Covalent Inhibition of the Peptidyl-Prolyl Isomerase Pin1 by Sulfopin Results in a Broad Impact on the Phosphoproteome of Human Osteosarcoma U2-OS Cells

Abstract

Peptidyl-prolyl isomerase, NIMA-interacting protein 1-(Pin1) catalyses the cis-trans interconversion of the inflexible bond between serine or threonine residues and proline at the +1 position (pSer/pThr-Pro). Although initially discovered as an essential regulator of cell division, Pin1 has since been identified as a regulator of many biological processes and is associated with numerous malignancies and neurodegenerative disorders. Pin1 has been shown to influence phosphorylation by modulating phosphatase accessibility. However, it can also indirectly regulate phosphorylation by isomerizing peptidyl-prolyl bonds on kinases, affecting their subcellular localization and/or substrate specificity. Here, SILAC-based mass spectrometry was employed to identify proteomic and phosphoproteomic changes in human osteosarcoma human osteosarcoma cell line (U2-OS) cells in response to treatment with the selective covalent Pin1 inhibitor Sulfopin. We confirmed that Sulfopin covalently binds Pin1 and profiled Pin1-dependent changes to the proteome and phosphoproteome, identifying 803 phosphosites that underwent significant Sulfopin-dependent changes. The identified phosphosites include substrates for a number of distinct kinases, including protein kinase B (AKT1), aurora kinase A (AURKA), cyclin-dependent kinase (CDK)1 and CK2. Overall, this study reveals the broad impact of Sulfopin on the phosphoproteome, improving our understanding of how Pin1 modulates complex regulatory kinase networks in living cells. SUMMARY: The peptidyl-prolyl isomerase (PPIase) Pin1 has emerged as a potential therapeutic target for numerous malignancies and neurodegenerative disorders based on its altered expression in several diseases. As the activity of Pin1 is phosphorylation-dependent, it is intimately involved with constituents of regulatory kinase networks within cells. To elucidate how Pin1 orchestrates regulatory signalling within cells, we performed quantitative proteomic and phosphoproteomic profiling of SILAC-labelled human osteosarcoma U2-OS cells treated with Sulfopin, a highly selective covalent Pin1 inhibitor. In addition to demonstrating that Pin1 inhibition alters the abundance and phosphorylation of proteins involved in a variety of fundamental cellular processes, these studies revealed that Pin1 inhibition modulates the phosphorylation of 803 phosphorylation sites, ultimately improving our understanding of how a PPIase regulates phosphorylation networks in complex biological systems.

Keywords: CK2; Pin1; Sulfopin; phosphoproteomics; protein kinase; proteomics.

FIGURE 1

Characterizing the effect of Sulfopin on Pin1‐binding and abundance in vitro and in human osteosarcoma (U2‐OS) cells. (A) Chemical structure of Sulfopin. (B) U2‐OS cells were treated with 10 µM Sulfopin for 72 h before lysis and incubation with maleimide‐PEG (mal‐PEG) [2:1 (m/m)] for 1 h at 37°C. The extent to which Pin1 and Sulfopin‐treated Pin1 were modified by PEG was assessed by immunoblotting with Pin1 antibodies. Cells treated with DMSO and lysates untreated with mal‐PEG were used as controls. (C) Recombinant WT and Cys113Asp (C113D) Pin1 were incubated in triplicate with 10 µM Sulfopin (Selleckchem) for 2 h at room temperature before 15% native‐PAGE. (D) Cells were treated in triplicate with 5.0–15.0 µM Sulfopin for 72 h, and Pin1 abundance was evaluated with immunoblotting. One‐way ANOVA and Dunnett's multiple comparisons tests (p < 0.05) were performed using GraphPad Prism (v10.0.2). Scale bar = 1000 µm. Vinculin was used as a loading control for immunoblotting. Cell media changed after 48 h. ANOVA, analysis of variance; U2‐OS, human osteosarcoma cell line.

FIGURE 2

The effect of Sulfopin on the human osteosarcoma (U2‐OS) proteome. Cells were treated in six biological replicates with 10 µM Sulfopin for 72 h. (A) Workflow for quantitative SILAC‐based proteomic and phosphoproteomic evaluations. (B) Volcano plot visualization of differential protein expression in Sulfopin versus DMSO‐treated cells. Green and blue dots indicate differential expression as determined by statistical (FDR < 0.05) or 1.5‐fold change (Log2 ± 0.585) filters, respectively. Red dots indicate differential expression as determined with both statistical significance and Log2FC cutoffs. (C) GO‐BP enrichment of differentially expressed proteins in U2‐OS cells treated with 10 µM Sulfopin for 72 h. PANTHER (v18.0) was used with default settings to identify enriched GO‐BPs, and the processes with the highest statistical significance were visualized with R Studio. GO‐BP, GO‐biological process; U2‐OS, human osteosarcoma cell line.

FIGURE 3

The effect of Sulfopin on the human osteosarcoma (U2‐OS) phosphoproteome. Cells were treated in six biological replicates with 10 µM Sulfopin for 72 h. (A) Volcano plot visualization of differential phosphorylation in Sulfopin versus DMSO‐treated cells. Green and blue dots indicate differential phosphorylation as determined by statistical (FDR < 0.05) or 1.5‐fold change (Log2 ± 0.585) filters, respectively. Red dots indicate differential phosphorylation, as determined by statistical significance and Log2FC cutoffs. (B) GO‐BP enrichment of differentially phosphorylated proteins in U2‐OS cells treated with 10 µM Sulfopin for 72 h. PANTHER (v18.0) was used with default settings to identify enriched GO‐BPs, and the processes with the highest statistical significance were visualized with R Studio. (C) STRING analysis and Molecular Complex Detection (MCODE) were employed within Cytoscape to identify the top three scoring protein–protein interaction clusters of differentially phosphorylated proteins, which were then individually analysed for GO‐BP enrichment. GO‐BP, GO‐biological process; U2‐OS, human osteosarcoma cell line.

FIGURE 4

Kinase‐substrate enrichment analysis (KSEA) of the Sulfopin‐dependent phosphoproteome. (A) The mean fold changes for kinase substrates (as determined by PhosphoSitePlus) were averaged and converted to a z.score to estimate kinase activity. Significantly upregulated or downregulated kinases due to Sulfopin treatment are identified with red and blue bars, respectively. (B) pLogo (https://plogo.uconn.edu, v1.2.0) motif analysis of upregulated (left) or downregulated (right) phosphosites after treatment with Sulfopin. Shortened sequence motifs of all identified phosphosites were used as background sequences. Redundant sequences were removed before analysis.