Dataset Information

An affinity-directed phosphatase (AdPhosphatase) system for targeted protein dephosphorylation

ABSTRACT: Reversible protein phosphorylation, catalysed by protein kinases and phosphatases, is a fundamental process that controls protein function and intracellular signalling. 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-stabilised 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.

INSTRUMENT(S): Orbitrap Fusion Lumos

ORGANISM(S): Homo Sapiens (human)

TISSUE(S): Epithelial Cell

SUBMITTER: Gopal Sapkota

LAB HEAD: Professor Gopal Sapkota

PROVIDER: PXD039200 | Pride | 2023-02-01

REPOSITORIES: Pride

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Publications

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

Simpson Luke M LM Fulcher Luke J LJ Sathe Gajanan G Brewer Abigail A Zhao Jin-Feng JF Squair Daniel R DR Crooks Jennifer J Wightman Melanie M Wood Nicola T NT Gourlay Robert R Varghese Joby J Soares Renata F RF Sapkota Gopal P GP

Cell chemical biology 20230130 2

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. He ...[more]

PMID: 36720221

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Project description:The mitogen-activated protein kinase (MAPK) pathway is one of the most altered pathways in cancer. It is involved in the control of cell proliferation, invasion, metabolism, and resistance to therapy. A number of aggressive malignancies, including melanoma, colon cancer and glioma, are driven by an activating missense mutation (V600E) in one component of the pathway, BRAF. BRAF V600E mutated cancers may respond initially to MEK inhibition, but may develop resistance mediated by increased reliance on mTOR signaling. We have previously demonstrated that the combination of the MEK inhibitor trametinib with the dual mTORC1/2 inhibitor TAK228 improved survival and decreased vascularization in a BRAFV600E mutant glioma model. To elucidate the mechanism of action of, and the changes in response to, MEK and mTOR inhibition, we performed comprehensive unbiased proteomic and phosphoproteomic characterization of BRAFV600E mutant glioma xenografts after short-course treatment with trametinib and TAK228, alone and in combination. We identified distinct response signatures for each monotherapy and combination therapy and validated that combination treatment inhibited activation of the MAPK and mTOR pathways, increased apoptotic signaling and suppressed angiogenesis signaling. Furthermore, we found that trametinib and TAK228 combination treatment broadly suppressed the activity of the cyclin-dependent kinases and increased the levels of proteins (and their activating phosphorylations) involved in glycolysis, the TCA cycle, nucleotide biosynthesis and DNA replication. We also demonstrated activation of both receptor tyrosine kinase and histone deacetylase proteins. This study reports a detailed (phospho)proteomic analysis of the response of BRAFV600E mutant glioma to combined MEK and mTOR pathway inhibition and identifies a number of targetable upregulated proteins and pathways, providing new avenues for the development of additional rational combination therapies for aggressive BRAF-driven tumors.

Dataset Information

An affinity-directed phosphatase (AdPhosphatase) system for targeted protein dephosphorylation

Publications

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

Similar Datasets

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