure to epidermal growth factor in association with filament reorganization [30]. In contrast K8 Ser-73 phosphorylation behaves like an on/off switch in cultured cells and in tissues, with phosphorylation being “on” during mitosis, a variety of cell stresses including heat and drug exposure, and during apoptosis [31]

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Conclusion