Zhang Wengang, Bradburn Devin, Heidebrink Gretchen, Liu Yonglan, Jang Hyunbum, Nussinov Ruth, Kõivomägi Mardo
Cancer Innovation Laboratory, National Cancer Institute, Frederick, MD 21702, USA.
Laboratory of Biochemistry and Molecular Biology, National Cancer Institute, Bethesda, MD 20892, USA.
J Mol Biol. 2025 Mar 31:169121. doi: 10.1016/j.jmb.2025.169121.
Cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) are key regulators of the G-S phase transition in the cell cycle. In cancer cells, CDK6 overexpression often outcompetes CDK4 in driving cell cycle progression, contributing to resistance against CDK4/6 inhibitors (CDK4/6i). This suggests distinct functional and conformational differences between these two kinases, despite their striking structural and sequence similarities. Understanding the mechanisms that differentiate CDK4 and CDK6 is crucial, as resistance to CDK4/6i-frequently linked to CDK6 overexpression-remains a significant therapeutic challenge. Notably, CDK6 is often upregulated in CDK4/6i-resistant cancers and rapidly proliferating hematopoietic stem cells, underscoring its unique regulatory roles. We hypothesize that their distinct conformational dynamics explain their differences in phosphorylation of retinoblastoma protein, Rb, inhibitor efficacy, and cell cycle control. This leads us to question how their dissimilar conformational dynamics encode their distinct actions. To elucidate their differential activities, molecular mechanisms, and inhibitor binding, we combine biochemical assays and molecular dynamics (MD) simulations. We discover that CDK4 and CDK6 have distinct allosteric networks connecting the β3-αC loop and the G-loop. CDK6 exhibits stronger coupling and shorter path lengths between these regions, resulting in higher kinase activity upon cyclin binding and impacting inhibitor specificity. We also discover an unrecognized role of the unstructured CDK6 C-terminus, which allosterically connects and stabilizes the R-spine, facilitating slightly higher activity. Our findings bridge the gap between the structural similarity and functional divergence of CDK4 and CDK6, advancing the understanding of kinase regulation in cancer biology.
细胞周期蛋白依赖性激酶4和6（CDK4和CDK6）是细胞周期中G-S期转换的关键调节因子。在癌细胞中，CDK6的过表达在驱动细胞周期进程方面常常胜过CDK4，导致对CDK4/6抑制剂（CDK4/6i）产生抗性。这表明这两种激酶之间存在明显的功能和构象差异，尽管它们在结构和序列上有显著的相似性。了解区分CDK4和CDK6的机制至关重要，因为对CDK4/6i的抗性（通常与CDK6过表达有关）仍然是一个重大的治疗挑战。值得注意的是，CDK6在对CDK4/6i耐药的癌症和快速增殖的造血干细胞中常常上调，突出了其独特的调节作用。我们假设它们不同的构象动力学解释了它们在视网膜母细胞瘤蛋白Rb磷酸化、抑制剂疗效和细胞周期控制方面的差异。这使我们质疑它们不同的构象动力学如何编码它们不同的作用。为了阐明它们的差异活性、分子机制和抑制剂结合情况，我们结合了生化分析和分子动力学（MD）模拟。我们发现CDK4和CDK6具有连接β3-αC环和G环的不同别构网络。CDK6在这些区域之间表现出更强的耦合和更短的路径长度，导致细胞周期蛋白结合后激酶活性更高，并影响抑制剂特异性。我们还发现了无结构的CDK6 C末端的一个未被认识的作用，它通过别构连接并稳定R脊柱，促进活性略高。我们的研究结果弥合了CDK4和CDK6在结构相似性和功能差异之间的差距，推进了对癌症生物学中激酶调节的理解。
