Assays for tyrosine phosphorylation in human cells

Abstract

Tyrosine kinases are important for many cellular processes and disruption of their regulation is a factor in diseases like cancer, therefore they are a major target of anticancer drugs. There are many ways to measure tyrosine kinase activity in cells by monitoring endogenous substrate phosphorylation, or by using peptide substrates and incubating them with cell lysates containing active kinases. However, most of these strategies rely on antibodies and/or are limited in how accurately they model the intracellular environment. In cases in which activity needs to be measured in cells, but endogenous substrates are not known and/or suitable phosphospecific antibodies are not available, cell-deliverable peptide substrates can be an alternative and can provide information on activation and inhibition of kinases in intact, live cells. In this chapter, we review this methodology and provide a protocol for measuring Abl kinase activity in human cells using enzyme-linked immunosorbent assay (ELISA) with a generic antiphosphotyrosine antibody for detection.

Keywords: Abl kinase; Cell penetrating peptide; K562 cells; Kinase substrate; Tyrosine kinase assay.

Figure 1.. Cell deliverable peptide kinase substrate design and assay strategies.

(A) Peptide kinase substrates for cell-based assays are designed with a kinase substrate sequence, a cell-penetrating peptide sequence, and other functionality to facilitate handling and read-out. (B) When incubated with cells, CPP-containing peptide substrates are taken up into the cell, phosphorylated according to the activation state of the kinase, and analyzed either using single cell or bulk cell approaches. Imaging can enable analysis of intact cells, while other methods require lysis and analysis of the cell contents.

Figure 2.. Balancing competing factors in cell-deliverable substrate kinase assay design.

In an optimal scenario, the delivered kinase substrate would be taken up and distributed easily by the cells, it would be stable enough to provide sufficient signal, and phosphorylated rapidly and efficiently by the desired kinase and slowly/poorly (if at all) by any others. There are a number of potential pitfalls to address, including issues of peptide/phosphopeptide stability and activity with the target kinase vs. others, all of which could lead to ambiguity in the measurement. Careful mitigation of pitfalls enables reliable, reproducible measurements of kinase activity.

Figure 3.. Plate map and timeline for Abl kinase assay in K562 cells.

(A) Example plate map for 12-well plate with each of six conditions (including controls) in duplicate. Randomizing conditions around the plate will help avoid systematic biases from plate position, therefore additional replicates should shuffle these positions. (B) Experiment timeline for planning purposes.