An affinity-directed phosphatase, AdPhosphatase, system for targeted protein dephosphorylation

Luke M. Simpson, Luke J. Fulcher, Gajanan Sathe, Abigail Brewer, Jin-Feng Zhao, Daniel R. Squair, Jennifer Crooks, Melanie Wightman, Nicola T. Wood, Robert Gourlay, Joby Varghese, Renata F. Soares, Gopal P. Sapkota (Lead / Corresponding author)

Research output: Contribution to journalArticlepeer-review

5 Citations (Scopus)
74 Downloads (Pure)

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 languageEnglish
Pages (from-to)188-202
Number of pages16
JournalCell Chemical Biology
Volume30
Issue number2
Early online date30 Jan 2023
DOIs
Publication statusPublished - 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

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