Biochemical mechanisms cyclin-dependent kinases use to control cell division

Division Of Basic Sciences - Nci

Investigators

Abstract

Determine the substrates and mechanisms of mammalian cyclin-Cdk complexes during the first steps in cell cycle Background: The cyclin-dependent kinases Cdk4 and Cdk6 form complexes with D-type cyclins to drive cell proliferation. A well-known target of cyclin D-Cdk4,6 is the retinoblastoma protein Rb, which inhibits cell-cycle progression until its inactivation by phosphorylation. Recently, we have shown that cyclin D-Cdk4,6 docks one side of an alpha-helix in the Rb C terminus, which is not recognized by downstream cell cycle cyclins. This helix-based docking mechanism for cyclin D is shared by the p107 and p130 Rb-family members across metazoans. Mutation of the Rb C-terminal helix prevents its phosphorylation, promotes G1 arrest, and enhances Rb's tumor suppressive function. However, because Rb can be phosphorylated by other cyclin-Cdks and cyclin D-Cdk4,6 may target other substrates involved in cell division, identifying novel targets of cyclin-Cdk complexes will deepen our understanding how Rb and cyclin D-Cdk4/6 regulate cell-cycle progression. Proposed aims: Our specific aims are: 1) Utilize novel analog-sensitive Cdk4,6 to identify cyclin D-Cdk4,6 substrates across the proteome 2) Identify substrates employing helix-based docking. Experimental systems: I have engineered chemically inhibitable versions of Cdk4 and Cdk6 kinases. While creating analog-sensitive versions of many kinases in the Cdk family has been relatively simple, Cdk4,6 versions have proven more challenging and were considered impossible. Using the developed Cdk4,6 analog-sensitive alleles, we will apply quantitative phosphoproteomic analysis to identify peptides with altered phosphorylation states following specific Cdk4,6 inhibition in vivo. As an alternative approach, we will employ improved proximity-labeling based methods coupled with mass spectrometry to validate the hits from bioinformatic analysis or to characterize entirely new ones in a cellular context. The combined proteomics techniques will allow the identification of proteins interacting with G1 cyclin-Cdk complexes potentially uncovering new docking mechanisms and providing a more comprehensive understanding of the role of G1 cyclin-Cdk complexes. Progress: This work has been started by a special volunteer and a post baccalaureate in the laboratory. In addition, we have successfully recruited a postdoctoral fellow who, along with the post baccalaureate (CRTA), will begin work on this project later in 2023. Moreover, all the essential equipment required for this research has been acquired and set up in the laboratory. Preparation of reagents for the quantitative biochemical assays is now complete, enabling us to conduct critical tests on our novel engineered version of Cdk4,6 kinase. Initial in vitro biochemical assays have already been performed, confirming the activity of the novel analog-sensitive Cdk4,6 kinases and their inhibition by bulky ATP analogs. Next, we are creating cell lines with modified Cdk4,6 kinases to first validate and then perform above proposed experiments to find novel targets G1 cyclin-Cdk complexes. To identify targets phosphorylated through the helix-based docking mechanism, we carried out a proteome-wide bioinformatic analysis. Currently, we are in the process of purifying the identified candidate proteins to evaluate their interactions with G1 cyclin-Cdk complexes. This will be achieved using quantitative biochemical assays to determine if these proteins are genuine substrates of the G1 cyclin-Cdk complexes. Determine promoter-specific RNA polymerase II regulation by cell cycle cyclin-Cdk complexes. Background: Cyclin dependent kinases (Cdks) and cyclins are the key regulators of proper cell cycle progression, phosphorylating their specific target proteins depending on the specific cell cycle phase. Recently, my work showed that in yeast, cell cycle G1 cyclin-Cdk complex directly phosphorylates and regulates RNA polymerase II activity at specific promoters, leading to transcriptional changes critical for the G1 to S phase transition. However, our understanding of how cyclin-Cdk complexes directly regulate transcription through modification of RNA polymerase II in cells during different cell cycle phases remains limited. Proposed aims: Our specific aims are: 1) Understand the molecular mechanisms by which cyclin-Cdk complexes regulate transcriptional changes in a promoter-specific manner during cell cycle, 2) Study how these changes in transcription affect cell cycle progression in normal and cancer cells. Alternatively, should the Cdk-dependent transcription not regulate cell cycle progression, we aim to study whether it is needed for other described non-canonical roles of cell cycle Cdks, including, but not limited to aging, cell differentiation, metabolism, and cell growth. Experimental systems: We will employ a combination of in vitro quantitative biochemical assays, state-of-the-art methods to determine protein-DNA and protein-protein interactions, transcriptomic methods, and genetic screens to determine functional interactions in different cell types. We will also use advanced imaging techniques to study the cyclin-Cdk-RNA polymerase II interactions and the effects of cyclin-Cdk binding on transcription kinetics in live cells. Progress: Work on this has been started by a pre-doctoral IRTA student doing rotation in the laboratory and by a summer student. In addition, we have successfully recruited two postdoctoral fellows who are set to join the laboratory in second part of 2023. They will be employing either the budding yeast or the mammalian system as a model to investigate our research questions proposed above. In preparation for their research, all the essential equipment required for this project has been procured and properly set up in our laboratory, ensuring a fully functional workspace. Moreover, we have made substantial progress in preparing the key reagents necessary for conducting quantitative biochemical assays essential for this project. During this, we have set up a workflow to biochemically reconstitute a key complex required to test our hypothesis described above. We have also generated a key tool, phospho-specific antibody, to critically test our ideas in cellular settings. In addition, as part of the initial biochemical proof of principle experiments, we have performed some crucial assays, testing validity of our proposed approach.