Rational Design of a Dephosphorylation-Resistant Reporter Enables Single-Cell Measurement of Tyrosine Kinase Activity

Abstract

Although peptide-based reporters of protein tyrosine kinase (PTK) activity have been used to study PTK enzymology in vitro, the application of these reporters to intracellular conditions is compromised by their dephosphorylation, preventing PTK activity measurements. Non-proteinogenic amino acids may be utilized to rationally design selective peptidic ligands by accessing greater chemical and structural diversity than is available using the native amino acids. We describe a peptidic reporter that, upon phosphorylation by the epidermal growth factor receptor (EGFR), is resistant to dephosphorylation both in vitro and in cellulo. The reporter contains a conformationally constrained phosphorylatable moiety (7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid) in the place of L-tyrosine and is efficiently phosphorylated in A431 epidermoid carcinoma cells. Dephosphorylation of the reporter occurs 3 orders of magnitude more slowly compared with that of the conventional tyrosine-containing reporter.

Selection and optimization of enzyme reporters for chemical cytometry

“…A wide variety of unnatural residues are phosphorylated by protein kinases, including a structurally constrained tyrosine residue [(7-(S)-hydroxy-1,2,3,4-tetrahydroisoquinoline-3carboxylic acid (Htc)]. (Kwon et al, 1994;Kwon et al, 1993;Lee et al, 1995;Prorok et al, 1989;Turner et al, 2016) Substrates containing Htc are particularly useful as probes of TPK activity since the corresponding phosphorylated product is resistant to dephosphorylation by intracellular protein phosphatases. (Turner et al, 2016) One of the key advantages of using peptides as protein kinase substrates is that unnatural residues are readily introduced during peptide synthesis.…”

Assays for tyrosine phosphorylation in human cells

“…Phosphorylation of the substrate peptide can then be measured using many types of read-outs, including single cell electrophoresis (Soughayer et al, 2004;Turner et al, 2016), mass spectrometry (Placzek, Plebanek, Lipchik, Kidd, & Parker, 2010;Yang, Eissler, Hall, & Parker, 2013), fluorescence lifetime imaging (Damayanti, Parker, & Irudayaraj, 2013), or generic antiphosphotyrosine antibodies (Lipchik, Killins, Geahlen, & Parker, 2012;Ouellette, Noel, & Parker, 2016), depending on the particular substrate design and application. A unique advantage of these kinds of synthetic substrates is the ability to incorporate non-natural amino acids and other chemical moieties that would not be compatible with genetic encoding into sensor proteins, such as D amino acids to improve peptide stability (Proctor, Wang, Lawrence, & Allbritton, 2012a) or constrained phosphotyrosine analogs that are resistant to dephosphorylation (Turner et al, 2016). In this chapter, we present a method to measure tyrosine kinase activity using an exogenous kinase substrate taken up in live cells, followed by extracting the substrate and quantifying phosphorylation by ELISA.…”

“…Again, depending on the cell type and conditions, phosphatase activity may outstrip kinase activity and make detection of any phosphorylated product challenging. One option is to use phosphatase inhibitors (as described below in section 2.2.2.1); a recent alternative was reported by the Allbritton and Lawrence labs using an unnatural tyrosine analog that is intrinsically resistant to dephosphorylation (Turner et al, 2016). Overall, taking these factors into account and using chemical biology principles to design cell-based substrates is a promising avenue for increasing stability, with the caveat that each substrate would still need to be individually validated as efficient and specific enough to the target kinase.…”

“…This has been observed by 1) reduction in apparent phosphopeptide signal upon kinase inhibitor addition (Damayanti et al, 2013), 2) increase in phosphopeptide signal upon incubation with phosphatase inhibitor (Lipchik et al, 2012;T. Y. Yang et al, 2013), and 3) stabilization of phosphopeptide signal when using a phosphatase-resistant tyrosine analog in the substrate (Turner et al, 2016). For cell-based tyrosine kinase assays, pervanadate can be used as a general phosphatase inhibitor, however it is toxic to most cells and so incubation time should be kept short (<5-10 min).…”

N-Gemini peptides: cytosolic protease resistance via N-terminal dimerization of unstructured peptides

“…We demonstrate that N-terminal dimerization improves the half-lives of unstructured peptides by up to 28-fold, which is on par with protection of an unstructured peptide by capping with N-terminal cyclic β-turn 14,15 or by incorporation and optimization of unnatural amino acids into the substrate sequence. 16,30 Importantly, the approach reported here is far more synthetically accessible, thus providing a significant advancement over previous methods and a new tool for achieving protease-resistant unstructured peptides.…”