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Critical reanalysis of the methods that discriminate the activity of CDK2 from CDK1

Nandini Sakurikar¹, Alan Eastman¹

¹a Department of Pharmacology and Toxicology, and Norris Cotton Cancer Center , Geisel School of Medicine at Dartmouth , Lebanon , NH , USA.

Cell Cycle (Georgetown, Tex.)

|March 18, 2016

Abstract:

Cyclin dependent kinases 1 and 2 (CDK1 and CDK2) play crucial roles in regulating cell cycle progression from G1 to S, through S, and G2 to M phase. Both inhibition and aberrant activation of CDK1/2 can be detrimental to cancer cell growth. However, the tools routinely employed to discriminate between the activities of these 2 kinases do not have the selectivity commonly attributed to them. Activation of these kinases is often assayed as a decrease of the inhibitory tyrosine-15 phosphorylation, yet the antibodies used cannot discriminate between phosphorylated CDK1 and CDK2. Inhibitors of these kinases, while partially selective against purified kinases, may lack selectivity when applied to intact cells. High levels of cyclin E are often considered a marker of increased CDK2 activity, yet active CDK2 targets cyclin E for degradation, hence high levels usually reflect inactive CDK2. Finally, inhibition of CDK2 does not arrest cells in S phase suggesting CDK2 is not required for S phase progression. Furthermore, activation of CDK2 in S phase can rapidly induce DNA double-strand breaks in some cell lines. The misunderstandings associated with the use of these tools has led to misinterpretation of results. In this review, we highlight these challenges in the field.

Insights

Commonly used methods to study cyclin-dependent kinases 1 and 2 (CDK1/2) lack selectivity, leading to misinterpretations. This review highlights challenges in assessing CDK1/2 activity and function in cell cycle research.

Area of Science:

Cell Biology
Molecular Biology
Biochemistry

Background:

Cyclin-dependent kinases 1 and 2 (CDK1/2) are critical regulators of the cell cycle.
Dysregulation of CDK1/2 activity is implicated in cancer progression.
Current tools for assessing CDK1/2 activity often lack the expected selectivity.

Purpose of the Study:

To review the limitations of commonly used methods for studying CDK1 and CDK2.
To highlight potential misinterpretations arising from these limitations.
To provide a clearer understanding of CDK1/2 activity and function.

Main Methods:

Review of existing literature and experimental methodologies.
Critical analysis of antibody specificity for detecting phosphorylated CDK1 and CDK2.
Evaluation of small molecule inhibitor selectivity in cellular contexts.
Examination of cyclin E levels as a proxy for CDK2 activity.

Main Results:

Antibodies used to detect tyrosine-15 phosphorylation cannot distinguish between CDK1 and CDK2.
Kinase inhibitors may exhibit different selectivity profiles in purified enzymes versus intact cells.
High cyclin E levels may indicate inactive CDK2 due to targeted degradation.
CDK2 inhibition does not consistently arrest cells in S phase, questioning its necessity for S phase progression.

Conclusions:

Misinterpretations of CDK1/2 activity are common due to tool limitations.
Re-evaluation of experimental approaches is necessary for accurate CDK1/2 research.
Understanding these challenges is crucial for advancing cancer cell cycle studies.

Read full analysis

Keywords:

CDK1 CDK2 CVT-313 Chk1 Ro3306 S phase progression cyclin E phospho-specific antibodies

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