Nodularin Exposure Induces SOD1 Phosphorylation and Disrupts SOD1 Co-localization with Actin Filaments

Linda Hjørnevik,Therese Solstad,Fiona Young,Kari Fladmark,Lise Fismen

doi:10.3390/toxins4121482

Linda Hjørnevik, Therese Solstad + Show 3 more

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https://doi.org/10.3390/toxins4121482

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Journal: Toxins	Publication Date: Dec 14, 2012
Citations: 41	License type: CC BY 3.0

Affiliation: University of Bergen, University of Dundee

Abstract

Apoptotic cell death is induced in primary hepatocytes by the Ser/Thr protein phosphatase inhibiting cyanobacterial toxin nodularin after only minutes of exposure. Nodularin-induced apoptosis involves a rapid development of reactive oxygen species (ROS), which can be delayed by the Ca2+/calmodulin protein kinase II inhibitor KN93. This apoptosis model provides us with a unique population of highly synchronized dying cells, making it possible to identify low abundant phosphoproteins participating in apoptosis signaling. Here, we show that nodularin induces phosphorylation and possibly also cysteine oxidation of the antioxidant Cu,Zn superoxide dismutase (SOD1), without altering enzymatic SOD1 activity. The observed post-translational modifications of SOD1 could be regulated by Ca2+/calmodulin protein kinase II. In untreated hepatocytes, a high concentration of SOD1 was found in the sub-membranous area, co-localized with the cortical actin cytoskeleton. In the early phase of nodularin exposure, SOD1 was found in high concentration in evenly distributed apoptotic buds. Nodularin induced a rapid reorganization of the actin cytoskeleton and, at the time of polarized budding, SOD1 and actin filaments no longer co-localized.

Highlights

The cyanobacterial-produced hepatotoxins, nodularin and microcystin, are potent inhibitors of the general Ser/Thr phosphatases (PP1 and PP2A)
We found that the initial co-localization of SOD1 and actin in hepatocytes was disrupted during nodularin exposure and that a high content of SOD1 was found in the apoptotic buds
Primary hepatocytes exposed to 5 μM nodularin developed apoptosis, involving membrane budding (Figure 1C) and actin reorganization (Figure 1E), in only 2–4 min after toxin addition (Figure 1)

Summary

Introduction

The cyanobacterial-produced hepatotoxins, nodularin and microcystin, are potent inhibitors of the general Ser/Thr phosphatases (PP1 and PP2A) Exposure to these toxins induces apoptotic cell death in isolated hepatocytes after only minutes of exposure [1,2]. Most known phosphorylated target proteins in nodularin and microcystin-exposed apoptotic hepatocytes are cytoskeletal, or proteins known to associate with the cytoskeleton, e.g., plectin [5], keratin 8/18 [6], myosin light chain [1], HSP27 and tau [7,8] Most likely, these phosphorylation events are coupled to the dramatic cytoskeletal reorganization occurring after phosphatase inhibitor exposure, starting with loss of cell-cell contacts [9], followed by disruption of intermediate filaments [10] and contraction and aggregation of actin microfilaments [11]. These cytoskeletal changes are reflected in the apoptotic morphology of the cell in which microvilli are lost, followed by the appearance of evenly distributed apoptotic buds, which eventually gather at one pole [4]

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