Abstract
Reversible protein phosphorylation, catalyzed by protein kinases and phosphatases, is a fundamental process that controls protein function and intracellular signaling. Failure of phospho-control accounts for many human diseases. While a kinase phosphorylates multiple substrates, a substrate is often phosphorylated by multiple kinases. This renders phospho-control at the substrate level challenging, as it requires inhibition of multiple kinases, which would thus affect other kinase substrates. Here, we describe the development and application of the affinity-directed phosphatase (AdPhosphatase) system for targeted dephosphorylation of specific phospho-substrates. By deploying the Protein Phosphatase 1 or 2A catalytic subunits conjugated to an antigen-stabilized anti-GFP nanobody, we can promote the dephosphorylation of two independent phospho-proteins, FAM83D or ULK1, knocked in with GFP-tags using CRISPR-Cas9, with exquisite specificity. By redirecting protein phosphatases to neo-substrates through nanobody-mediated proximity, AdPhosphatase can alter the phospho-status and function of target proteins and thus, offers a new modality for potential drug discovery approaches.
Original language | English |
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Pages (from-to) | 188-202 |
Number of pages | 16 |
Journal | Cell Chemical Biology |
Volume | 30 |
Issue number | 2 |
Early online date | 30 Jan 2023 |
DOIs | |
Publication status | Published - 16 Feb 2023 |
Keywords
- Affinity-directed phosphatase
- AdPhosphatase
- Targeted dephosphorylation
- PPP1CA
- PPP2CA
- Nanobody
- ULK1
- FAM83D
ASJC Scopus subject areas
- Drug Discovery
- Molecular Medicine
- Molecular Biology
- Biochemistry
- Clinical Biochemistry
- Pharmacology