title: Identification of phosphorylation sites within the signaling adaptor APPL1 by mass spectrometry.
abstract: APPL1 is a membrane-associated adaptor protein implicated in various cellular processes, including apoptosis, proliferation, and survival. Although there is increasing interest in the biological roles as well as the protein and membrane interactions of APPL1, a comprehensive phosphorylation profile has not been generated. In this study, we use mass spectrometry (MS) to identify 13 phosphorylated residues within APPL1. By using multiple proteases (trypsin, chymotrypsin, and Glu C) and replicate experiments of linear ion trap (LTQ) MS and LTQ-Orbitrap-MS, a combined sequence coverage of 99.6% is achieved. Four of the identified sites are located in important functional domains, suggesting a potential role in regulating APPL1. One of these sites is within the BAR domain, two cluster near the edge of the PH domain, and one is located within the PTB domain. These phosphorylation sites may control APPL1 function by regulating the ability of APPL1 domains to interact with other proteins and membranes.
authors.affiliation: Department of Chemistry, Vanderbilt Institute for Chemical Biology (VICB), Vanderbilt University, Nashville, Tennessee 37235, USA.
authors.forename: Randi L
authors.initials: RL
authors.lastname: Gant-Branum
authors.forename: Joshua A
authors.initials: JA
authors.lastname: Broussard
authors.forename: Ablatt
authors.initials: A
authors.lastname: Mahsut
authors.forename: Donna J
authors.initials: DJ
authors.lastname: Webb
authors.forename: John A
authors.initials: JA
authors.lastname: McLean
mesh_terms: D048868:Adaptor Proteins, Signal Transducing / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D006801:Humans; D008969:Molecular Sequence Data; D010447:Peptide Hydrolases / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D010750:Phosphoproteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D010766:Phosphorylation; D010770:Phosphotransferases / Q000378:metabolism; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D053719:Tandem Mass Spectrometry / Q000379:methods*
chemical_list: C464801:APPL1 protein, human; D048868:Adaptor Proteins, Signal Transducing; D010750:Phosphoproteins; D010770:Phosphotransferases; D010447:Peptide Hydrolases