Study Reveals CDK2 Activity Crosstalk in ERK Kinase Translocation Reporter

Oncology Medicine Biochemistry Life Sciences Cell Biology
ERK kinase CDK2 activity single-cell imaging signaling pathway computational methods

CDK2 Activity Interference on ERK and p38 KTR Signals and Computational Correction Methods

Recently, a paper published in Cell Systems by authors Timothy E. Hoffman, Chengzhe Tian, Varuna Nangia, and others revealed the interference of cyclin-dependent kinase 2 (CDK2) on the kinase translocation reporter (KTR) signals in the extracellular signal-regulated kinase (ERK) and p38 signaling pathways. The study also proposed computational methods to eliminate this interference. This research not only provides new insights into the complexity of cellular signaling but also offers essential tools and methods for future studies.

Research Background

The mitogen-activated protein kinase (MAPK) pathway plays a crucial role in cell growth, differentiation, and survival. Among them, the ERK and p38 signaling pathways respond to growth factors and stress signals, respectively, regulating cell proliferation and stress responses. To monitor ERK and p38 activity in real time, researchers developed the kinase translocation reporter (KTR) system, which reflects kinase activity through the translocation of fluorescent proteins between the nucleus and cytoplasm. However, recent studies have shown that these KTR systems may be interfered with by other kinases, particularly CDK2. CDK2 is a key kinase in cell cycle regulation, and its activity gradually increases during the cell cycle, which may lead to misinterpretation of KTR signals.

Although researchers have been aware of potential cross-interference issues in KTR systems, previous studies have primarily focused on CDK1 interference. The issue of CDK2 interference has not been thoroughly investigated, especially the residual signal in ERK KTR when the MAPK pathway is completely inhibited. This study aims to reveal the interference of CDK2 on ERK and p38 KTR signals and proposes computational methods to eliminate this interference.

Researchers and Publication Information

The research was led by Sabrina L. Spencer’s team from the Department of Biochemistry and BioFrontiers Institute at the University of Colorado Boulder, with collaborators from Johns Hopkins University School of Medicine and Genentech. The paper was published on January 15, 2025, in Cell Systems, titled CDK2 Activity Crosstalk on the ERK Kinase Translocation Reporter Can Be Resolved Computationally.

Research Process

Experimental Design and Methods

  1. Cell Model Construction
    The research team first constructed A375 BRAF V600E mutant melanoma cells expressing DHB-mCherry (CDK2 reporter) and Elk1-mClover (ERK KTR). These cells also expressed H2B-miFP (nuclear marker) for nuclear segmentation and single-cell tracking. Through time-lapse imaging, researchers were able to quantify changes in ERK KTR signals in real time.

  2. Drug Treatment and Signal Observation
    Researchers treated A375 cells with Dabrafenib (BRAF inhibitor) and observed a rapid drop in ERK KTR signal, but residual signal persisted. To further validate this phenomenon, they treated cells with Trametinib (MEK inhibitor) and SCH772984 (ERK inhibitor) and found that the residual signal remained. This indicates that even when the MAPK pathway is completely inhibited, residual ERK KTR signal persists.

  3. CDK2 Inhibitor Validation
    To verify whether the residual signal resulted from CDK2 interference, researchers treated cells with selective CDK2 inhibitors PF3600 and PF4091 and found that the residual signal was completely eliminated. This confirmed that the residual signal was indeed due to cross-interference from CDK2.

  4. FRET Sensor Validation
    To further validate this finding, researchers used the improved fluorescence resonance energy transfer (FRET) sensor EKARen5, which had been modified to reduce CDK1 sensitivity. Results showed that the EKARen5 sensor was not affected by CDK2 interference, further supporting the interference of CDK2 on ERK KTR signals.

  5. Interference Study on p38 KTR
    Researchers also tested p38 KTR and found that it was similarly affected by CDK2 interference. By using CDK2 inhibitors, they successfully eliminated the residual signal in p38 KTR.

  6. Development of Computational Methods
    To eliminate CDK2 interference on ERK and p38 KTR signals, researchers developed linear and nonlinear computational methods. These methods subtract CDK2 signals from ERK and p38 KTR signals to more accurately quantify MAPK activity.

Key Results

  1. Residual ERK KTR Signal After MAPK Inhibition
    Even when the MAPK pathway is completely inhibited, residual signal persists in ERK KTR, which is highly correlated with CDK2 activity.

  2. CDK2 Inhibitors Eliminate Residual Signal
    By using selective CDK2 inhibitors PF3600 and PF4091, researchers successfully eliminated the residual signal in ERK KTR, confirming CDK2 interference.

  3. EKARen5 Sensor Free from CDK2 Interference
    The improved FRET sensor EKARen5 was not affected by CDK2 interference, indicating that modifying the sensor can prevent cross-interference from CDK2.

  4. p38 KTR Also Affected by CDK2 Interference
    p38 KTR signal was also affected by CDK2 interference, but this interference could be eliminated using CDK2 inhibitors.

  5. Computational Methods Eliminate CDK2 Interference
    Researchers developed linear and nonlinear computational methods that subtract CDK2 signals from ERK and p38 KTR signals to more accurately quantify MAPK activity.

Research Conclusion

This study reveals the interference of CDK2 on ERK and p38 KTR signals and proposes computational techniques to eliminate this interference. This finding not only provides new insights into the complexity of cellular signaling but also offers essential tools and methods for future research. By improving sensors and developing computational methods, researchers can more accurately monitor ERK and p38 activity, thereby advancing the study of cellular signaling.

Research Highlights

  1. CDK2 Interference on KTR Signals
    This study is the first to systematically reveal the interference of CDK2 on ERK and p38 KTR signals, filling gaps in previous research.

  2. Improved FRET Sensor
    The improved FRET sensor EKARen5 demonstrates the potential to avoid CDK2 interference, providing new insights for future sensor design.

  3. Innovation in Computational Methods
    The linear and nonlinear computational methods developed by researchers provide effective solutions for eliminating cross-interference in KTR signals, with broad application prospects.

Research Significance

This study is not only scientifically significant but also provides new technical means for cellular signaling research. By revealing the interference of CDK2 on KTR signals, researchers can more accurately monitor ERK and p38 activity, offering new tools for studies in cell cycle regulation, cancer treatment, and other fields. Additionally, the improved sensors and computational methods provide important references for future research.

This study not only addresses cross-interference issues in KTR systems but also opens new directions for cellular signaling research. By continuously improving and optimizing research tools, researchers will be able to gain a deeper understanding of the complex mechanisms of cellular signaling, thereby providing critical theoretical support for disease treatment and drug development.