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Identification of phosphorylation sites within the signaling adaptor APPL1 by mass spectrometry
Randi L Gant-Branum1, Joshua A Broussard, Ablatt Mahsut
1Department of Chemistry, Vanderbilt Institute for Chemical Biology (VICB), Vanderbilt University, Nashville, Tennessee 37235, USA.
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.
Insights
Researchers identified 13 phosphorylation sites on the APPL1 protein using mass spectrometry. Four sites in key functional domains may regulate APPL1
Area of Science:
- Cellular Biology
- Molecular Biology
- Biochemistry
Background:
- APPL1 (Adaptor Protein, Phosphotyrosine Interacting with PH Domain and Leucine Rich Repeats) is a membrane-associated adaptor protein involved in crucial cellular processes.
- Existing research highlights APPL1's roles in apoptosis, proliferation, and survival, alongside its protein and membrane interactions.
- A comprehensive understanding of APPL1's phosphorylation landscape, critical for its function, has been lacking.
Purpose of the Study:
- To generate a comprehensive phosphorylation profile of the APPL1 protein.
- To identify specific phosphorylated residues within APPL1 and map their locations.
- To investigate the potential functional implications of identified phosphorylation sites on APPL1 domains and interactions.
Main Methods:
- Utilized mass spectrometry (MS) for the identification of phosphorylated residues in APPL1.
- Employed multiple proteases (trypsin, chymotrypsin, and Glu C) to enhance peptide coverage.
- Conducted replicate experiments using both linear ion trap (LTQ) MS and LTQ-Orbitrap-MS for robust data acquisition.
Main Results:
- Identified a total of 13 phosphorylated residues within the APPL1 protein.
- Achieved high sequence coverage (99.6%) through the combined proteomic analysis.
- Located four critical phosphorylation sites within functionally important domains: one in the BAR domain, two near the PH domain, and one in the PTB domain.
Conclusions:
- The identified phosphorylation sites, particularly those in functional domains, suggest a regulatory role in APPL1 activity.
- Phosphorylation may modulate the interaction capabilities of APPL1 domains with other proteins and cellular membranes.
- This study provides a foundational phosphorylation map for APPL1, paving the way for future functional investigations.
