Identification of differentially regulated proteins associated with the cellular cytoskeleton in okadaic acid-exposed neuroblastoma cells

Jill A. Opsahl1, Sonja Ljostveit2, Therese Solstad3, Kristin Risa4, Peter Roepstroff5, Kari E. Fladmark6. 1Proteomic Unit (PROBE), Departement of Biomedicine, University of Bergen, Jonas Lies Vei 91, N-5009 Bergen, Norway, 2Departement for Molecular Biology, University of Bergen, Thormøhlensgate 55, N5008 Bergen, Norway, 3The Biotechnology Center, University of Oslo, Gaustas alleen 21, N-0349 Oslo, Norway, 4Departement for Molecular Biology, University of Bergen, Thormøhlensgate 55, N5008 Bergen, Norway, 5Department of Biochemistry and Molecular Biology, University of Southern Denmark, Odense, Denmark, 6Departement for Molecular Biology, University of Bergen, Thormøhlensgate 55, N5008 Bergen, Norway

The diarrhetic shellfish poison okadaic acid acts both as tumor promotor and as an apoptosis inducer (Gehringer, 2004). The dualistic function of this algal toxin seems dose dependent. Okadaic acid targets the serine/threonine protein phosphatases 1 and 2A which control more than 90% of all serine/threonine dephosphorylation in mammalian cells, thereby resulting in hyperphosphorylation of a large number of proteins. Exposure to okadaic acid in the 100-500 nM range induces apoptotic cell death in all hitherto tested cell lines and the toxin is frequently used to study neural cell death as it reproduces the characteristic tau-hyperphosphorylation found in Alzheimer’s disease.

Exposure of neuroblastoma SH-SY5Y cells to 400 nM okadaic acid leads to a dramatic and rapid reorganization of the cytoskeleton architecture and disruption of cell-cell interaction. The aim of the study was to identify the phosphoproteins involved in this reorganization. In order to enrich for these phosphoproteins we isolated lipid rafts which are known to play a role in cell adhesion and cytoskeleton to cell membrane coupling (Levitan and Gooch, 2007).

Stable isotope labeling of amino acids in cell culture (SILAC) was used to enable to combine okadaic acid-treated and control SH-SY5Y cell lysates prior to the isolation of lipid rafts. Proteins of the lipid rafts were then separated by SDS-PAGE, trypsinated and phosphopeptides were enriched by IMAC and TiO2. Phosphopeptides were then analyzed by LC-MS/MS using a LTQ-Orbitrap XL. The general lipid raft protein was also analyzed to identify okadaic acid-induced protein translocations.

We identified 167 phosphopeptides belonging to 67 different proteins. A functional classification (GeneOntology-index) showed that 77% of these proteins were membrane proteins and 75% cytosolic. Most importantly, 23% had previously been shown to be involved in the regulation of the cell cytoskeleton and cell adhesion. We found that okadaic acid regulated the phosphorylation status and translocation of proteins associated with the actin cytoskeleton, microtubuli and cell adhesion structures. The majority of the identified okadaic acid-regulated phosphorylation events have also been shown to be stimulated in proliferating cells (Olsen 2006; Dephoure 2006) and some of them are known to be calpain substrates that must be cleaved prior to cell proliferation and migration.

Our results suggest that okadaic acid activates cell signaling pathways that induce breakdown of the cortical cytoskeleton and cell adhesion that are common for both apoptotic and proliferating cells.