CDK Substrate Phosphorylation and Ordering the Cell Cycle Dipartimento di Biologia e Biotecnologie Charles Darwin Corso di Laurea Magistrale in Genetica e Biologia Molecolare A.A.2017/2018 CDK Substrate Phosphorylation and Ordering the Cell Cycle (Nurse et al., 2017) Joel Gladio Giannitelli

Introduction: Cyclin/CDK complexes Cyclins are proteins with a cyclic expression pattern that drive the progression of the cell cycle in eucaryotic cells They form heterodimers with CDKs (cyclin dependent kinase) to selectively phosphorilate key factors in each phase After their activity, is completed cyclins are swiftly degraded until the next cell cycle The number of different cyc/CDK complexes increases with the complexity of the cell cycle It is commonly accepted as a fact that cyclins regulate the progression of events through specific recognition of their substrates However, in recent years this paradigm has been challenged…

Background – The mono-oscillator system In 2010, a study showed that the whole cell cycle could be directed in S. pombe by a single cyclin/CDK complex In absence of canonical regulation, this “mono-oscillator” can apparently control the cell cycle progression only by the means of different “activity thresholds” The system still had to be fully characterized, since it was not completely understood how a a single cyclin could guide the complex sequence of events that go from G1 to mitosis

Background – The mono-oscillator system Features of the system Deletion of all cyclin genes (Δcig1, Δcig2, Δcdc13, Δpuc1) and of the only CDK gene (Δcdc2) Insertion of a fusion construct (cdc2-L-cdc13) including both the CDK and the mitotic cyclin sequences and under transcriptional control of the mitotic cyclin promoter Cdc2 modified to bind NmPP1 (ATP analog) Cdc2 modified to lose binding to regulatory loop factors and yield a linear activity

Novel Approach The present study hypothesizes that the activity level of cyc/CDK complexes, rather than the specificity of cyclins for their substrates, is the driving force behind the progression of the cell cycle The study has used the genetically simplified S. pombe model for a series of in vivo assays, that, through an innovative phosphoproteomics approach, aim at characterizing the mono-oscillator model and the way in which a single cyc/CDK complex can determine differential phosphorylation of different substrates and, therefore, guide the whole cell cycle machinery

Methods: Phosphoproteomics SILAC encoding in cell culture Protein extraction through cell lysis Phosphopeptides enriched through Ti-dioxide chromatography Mass spectrometry analysis of isolated proteins Bioinformatics for protein identification and phospho-site characterization

Results – Identification of CDK substrates To identify putative CDK substrates, the study observed dephosphorylation rates after addition of NmPP1 CDK inhibitor to the medium of synchronized cells at different cell cycle stages Among candidates, the study selected only substrates that showed: - minimal consensus sequence (S/T-P) - halvening of phosphorylation within 24 minutes - a dephosphorilation rate fitting an exponential decay More than 250 candidates were selected. Several substrates were already characterized for their involvement in the cell cycle, or had characterized homologues in other species

Results – Phosphorylation dynamics Phosphorylation rates of selected substrates were studied during two complete cell cycles Three different patterns of phosphorylation were observed, identifying three classes of substrates: Early substrates: increase in phosphorylation mainly during G1/S transition and constant increase during the rest of the cell cycle Intermediate substrates: Increase in both G1/S transition and in G2/M transition Late substrates: phosphorylated only from G2/M transition Late substrates are 10 times more than early substrates After mitotic exit, all substrates are completely dephosphorylated The ordering of substrate phosphorylation is remarkable, considering it is carried out by a single cyc/CDK complex

Results – Activity of CDK Analysis of final CDK activity, considered as a ratio between CDK activity and phosphatase activity CDK phosphorylation rates were obtained by measuring substrate increase in phosphorylation at different time intervals after release from CDK inhibitor (NmPP1) Phosphorylation rates increase during the cell cycle progression: G1: 1.7% S: 7.3% G2: 36% M: 100% CDK activity seems to rise during the cell cycle. Could the patterns of early, intermediate and late phosphorylation be a result of a differential sensitivity to CDK activity?

Results – Substrate sensitivity It is possible that phosphorylation timing depends on a differential sensitivity of substrates to CDK activity To confirm this, CDK activity was modulated with varying amounts of NmPP1 inhibitor and the substrate’s half maximal inhibitory concentration (IC50) was calculated Early substrates: less susceptible to inhibitor (IC50 = 3.7 μM) Intermediate substrates: average susceptibility (IC50 = 1.7 μM) Late substrates: high susceptibility (IC50 = 0.2 μM) The timing of phosphorylation for different substrates correlates with their sensitivity to CDK activity

Results – Dephosphorylation rates Differential phosphorylation might depend on phosphatases targeting preferentially late substrates To verifiy this, phosphatase activity was measured after critical NmPP1 concentrations were used to block CDK Early and late substrates were found to be dephosphorylated at comparable rates, with no significant difference between S and M phases Furthermore, alteration of RxL putative CDK docking sites on selected early substrates showed to significantly impact phosphorylation efficiency, bringing the sensitivity of early substrates to late substrate levels Phosphorylation dynamics correlate to CDK activity and substrate sensitivity, not to phosphatase activity Results indicate that early and late substrates have an intrinsic differential sensitivity that is largely due to the structure of the interaction domain

Results – Reordering the cell cycle The activity of CKD was artificially deregulated in order to alter substrate phosphorylation patterns and observe their effect on the events of the cell cycle CDK was inactivated in G2, and then only partially reactivated to G1 levels of activity using different NmPP1 concentrations, inducing cells to re-enter DNA synthesis CDK was inactivated in G1 and then reactivated at mitotic levels of activity, inducing cells to simultaneously activate DNA synthesis and mitosis Results show that perturbation of phosphorylation patterns induces a complete deregulation of the cell cycle, causally linking differential activity of CDK, substrate sensitivity, and timing of substrate phosphorylation with the ordering of the cell cycle

Results – Comparison with wild-type To verify whether the presence of other cyc/CDK complexes has an impact on phosphorilation pattern, ΔCCP cell phosphorylation rates were compared to wild-type cells The presence of a specific G1/S cyclin has a modest effect on early substrates, but significantly enhances expression of intermediate substrates during G1/S transition Deletion of CDK inhibitor Rum1 in ΔCCP cells removes differences for early substrates, but not for intermediate substrates No substrates were found to be exclusively phosphorylated by G1/S cyc/CDK complexes These results indicate that the presence of specific G1/S transition cyclins has a generic effect on early substrates, but targets intermediate substrates with specificity

Discussion Founded on pre-existing evidence, the present study shows that a single mitotic cyc/CDK complex is sufficient to drive progression of the cell cycle in a simplified S. pombe model. A thorough characterization of the molecular and biochemical features of the model reveals the existence of early and late phosphorylation patterns, which result from the combination of differential substrate sensitivity and differential CDK activity during the course of the cell cycle. Results indicate that CDK activity thresholds have a dominant role in ordering of the cell cycle, redifining the paradigm of substrate specificity as the main driving force behind phase-specific activities.

Critical analysis and perspectives Evaluation of the study Impact: The study offers a new perspectives on the basic principles that regulate cell cycle Deep characterization: Thorough study of yeast model that covers many relevant aspects of CDK regulation Methodology: “Omics” analysis offers a high-throughput method of investigation Model organism: Not all variables can be accounted for (e.g. regulation of cyc/CDK interaction) Perspectives Evolution: Model of basic cell cycle regulation based on activity thresholds, importance of cyclin specificity gradually increases with organism complexity Integrating specificity and activity thresholds: Are there other factors that regulate substrate sensitivity to CDK? How does cyclin specificity contribute to the overall regulation of cell cycle? Other models: Can this paradigm be applied to other systems? In higher eucaryotes CDK activity levels are important (e.g. Cyc-B gradual inactivation during mitotic exit), but many processes are directed by substrate specificity (e.g. Cyc-E/Cyc-A switch during mid-G1 progression)