| Summary: | The binding of integrin adhesion receptors to their extracellular matrix (ECM) ligands activates intracellular signalling pathways that control diverse and fundamental aspects of cell behaviour. While it is clear that protein kinases and phosphatases play an integral role in such adhesion-mediated signalling, current knowledge of the phosphorylation events regulated downstream of integrin ligation is limited and prohibits a systems-level understanding of the molecular mechanisms through which ECM engagement influences cell phenotype. Here, mass spectrometry (MS)-based phosphoproteomic approaches were used to globally characterise the phosphorylation-dependent signalling networks activated by integrin-mediated adhesion. A phosphoproteomic workflow was developed to detect phosphorylation sites from the lysates of cells adherent to the ECM protein fibronectin (FN). Optimisation of chromatographic techniques for the enrichment of phosphopeptides prior to mass spectrometric analyses resulted in the detection of 735 phosphopeptides from FN-engaged cells, including a number that were derived from key adhesion-related proteins. However, several phosphorylation sites that are well-documented to be upregulated in response to integrin-mediated adhesion were not detected by analyses using cell lysates, indicating that a comprehensive analysis of adhesion-regulated signalling was not being achieved. To specifically analyse integrin-proximal signalling and thereby improve the identification of adhesion-regulated phosphorylation events, the phosphoproteomic workflow was applied to the analysis of isolated integrin-based adhesion complexes. The coverage of phosphopeptides from these samples was incrementally improved through optimisation of various stages of the workflow (including adhesion complex isolation, proteolytic digestion and MS-based data acquisition strategies), culminating in a fully optimised workflow facilitating the detection of 2049 phosphopeptides. Notably, compared to analyses using celllysates, these data represented a major improvement in the detection of phosphorylation sites from well-characterised adhesion complex proteins, and included many known components of adhesion-induced signalling pathways. A semi-quantitative analysis of MS data collected from adhesion complexes compared to complexes isolated from cells engaging a non-integrin ligand identified 1109 phosphorylation sites that were specific to integrin-based adhesion complexes. This dataset provides a detailed and global depiction of the phosphorylation sites found specifically on proteins recruited to sites of integrinmediated adhesion. Moreover, validation of a number of phosphorylation sites confirmed the specificity of the dataset and suggested that two classes of phosphorylation sites reside at adhesion complexes: sites induced by adhesion and sites that are constitutively phosphorylated but recruited by adhesion. The workflow described here represents a novel approach for analyses of the signalling networks that function at the cell-ECM interface, and will provide a useful tool for future analyses aiming to elucidate specific mechanisms through which adhesion receptors control cell behaviour.
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