Glycoprotein capture and quantitative phosphoproteomics indicate coordinated regulation of cell migration upon lysophosphatidic acid stimulation.
The lipid mediator lysophosphatidic acid (LPA) is a serum component that regulates cellular functions such as proliferation, migration, and survival via specific G protein-coupled receptors. The underlying signaling mechanisms are still incompletely understood, including those that operate at the plasma membrane to modulate cell-cell and cell-matrix interactions in LPA-promoted cell migration. To explore LPA-evoked phosphoregulation with a focus on cell surface proteins, we combined glycoproteome enrichment by immobilized lectins with SILAC-based quantitative phosphoproteomics. We performed biological replicate analyses in SCC-9 squamous cell carcinoma cells and repeatedly quantified the effect of 1.5- and 5-min LPA treatment on more than 700 distinct phosphorylations in lectin-purified proteins. We detected many regulated phosphorylation events on various types of plasma membrane proteins such as cell adhesion molecules constituting adherens junctions, desmosomes, and hemidesmosomes. Several of these LPA-regulated phosphorylation sites have been characterized in a biological context other than G protein-coupled receptor signaling, and the transfer of this functional information suggests coordinated and multifactorial cell adhesion control in LPA-induced cell migration. Additionally, we identified LPA-mediated activation loop phosphorylation of the serine/threonine kinase Wnk1 and verified a role of Wnk1 for LPA-induced cell migration in knock-down experiments. In conclusion, the glycoproteome phosphoproteomics strategy described here sheds light on incompletely understood mechanisms in LPA-induced cell migratory behavior.
Mäusbacher
Nina
N
Project Group Cell Signaling, Department of Molecular Biology, Max Planck Institute of Biochemistry, Am Klopferspitz 18, Martinsried, Germany.
Schreiber
Thiemo B
TB
Daub
Henrik
H
D000595:Amino Acid Sequence; D000818:Animals; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000187:drug effects*; D019295:Computational Biology; D066246:ErbB Receptors / Q000235:genetics / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D008246:Lysophospholipids / Q000494:pharmacology*; D008969:Molecular Sequence Data; D010750:Phosphoproteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods*; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism; D049368:Receptors, Lysophosphatidic Acid / Q000378:metabolism
D006023:Glycoproteins; D008246:Lysophospholipids; D010750:Phosphoproteins; D043562:Receptors, G-Protein-Coupled; D049368:Receptors, Lysophosphatidic Acid; D066246:ErbB Receptors; C032881:lysophosphatidic acid