title: Probing protein phosphatase substrate binding: affinity pull-down of ILKAP phosphatase 2C with phosphopeptides.
abstract: Proteomics and high throughput analysis for systems biology can benefit significantly from solid-phase chemical tools for affinity pull-down of proteins from complex mixtures. Here we report the application of solid-phase synthesis of phosphopeptides for pull-down and analysis of the affinity profile of the integrin-linked kinase associated phosphatase (ILKAP), a member of the protein phosphatase 2C (PP2C) family. Phosphatases can potentially dephosphorylate these phosphopeptide substrates but, interestingly, performing the binding studies at 4 °C allowed efficient binding to phosphopeptides, without the need for phosphopeptide mimics or phosphatase inhibitors. As no proven ILKAP substrates were available, we selected phosphopeptide substrates among known PP2Cδ substrates including the protein kinases: p38, ATM, Chk1, Chk2 and RSK2 and synthesized directly on PEGA solid supports through a BAL type handle. The results show that phosphopeptides tethered to a flexible solid support bind with high affinity and specificity to ILKAP, which is pulled down from lysates of cells transfected with ILKAP cDNA. Phosphorylation on Ser or Thr residues is important for binding of ILKAP, but sequences around the phosphorylated residue are important for the binding affinity of ILKAP. We conclude that solid-phase affinity pull-down of proteins from complex mixtures can be applied in phosphoproteomics and systems biology.
authors.affiliation: Department of Clinical Biochemistry, Glostrup Hospital, 2600 Glostrup, Denmark. kimlar01@regionh.dk
authors.forename: Kim B
authors.initials: KB
authors.lastname: Højlys-Larsen
authors.forename: Kasper K
authors.initials: KK
authors.lastname: Sørensen
authors.forename: Knud J
authors.initials: KJ
authors.lastname: Jensen
authors.forename: Steen
authors.initials: S
authors.lastname: Gammeltoft
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D019556:COS Cells; D018797:Cell Cycle Proteins / Q000378:metabolism; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D002522:Chlorocebus aethiops; D002846:Chromatography, Affinity / Q000379:methods*; D004268:DNA-Binding Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D008969:Molecular Sequence Data; D010748:Phosphopeptides / Q000737:chemistry / Q000378:metabolism*; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D011494:Protein Kinases / Q000378:metabolism; D000071636:Protein Phosphatase 2C; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D040901:Proteomics; D038744:Ribosomal Protein S6 Kinases, 90-kDa / Q000378:metabolism; D012394:Rosaniline Dyes; D060327:Solid-Phase Synthesis Techniques; D013194:Staining and Labeling; D013379:Substrate Specificity; D049490:Systems Biology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D025521:Tumor Suppressor Proteins / Q000378:metabolism; D048051:p38 Mitogen-Activated Protein Kinases / Q000378:metabolism
chemical_list: D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D010748:Phosphopeptides; D012394:Rosaniline Dyes; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; C048139:Coomassie blue; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; D064007:Ataxia Telangiectasia Mutated Proteins; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D038744:Ribosomal Protein S6 Kinases, 90-kDa; C069330:ribosomal protein S6 kinase, 90kDa, polypeptide 3; D048051:p38 Mitogen-Activated Protein Kinases; D010749:Phosphoprotein Phosphatases; D000071636:Protein Phosphatase 2C