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Updated: Oct 13, 2025
A Mass Spectrometry-Based Approach to Identify Phosphoprotein Phosphatases and their Interactors
Published on: April 29, 2022
Modulation of Phosphoprotein Activity by Phosphorylation Targeting Chimeras (PhosTACs)
Po-Han Chen1, Zhenyi Hu1, Elvira An2
1Department of Molecular, Cellular, and Developmental Biology, Yale University, Yale Science Building, 260 Whitney Avenue, P.O. Box 208103, New Haven, Connecticut 06520-8103, United States.
Abstract:
Protein phosphorylation, which regulates many critical aspects of cell biology, is dynamically governed by kinases and phosphatases. Many diseases are associated with dysregulated hyperphosphorylation of critical proteins, such as retinoblastoma protein in cancer. Although kinase inhibitors have been widely applied in the clinic, growing evidence of off-target effects and increasing drug resistance prompts the need to develop a new generation of drugs. Here, we propose a proof-of-concept study of phosphorylation targeting chimeras (PhosTACs). Similar to PROTACs in their ability to induce ternary complexes, PhosTACs focus on recruiting a Ser/Thr phosphatase to a phosphosubstrate to mediate its dephosphorylation. However, distinct from PROTACs, PhosTACs can uniquely provide target gain-of-function opportunities to manipulate protein activity. In this study, we applied a chemical biology approach to evaluate the feasibility of PhosTACs by recruiting the scaffold and catalytic subunits of the PP2A holoenzyme to protein substrates such as PDCD4 and FOXO3a for targeted protein dephosphorylation. For FOXO3a, this dephosphorylation resulted in the transcriptional activation of a FOXO3a-responsive reporter gene.
Insights
Researchers developed novel phosphorylation targeting chimeras (PhosTACs) to recruit phosphatases for targeted protein dephosphorylation. This approach offers a new therapeutic strategy for diseases linked to protein hyperphosphorylation.
Area of Science:
- Biochemistry
- Molecular Biology
- Chemical Biology
Background:
- Protein phosphorylation is crucial for cell biology, regulated by kinases and phosphatases.
- Dysregulated phosphorylation, like retinoblastoma protein hyperphosphorylation in cancer, is linked to diseases.
- Existing kinase inhibitors face challenges like off-target effects and drug resistance.
Purpose of the Study:
- To introduce and evaluate the proof-of-concept for phosphorylation targeting chimeras (PhosTACs).
- To explore PhosTACs as a novel therapeutic strategy for diseases involving protein hyperphosphorylation.
- To demonstrate PhosTACs' ability to recruit phosphatases for targeted protein dephosphorylation.
Main Methods:
- Utilized a chemical biology approach to design and test PhosTACs.
- Recruited PP2A holoenzyme (scaffold and catalytic subunits) to protein substrates.
- Assessed PhosTAC efficacy on protein substrates like PDCD4 and FOXO3a.
Main Results:
- Successfully demonstrated targeted protein dephosphorylation using PhosTACs.
- PhosTACs recruited PP2A to PDCD4 and FOXO3a substrates.
- Dephosphorylation of FOXO3a by PhosTACs led to transcriptional activation of a reporter gene.
Conclusions:
- PhosTACs represent a viable new modality for targeted protein dephosphorylation.
- This technology offers potential gain-of-function opportunities by manipulating protein activity.
- PhosTACs provide a promising alternative to kinase inhibitors for treating diseases associated with aberrant phosphorylation.
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