Increased Phospho-Keratin 8 Isoforms in Colorectal Tumors Associated with EGFR Pathway Activation and Reduced Apoptosis.

Georgia Arentz,Peter Hoffmann,Timothy J Price,Tim Chataway,Jennifer E Hardingham,Mark R Condina

doi:10.5402/2012/706545

Georgia Arentz, Peter Hoffmann + Show 4 more

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https://doi.org/10.5402/2012/706545

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Abstract

Hyperphosphorylated keratin (K) 8 acts as a phosphate “sponge” for stress-activated protein kinases thereby inhibiting pro-apoptotic molecules and thus apoptosis. MAP kinase/ERK1 has increased activity in colorectal cancer (CRC) and is known to phosphorylate K8. The aims were to identify the K8 isoforms abundantly present in colon tumors, using 2D difference gel electrophoresis (DIGE), to identify the modifications using mass spectrometry, and to validate the differential abundance of these isoforms in tumors relative to matched normal mucosae. 2D DIGE showed 3 isoforms of K8 significantly increased in tumor ≥2-fold in 6/8 pairs. Metal oxide affinity chromatography mass spectrometry and bioinformatics were used to identify phosphorylated serine residues. Levels of PS24, PS432, and PS74 by western blotting were found to be significantly increased in tumor versus matched normal. Blocking of EGFR signaling in Caco2 cells showed a significant decrease (P < 0.0001) in K8 PS74 and PS432 levels by 59% and 66%, respectively, resulting in increased apoptosis.

Highlights

K8 and K18 are the major intermediate filament (IF) components of simple epithelia of the gastrointestinal tract, liver, pancreas, and mammary glands [1]
The aims were to identify the K8 isoforms abundantly present in colon tumors, using 2D difference gel electrophoresis (DIGE), to identify the modifications using mass spectrometry, and to validate the differential abundance of these isoforms in tumors relative to matched normal mucosae. 2-Dimensional Difference Gel Electrophoresis (2D DIGE) showed 3 isoforms of K8 significantly increased in tumor ≥2fold in 6/8 pairs
The K8 residue serine 73 is known to be phosphorylated by c-Jun Nterminal kinase (JNK) and p38 kinases during cellular stress [10,11,12] and serine residue 431 is phosphorylated by ERK1 in response to EGFR stimulation [10, 12, 13]. (Phosphoserine (PS) 73 is subsequently referred to in this paper as PS74, PS431 as PS432 and PS23 as PS24, in accordance with the nomenclature adopted by UniProtKB, taking into account the first Met.) Increased signaling via the EGFR pathway has been well documented in colorectal cancer (CRC) and is due to upregulation of activating ligands such as EGF, epiregulin, amphiregulin, and TGFα or by activating mutations in EGFR itself [14]

Summary

Introduction

K8 and K18 are the major intermediate filament (IF) components of simple epithelia of the gastrointestinal tract, liver, pancreas, and mammary glands [1]. The phosphorylation of IF proteins is of primary importance in their function, regulating assembly, disassembly, and organisation in vitro and in vivo [7, 8]. Toivola et al (1997) showed that phosphorylation of IF is essential for the correct function of keratins, and when serine/threonine phosphatase activity is downregulated, cell junctions and the organisation of IF and microfilament assembly are disrupted. Constitutive activation of the RAS/RAF/MEK/ERK pathway occurs in almost 50% of CRC patients due to mutations in the KRAS and BRAF genes [15,16,17]. Little is known of the frequency and type of K8 phospho-isoforms in CRC. We sought to determine the effect of blocking MAP kinase activity on the level of K8 phosphorylation and level of induced apoptosis

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