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Protein Sci. 2019 Mar;28(3):654-662. doi: 10.1002/pro.3570. Epub 2019 Jan 31.

Chemically reprogramming the phospho-transfer reaction to crosslink protein kinases to their substrates.

Author information

1
Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, California.
2
Department of Pathology, University of California San Francisco, San Francisco, California.
3
Department of Pharmaceutical Chemistry, University of California San Francisco, San Francisco, California.
4
Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California.

Abstract

The proteomic mapping of enzyme-substrate interactions is challenged by their transient nature. A method to capture interacting protein kinases in complexes with a single substrate of interest would provide a new tool for mapping kinase signaling networks. Here, we describe a nucleotide-based substrate analog capable of reprogramming the wild-type phosphoryl-transfer reaction to produce a kinase-acrylamide-based thioether crosslink to mutant substrates with a cysteine nucleophile substituted at the native phosphorylation site. A previously reported ATP-based methacrylate crosslinker (ATP-MA) was capable of mediating kinase crosslinking to short peptides but not protein substrates. Exploration of structural variants of ATP-MA to enable crosslinking of protein substrates to kinases led to the discovery that an ADP-based methacrylate (ADP-MA) crosslinker was superior to the ATP scaffold at crosslinking in vitro. The improved efficiency of ADP-MA over ATP-MA is due to reduced inhibition of the second step of the kinase-substrate crosslinking reaction by the product of the first step of the reaction. The new probe, ADP-MA, demonstrated enhanced in vitro crosslinking between the Src tyrosine kinase and its substrate Cortactin in a phosphorylation site-specific manner. The kinase-substrate crosslinking reaction can be carried out in a complex mammalian cell lysate setting, although the low abundance of endogenous kinases remains a significant challenge for efficient capture.

KEYWORDS:

chemical genetics; kinase-substrate interactions; mass spectrometry; phosphorylation; protein kinases; signaling networks

PMID:
30636329
PMCID:
PMC6371225
[Available on 2020-03-01]
DOI:
10.1002/pro.3570

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