Identification of dynamic changes in proteins associated with the cellular cytoskeleton after exposure to okadaic acid

Abstract

Exposure of cells to the diarrhetic shellfish poison, okadaic acid, leads to a dramatic reorganization of cytoskeletal architecture and loss of cell-cell contact. When cells are exposed to high concentrations of okadaic acid (100-500 nM), the morphological rearrangement is followed by apoptotic cell death. Okadaic acid inhibits the broad acting Ser/Thr protein phosphatases 1 and 2A, which results in hyperphosphorylation of a large number of proteins. Some of these hyperphosphorylated proteins are most likely key players in the reorganization of the cell morphology induced by okadaic acid. We wanted to identify these phosphoproteins and searched for them in the cellular lipid rafts, which have been found to contain proteins that regulate cytoskeletal dynamics and cell adhesion. By using stable isotope labeling by amino acids in cell culture cells treated with okadaic acid (400 nM) could be combined with control cells before the isolation of lipid rafts. Protein phosphorylation events and translocations induced by okadaic acid were identified by mass spectrometry. Okadaic acid was shown to regulate the phosphorylation status and location of proteins associated with the actin cytoskeleton, microtubules and cell adhesion structures. A large number of these okadaic acid-regulated proteins have previously also been shown to be similarly regulated prior to cell proliferation and migration. Our results suggest that okadaic acid activates general cell signaling pathways that induce breakdown of the cortical actin cytoskeleton and cell detachment.

Figure 1

Actin re-organization in okadaic acid-exposed cells. SH-SY5Y cells were left untreated (A,B) or exposed to 400 nM okadaic acid for 25 (C,D) or 50 (E–H) min. Fixed cells were labeled with Rhodamine-conjugated phalloidin, and actin distribution (red) was visualized using confocal microscopy. Nuclei were labeled using DAPI (blue).

Figure 2

Workflow to identify lipid raft-associated proteins. Stable isotopic labeling of amino acids in cell culture (SILAC) labeled SH-SY5Y cells left untreated or exposed to 400 nM okadaic acid for 50 min before mixed in a ratio 1:1 in 0.7% Triton X-100/MES buffer. After centrifugation in a sucrose gradient, 10 fractions of 0.5 mL were collected. The lipid raft fractions were identified by Western blotting using flotillin as a lipid raft marker. Fractions containing flotillin were pooled prior to removal of lipids by chloroform/methanol extraction, followed by acetone precipitation. Proteins were separated by SDS-PAGE. Proteins were in-gel digested with trypsin, and peptides were purified either with (1) C18 or (2) IMAC/TiO₂ and analyzed on LTQ-Orbitrap. Proteins were identified with Mascot (ver. 2.2) and quantified using MaxQuant (ver. 1.13).

Figure 3

Distribution of the identified phosphoproteins according to cellular compartment. 167 unique phosphopeptides belonging to 67 proteins were categorized according to cellular compartment (GeneOntology). Note that a protein can be located in more than one cellular component.

Figure 4

Verification of mass spectrometry-based results by Western blots of representative proteins. SH-SY5Y cells were treated with 400 nM okadaic acid for 50 min or left untreated two days after they had reached 100% confluency. Lipid rafts were isolated as described in the Materials and Methods sections, and proteins from all fractions (fractions 1–10) were separated by SDS-PAGE and immunoblotted using specific MARCKS-related protein, α-spectrin, β-spectrin, GAP43 and flotillin-1 antibodies. Arrows point to full-length spectrins and asterisks to the okadaic acid-induced cleavage product. Flotillin was used as a marker for lipid rafts (fractions 4–6).

Figure 5

Okadaic acid disrupts the microfilament-associated structure of α-actinin-4 and induces cytoplasm to nucleus shuttling. Confocal images of SH-SY5Y cells left untreated (A,B); or exposed to 400 nM okadaic acid for 50 min (C,D). Cells were immunostained with anti-α-actinin-4 (B,D), and nuclei were labeled with DAPI (A,C).

Figure 6

Protein interaction map of identified okadaic acid-regulated proteins. The map was constructed using the STRING 8.3 web tool [55]. Key proteins of cell adhesion platforms, cadherin (CDH1), tight junction protein (TJP1), desmoplakin (DSP), actin (ACTB) and tubulins (TUBA1A and TUBB1), were submitted to the database, together with the okadaic acid-regulated proteins (Table 1, Table 2). Colored lines denote interactions: green (text mining), red (gene fusion), blue (database) and grey (experiment).

Figure 7

Okadaic acid-induced cell rounding is not directly coupled to cell death. Cells were exposed to 400 nM okadaic acid for 30, 60, 75 and 90 min before they were either fixed (A,C,E,G) or the toxin was removed (B,D,F,H). After removal of the toxin, cells were left to grow for two more days.