Specificity and commonality of the phosphoinositide-binding proteome analyzed by quantitative mass spectrometry.
Phosphoinositides (PIPs) play key roles in signaling and disease. Using high-resolution quantitative mass spectrometry, we identified PIP-interacting proteins and profiled their binding specificities toward all seven PIP variants. This analysis revealed 405 PIP-binding proteins, which is greater than the total number of phospho- or ubiquitin-binding domains. Translocation and inhibitor assays of identified PIP-binding proteins confirmed that our methodology targets direct interactors. The PIP interactome encompasses proteins from diverse cellular compartments, prominently including the nucleus. Our data set revealed a consensus motif for PI(3,4,5)P3-interacting pleckstrin homology (PH) domains, which enabled in silico identification of phosphoinositide interactors. Members of the dedicator of cytokinesis family C exhibited specificity toward both PI(3,4,5)P3 and PI(4,5)P2. Structurally, this dual specificity is explained by a decreased number of positively charged residues in the L1 subdomain compared with DOCK1. The presented PIP-binding proteome and its specificity toward individual PIPs should be a valuable resource for the community.
Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.
Stephanie
S
Jungmichel
Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.
Kathrine B
KB
Sylvestersen
Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark.
Chunaram
C
Choudhary
Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Martinsried 82152, Germany.
Steve
S
Nguyen
Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark; Department of Proteomics and Signal Transduction, Max Planck Institute for Biochemistry, Martinsried 82152, Germany. Electronic address: mmann@biochem.mpg.de.
Matthias
M
Mann
Department of Proteomics, The Novo Nordisk Foundation Center for Protein Research, Faculty of Health Sciences, University of Copenhagen, Blegdamsvej 3, 2200 Copenhagen, Denmark. Electronic address: michael.lund.nielsen@cpr.ku.dk.
Michael L
ML
Nielsen
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D003198:Computer Simulation; D006367:HeLa Cells; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D013058:Mass Spectrometry / Q000379:methods*; D008565:Membrane Proteins / Q000378:metabolism; D008969:Molecular Sequence Data; D010716:Phosphatidylinositols / Q000378:metabolism*; D011485:Protein Binding; D025941:Protein Interaction Mapping; D017434:Protein Structure, Tertiary; D020543:Proteome / Q000737:chemistry / Q000378:metabolism*; D014162:Transfection
D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; C435927:PLEKHA1 protein, human; C461494:PLEKHA2 protein, human; D010716:Phosphatidylinositols; D020543:Proteome