Assays for tyrosine phosphorylation in human cells

Monica Kruk; Naomi Widstrom; Sampreeti Jena; Nicole L. Wolter; John F. Blankenhorn; Ibrahim Abdalla; Tzu Yi Yang; Laurie L. Parker

doi:10.1016/bs.mie.2019.06.026

Assays for tyrosine phosphorylation in human cells

Monica Kruk, Naomi Widstrom, Sampreeti Jena, Nicole L. Wolter, John F. Blankenhorn, Ibrahim Abdalla, Tzu Yi Yang, Laurie L. Parker

Research output: Chapter in Book/Report/Conference proceeding › Chapter

3 Scopus citations

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.

Original language	English (US)
Title of host publication	Post-translational Modifications That Modulate Enzyme Activity
Editors	Benjamin A. Garcia
Publisher	Academic Press Inc.
Pages	375-406
Number of pages	32
Volume	626
ISBN (Print)	9780128186695
DOIs	https://doi.org/10.1016/bs.mie.2019.06.026
State	Published - 2019

Publication series

Name	Methods in enzymology
Publisher	Academic Press Inc.
ISSN (Print)	0076-6879

Bibliographical note

Funding Information:
We are very grateful for funding support from the National Institutes of Health through grants R01CA182543 and R33CA217780 (PI: L.L.P.), and Physical Science Oncology Center grant U54CA210190 (PI: David Odde, Paolo Provenzano).

Publisher Copyright:
© 2019 Elsevier Inc.

Keywords

Abl kinase
Cell penetrating peptide
K562 cells
Kinase substrate
Tyrosine kinase assay

PubMed: MeSH publication types

Journal Article
Research Support, N.I.H., Extramural

UN SDGs

This output contributes to the following UN Sustainable Development Goals (SDGs)

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10.1016/bs.mie.2019.06.026

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Kruk, M., Widstrom, N., Jena, S., Wolter, N. L., Blankenhorn, J. F., Abdalla, I., Yang, T. Y., & Parker, L. L. (2019). Assays for tyrosine phosphorylation in human cells. In B. A. Garcia (Ed.), Post-translational Modifications That Modulate Enzyme Activity (Vol. 626, pp. 375-406). (Methods in enzymology). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2019.06.026

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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.",

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author = "Monica Kruk and Naomi Widstrom and Sampreeti Jena and Wolter, {Nicole L.} and Blankenhorn, {John F.} and Ibrahim Abdalla and Yang, {Tzu Yi} and Parker, {Laurie L.}",

note = "Funding Information: We are very grateful for funding support from the National Institutes of Health through grants R01CA182543 and R33CA217780 (PI: L.L.P.), and Physical Science Oncology Center grant U54CA210190 (PI: David Odde, Paolo Provenzano). Publisher Copyright: {\textcopyright} 2019 Elsevier Inc.",

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AU - Widstrom, Naomi

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AU - Wolter, Nicole L.

AU - Blankenhorn, John F.

AU - Abdalla, Ibrahim

AU - Yang, Tzu Yi

AU - Parker, Laurie L.

N1 - Funding Information: We are very grateful for funding support from the National Institutes of Health through grants R01CA182543 and R33CA217780 (PI: L.L.P.), and Physical Science Oncology Center grant U54CA210190 (PI: David Odde, Paolo Provenzano). Publisher Copyright: © 2019 Elsevier Inc.

PY - 2019

Y1 - 2019

N2 - 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.

AB - 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.

KW - Abl kinase

KW - Cell penetrating peptide

KW - K562 cells

KW - Kinase substrate

KW - Tyrosine kinase assay

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ER -

Assays for tyrosine phosphorylation in human cells

Abstract

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Bibliographical note

Keywords

PubMed: MeSH publication types

UN SDGs

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