Schwarz2018-Cdk Activity Threshold Determines Passage through the Restriction Point 

July 2020, model of the month by Ahmad Zyoud
Original model: BIOMD0000000918

Background

The cell cycle has five distinct phases namely G0, G1, S, G2, and M phase. G1 and G2 represent the “gap” period between two discernible phases, DNA synthesis and mitosis. In G1 (the first gap) phase, cells start to prepare for DNA synthesis. Moreover in this phase cell make the irreversible  decision for division  (Schafer 1998). G0 phase is where G1 cells remain in a resting (quiescent) state (Harper and Brooks 2005). S-phase marks the DNA replication (Harper and Brooks 2005). During this phase the cell will have aneuploid DNA content between 2N and 4N. G2 (the second gap) phase is where the cell prepares for mitosis (Schafer 1998) and M-phase is where the cell undergoes cell division (mitosis and cytokinesis) (Harper and Brooks 2005).  

Cell cycle is a strictly regulated process (Satyanarayana and Kaldis 2009). Two classes of molecules are found to control the ability of cells to divide; cyclin-dependent kinases (Cdks), a family of serine/threonine kinases and their binding partners, cyclins (Morgan 1997).

In addition, retinoblastoma protein (Rb) is a key element in regulating cell cycle progression by binding to several type of cellular proteins. The binding ability of the Rb protein is regulated through its phosphorylation states. Rb is deactivated when hyperphosphorylated and activated when dephosphorylated (Grafstrom, Pan and Hoess 1999) .

Rb-E2F signalling is considered as a critical pathway to regulate cell cycle progression. E2F transcriptional factor activates genes encoding proteins involved in DNA replication and cell-cycle progression. It also plays a key role in controlling cell proliferation. In resting state, E2F is deactivated by binding to Rb protein. The phosphorylation of the bound Rb by CycD-Cdk4,6 complex terminate its suppression and reactivates E2F. This phosphorylation process is triggered by sufficient growth factor stimulation. Subsequently, the activation of E2F will establish a positive feedback loop by activating CycE that forms a complex with Cdk2 to further phosphorylate Rb and remove its repression (Yao et al. 2008), and initiate E2F-dependent transcriptional activation (Schwarz et al. 2018).

Cell cycle progression in mammals is driven by growth factor stimulation until a certain point referred to as restriction point (R) (Schwarz et al. 2018). Past this point, cells will progress through the division and growth factor stimulation will not be required.  Therefore, the restriction point, R marks the point of irreversible commitment to division.  It is considered as a critical point in normal cell development and diseases, due to its importance in the regulation of cell proliferation and prevent accumulation of mutations (Schwarz et al. 2018).

Experimental observation

Schwarz et al. 2018 model is derived from the already established ordinary differential equation model of R  (Yao et al. 2008), that demonstrates a bistability in the E2F activation generated from Rb-E2F pathway. Yao et al. 2008 predicted the activation of the inactive E2F when the serum concentration reaches a critical threshold. Once activated, E2F can maintain this state even when the serum concentration falls below the initial threshold (Yao et al. 2008).  However, multiple experimental reports have doubted this model, which suggested that R threshold and E2F-Rb-cyclin E feedback loop are considered as separate processes. (Martinsson et al. 2005, Spencer et al. 2013)

Schwarz et al. 2018 redefined R commitment threshold as the first increase in Cdk activity. They demonstrated their results by measuring the change in fluorescent reporters of specific protein activities in single-cells. Cdk activity were analysed using live-cell sensor (HDHB-EGFP), this sensor is phosphorylated in vitro by Cdk1 and Cdk2 in complex with either cyclin E or cyclin A. the export HDHB-EGFP from nucleus to the cytoplasm is dependent on the increasing of phosphorylation status of the sensor through the cell cycle. At the time of serum removal, the relationship between Cdk activity and R were found to be highly correlated. The results were concluded by measuring the response of cells expressing HDHB-EGFP sensor to serum removal.

In addition, this study allowed to assess the contribution of mitogen-activated protein kinase (MAPK) pathway to the Cdk activity threshold. Since MAPK activity is essential for primary cell proliferation. The activity of this pathway has been inhibited using MEK inhibitor. The resulting data showed that the threshold of Cdk activity was lower than that defining R in response to serum removal (Schwarz et al. 2018)

 

The model

Therefore, new interaction has been incorporated to the Yao et al. 2008 model (figure 1) to show that once Cdk activity raises above a threshold it will remain high even if serum (growth signal) is removed.

 

Figure 1: Schematics indicating the molecular interactions included in the model Adapted from (Schwarz et al. 2018)

Schwarz et al 2018 modified the Yao et al. 2008 model in two ways indicated by orange arrows in Figure 1 to bring together the previous experimental observation with R model proposed by Yao et al. 2008. First, serum-independent E2F transcription rate, kpfb was added to allow the cells post-R threshold to continue through the cell cycle despite complete serum removal. In the original model, all E2F transcription depends on serum so that the model is not bistable at 0% serum (Figure 1).

Second, as the experimental data have revealed that R commitment threshold is decreasing when cells are exposed to MEK inhibitor after serum removal. To account for this decline, the growth signals has been divided into a MEK-dependent and a MEK-independent part in the model. MEK-independent signalling pathway will maintain the reduction that might occur in the MEK-dependent pathway due to MEK inhibitor.   

 

Table 1: The differential equation used in this model, adapted from (Schwarz et al. 2018), S = growth signals (e.g. serum), M = Myc, E = E2F, CD = CycD, CE = CycE, R = unphosphorylated (active) Rb, RP = phosphorylated (inactive) Rb, RE = Rb-E2F complex. Except the kpfb term, the equations are the same the original model (Yao et al. 2008)

 

Results and Discussion

Schwarz et al 2018 model provided more precise way to predict R threshold using the aforementioned experimental data. The model was simulated with the initial conditions of [Rb] = 0.55 μM. Rb here represent the phosphorylated form (inactive). This initial value allows the model to trigger the Rb-E2F signalling pathway. All other concentrations were set to zero.  

In the model, the term ‘CE’ represents a composite of Cdk2 and Cdk1 activities with cyclins E and A. 2% serum were added initially and lowered to zero in variable times. CE eventually reached a high steady state value in cells which passed the point of commitment before removal of 2% serum. (Figure 2B). If the R threshold was not passed, CE returned to its initial value of zero (Figure 2A). The same serum concentration (2%)  was used as in (Yao et al. 2008). Figure 2 showed that CE will return to its initial value of  zero when serum has been added for 9.55 hours or less (Figure 2A), however, CE will  reach a steady state level if the serum been added for more than 10 hours (Figure 2B). This suggested that after 10 hours of 2% serum, R threshold will be reached; the cells will commit to divide and serum stimulation will no longer be required.

To simulate the effect of the MEK inhibitor, Cdk activity threshold for commitment to division has been reduced by lowering the serum concentration to 0.28% instead of 0%. The increase in the serum value (upstream stimulation) will result in decreasing the amount of Cdk activity required for cell to commit for division. This process mimics the behaviour of the positive feedback loop of the Cdk activity in the presence of MEK inhibitor

Figure 2: Schematic for CE behaviour after serum removal at A) 9.55 hours, B) 10 hours. Reproduced from (Schwarz et al. 2018)

This model unified multiple contrasting reports regarding the role of R in G1/S transition by demonstrating its behaviour in primary fibroblast (Schwarz et al. 2018). Previous reports have suggested that the phosphorylation of Rb  and cyclin E accumulation are not directly related to the molecular mechanism behind the passage through R (Martinsson et al. 2005). However, Yao et al. 2008 provided an evidence for the direct role of Rb hyperphosphorylation in R determination using bistable E2F activation process. Herein, the mathematical model along with the experimental evidence described in (Schwarz et al. 2018)  suggested the association of R with activation of a positive feedback loop of Cdk activity, E2F-dependent transcription, and Rb hyperphosphorylation. The experimental data provided with this study demonstrated that the timing required for the majority of cells to pass R is matching the timing at which individual cells pass the threshold level of Cdk activity required to commit to cell division, as well as, it matches the timing of cyclin E accumulation mentioned in another study (Ekholm et al. 2001). In addition, in consistent with a previous report (Yen and Pardee 1978), this model predicted that R cell passage during G1 will occur one to two hours before DNA replication phase. However, the thresholds determined here may not be the most predictive in other conditions or in different cell types.  

In conclusion, Schwarz et al 2018 provided a comprehensive simple threshold model that accurately predict R through detecting Cdk activity before serum removal. Also, they presented a key modification to the Rb-E2F model provided by Yao et al. 2008 to fit with the results of the contrasting previous studies. In addition, this model demonstrates the ability of a few critical protein activity such as Cdk to control cell division and fate.

 

Reference

Ekholm, S. V., P. Zickert, S. I. Reed & A. Zetterberg (2001) Accumulation of cyclin E is not a prerequisite for passage through the restriction point. Molecular and cellular biology, 21, 3256-3265.

Grafstrom, R. H., W. Pan & R. H. Hoess (1999) Defining the substrate specificity of cdk4 kinase–cyclin D1 complex. Carcinogenesis, 20, 193-198.

Harper, J. V. & G. Brooks. 2005. The mammalian cell cycle. In Cell Cycle Control, 113-153. Springer.

Martinsson, H.-S., M. Starborg, F. Erlandsson & A. Zetterberg (2005) Single cell analysis of G1 check points—the relationship between the restriction point and phosphorylation of pRb. Experimental cell research, 305, 383-391.

Morgan, D. O. (1997) Cyclin-dependent kinases: engines, clocks, and microprocessors. Annual review of cell and developmental biology, 13, 261-291.

Satyanarayana, A. & P. Kaldis (2009) Mammalian cell-cycle regulation: several Cdks, numerous cyclins and diverse compensatory mechanisms. Oncogene, 28, 2925-2939.

Schafer, K. (1998) The cell cycle: a review. Veterinary pathology, 35, 461-478.

Schwarz, C., A. Johnson, M. Kõivomägi, E. Zatulovskiy, C. J. Kravitz, A. Doncic & J. M. Skotheim (2018) A precise Cdk activity threshold determines passage through the restriction point. Molecular cell, 69, 253-264. e5.

Spencer, S. L., S. D. Cappell, F.-C. Tsai, K. W. Overton, C. L. Wang & T. Meyer (2013) The proliferation-quiescence decision is controlled by a bifurcation in CDK2 activity at mitotic exit. Cell, 155, 369-383.

Yao, G., T. J. Lee, S. Mori, J. R. Nevins & L. You (2008) A bistable Rb–E2F switch underlies the restriction point. Nature cell biology, 10, 476-482.

Yen, A. & A. B. Pardee (1978) Exponential 3T3 cells escape in mid-G1 from their high serum requirement. Experimental cell research, 116, 103-113.