Cell Cycle-Specific Protein Phosphatase 1 (PP1) Substrates Identification Using Genetically Modified Cell Lines

Area of Science:

• Molecular Biology
• Cell Biology
• Biochemistry

Background:

• Identifying protein phosphatase 1 (PP1) holoenzyme substrates is challenging due to diverse regulatory subunits and cell cycle regulation.
• Existing methods lack cell cycle specificity for PP1 substrate identification.

Purpose of the Study:

• To develop a novel strategy for investigating PP1 substrates throughout the cell cycle.
• To generate cell lines with endogenously tagged PP1 regulatory subunits (RIPPOs) for substrate identification.

Main Methods:

• Utilized CRISPR-Cas9 genome editing to tag RIPPOs with auxin-inducible degron (AID) or ascorbate peroxidase 2 (APEX2) modules.
• Employed SILAC proteomic-based approaches, including mass spectrometry (MS) with APEX2 for proximity labeling and SILAC phospho-MS after RIPPO degradation via AID.
• Performed in silico overlap analysis between proximity-labeled and differentially phosphorylated proteomes to identify putative PP1 substrates.

Main Results:

• Successfully generated cell lines with endogenously tagged RIPPOs using CRISPR-Cas9.
• Established a workflow combining proximity labeling and degradable protein strategies for substrate identification.
• Identified putative PP1 substrates enriched by the overlap of proteomic datasets.

Conclusions:

• The presented strategy enables the investigation of PP1 substrates with cell cycle specificity.
• This approach provides a powerful tool for understanding PP1 function in dynamic cellular processes.
• Further validation using methods like FRET, PLA, and in vitro assays is recommended.