K-Ras Binds Calmodulin-Related Centrin1 with Potential Implications for K-Ras Driven Cancer Cell Stemness
Abstract
:Simple Summary
Abstract
1. Introduction
2. Experimental Procedures
2.1. Plasmids, siRNAs and Inhibitors
2.2. Protein Sequence Analyses
2.3. Protein Purification
2.4. Fluorescence Polarisation Binding Assay
2.5. Co-Immunoprecipitation Experiments
2.6. BRET Donor Saturation Titration Assays
2.7. Dose Response Analysis of Inhibitors and siRNA Knockdown in BRET Assays
2.8. siRNA-Mediated Knockdown and Western Blotting
2.9. Three-Dimensional Spheroid Assay
2.10. Confocal Microscopy
2.11. Data and Statistical Analysis
3. Results and Discussion
3.1. Binding Studies Support Specific Canonical Target Peptides for CaM or Centrin1
3.2. Cellular BRET Data Suggest That the K-Ras G-Domain Participates in Complexes with Either CaM or Centrin1
3.3. CaM Inhibitors Bind to Centrin
3.4. Inhibition of Prenylation Does Not Disrupt the BRET-Interaction of K-Ras with CaM or Centrin1 in Cells
3.5. Membrane Targeting and Anchorage of K-Ras Depends More on CaM Than on Centrin1
3.6. Centrin1 Co-Distributes with CaM during the Cell Cycle
4. Conclusions
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Conflicts of Interest
References
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Inhibitor | Centrin1 | CaM |
---|---|---|
Mean Kd (Repeat Values) | Kd (References) | |
calmidazolium | 1.6 (1.4; 1.8) µM | 13.5 nM [26] |
W-7 | 18.2 (17.8; 18.5) µM | 1.47 µM [26] |
ophiobolin A | 49 (58; 39) µM | 3.5 µM [9] |
calmirasone1 | 0.9 (1.0; 0.8) µM | 0.87 µM [9] |
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Manoharan, G.b.; Laurini, C.; Bottone, S.; Ben Fredj, N.; Abankwa, D.K. K-Ras Binds Calmodulin-Related Centrin1 with Potential Implications for K-Ras Driven Cancer Cell Stemness. Cancers 2023, 15, 3087. https://doi.org/10.3390/cancers15123087
Manoharan Gb, Laurini C, Bottone S, Ben Fredj N, Abankwa DK. K-Ras Binds Calmodulin-Related Centrin1 with Potential Implications for K-Ras Driven Cancer Cell Stemness. Cancers. 2023; 15(12):3087. https://doi.org/10.3390/cancers15123087
Chicago/Turabian StyleManoharan, Ganesh babu, Christina Laurini, Sara Bottone, Nesrine Ben Fredj, and Daniel Kwaku Abankwa. 2023. "K-Ras Binds Calmodulin-Related Centrin1 with Potential Implications for K-Ras Driven Cancer Cell Stemness" Cancers 15, no. 12: 3087. https://doi.org/10.3390/cancers15123087