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Updated: May 28, 2026
Assaying Protein Kinase Activity with Radiolabeled ATP
Published on: May 26, 2017
Protein tyrosine kinase activity assays
1Schering-Plough Research Institute, Kenilworth, New Jersey, USA.
Abstract:
Protein tyrosine kinases (PTKs) are ubiquitous enzymes that are integrally involved in the regulation of transformation mechanisms, normal and pathological growth, cell cycle regulation, immune responses, and a variety of intracellular signaling mechanisms. This rapidly growing family of enzymes is generally divided into two groups: receptor PTKs (with more than twelve distinct families) and nonreceptor PTKs (with more than nine distinct families). PTKs mediate the enzymatic transfer of the gamma phosphate of ATP to the phenolic groups on tyrosine residues to generate phosphate monoesters. In this unit, several assays are provided to measure the ability of PTKs to transphosphorylate protein and peptide substrates, and to autophosphorylate. Phosphorylation of exogenous substrates or autophosphorylation is detected using a ³²P- or ³³P-phosphorylated protein. Alternatively, antibodies recognizing phosphorylated tyrosine residues can be used to quantify PTK activity. In some cases, antibodies are available for context-specific phosphotyrosine residues, thereby enabling the detection of PTK-specific substrate phosphorylation.
Insights
Protein tyrosine kinases (PTKs) are crucial enzymes regulating cell processes. Assays are presented to measure PTK activity through phosphorylation of substrates or autophosphorylation, aiding in understanding these vital signaling proteins.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Signaling
Background:
- Protein tyrosine kinases (PTKs) are essential enzymes regulating critical cellular functions.
- PTKs are broadly classified into receptor and nonreceptor types, with numerous families within each group.
- They catalyze the transfer of a phosphate group from ATP to tyrosine residues on proteins.
Purpose of the Study:
- To provide assays for measuring protein tyrosine kinase (PTK) activity.
- To enable the detection of both substrate phosphorylation and autophosphorylation by PTKs.
- To offer methods for quantifying PTK activity using radiolabeled or antibody-based detection.
Main Methods:
- Assays utilizing radioactive isotopes (³²P or ³³P) to detect protein phosphorylation.
- Employing antibodies that recognize phosphorylated tyrosine residues for quantification.
- Utilizing context-specific antibodies for detecting PTK-specific substrate phosphorylation.
Main Results:
- Established methods for measuring PTK transphosphorylation and autophosphorylation.
- Demonstrated the utility of both radiolabeled detection and antibody-based quantification.
- Highlighted the potential for specific PTK activity detection using targeted antibodies.
Conclusions:
- The provided assays offer versatile tools for studying PTK function.
- These methods facilitate the investigation of PTK roles in normal and pathological cellular processes.
- Accurate measurement of PTK activity is crucial for understanding cell signaling and disease mechanisms.
