CDK4: Linking cell size to cell cycle control

A size checkpoint active during cell proliferation ensures that cells reach a certain target size before transitioning into S phase. In this issue of Developmental Cell, Tan et al. identify a distinct function of cyclin-dependent kinase 4 (CDK4) in determining the target cell size for cell cycle progression. A size checkpoint active during cell proliferation ensures that cells reach a certain target size before transitioning into S phase. In this issue of Developmental Cell, Tan et al. identify a distinct function of cyclin-dependent kinase 4 (CDK4) in determining the target cell size for cell cycle progression. Cell growth, the process of increase in biomass and cell size, is closely linked to cell cycle progression in many types of growing cells. As first observed in cultured mouse fibroblasts, cells typically need to reach a certain target cell size before entering S phase, when DNA is replicated (Killander and Zetterberg, 1965Killander D. Zetterberg A. Quantitative cytochemical studies on interphase growth: I. Determination of DNA, RNA and mass content of age determined mouse fibroblasts in vitro and of intercellular variation in generation time.Exp. Cell Res. 1965; 38: 272-284Crossref PubMed Scopus (144) Google Scholar). This phenomenon later came to be viewed as being under the control of a cell size “checkpoint” that would instruct cells with a small birth size to spend a longer time in G1 in order to reach the permissive cell size for cell division. Recent studies have suggested molecular mechanisms that could help explain this cell size checkpoint. In one model, increasing cell volume reduces the concentration of cell cycle repressors, such as Rb in mammalian cells (Zatulovskiy et al., 2020Zatulovskiy E. Zhang S. Berenson D.F. Topacio B.R. Skotheim J.M. Cell growth dilutes the cell cycle inhibitor Rb to trigger cell division.Science. 2020; 369: 466-471Crossref PubMed Scopus (22) Google Scholar) or Whi5 in yeast (Schmoller et al., 2015Schmoller K.M. Turner J.J. Kõivomägi M. Skotheim J.M. Dilution of the cell cycle inhibitor Whi5 controls budding-yeast cell size.Nature. 2015; 526: 268-272Crossref PubMed Scopus (151) Google Scholar), until the cells can surpass a repression threshold and enter S phase. Another model invokes the physical segregation of cell cycle control factors within the cell as it grows (Moseley et al., 2009Moseley J.B. Mayeux A. Paoletti A. Nurse P. A spatial gradient coordinates cell size and mitotic entry in fission yeast.Nature. 2009; 459: 857-860Crossref PubMed Scopus (275) Google Scholar). A number of signaling pathways, such as mTORC1, PI3K, RAS/ERK, Hippo, etc., regulate cell growth. However, only recently have molecular sensors been identified that monitor the difference between actual cell size and target cell size for cell division. p38MAPK (a mitogen-activated protein kinase) has been found to play a critical role in small cells, where it prevents them from entering S phase prematurely (Liu et al., 2018Liu S. Ginzberg M.B. Patel N. Hild M. Leung B. Li Z. Chen Y.-C. Chang N. Wang Y. Tan C. et al.Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length.eLife. 2018; 7: e26947Crossref PubMed Scopus (33) Google Scholar). Despite advances in research on cell size control, our understanding of how target cell size is determined for a given cell type is still limited. Rates of cell division and rates of biosynthetic activity must be coordinated to ensure that cells enter S phase only when they reach an appropriate biomass, typically twice their mass at birth during mitosis. In this issue of Developmental Cell, Tan et al., 2021Tan C. Ginzberg M.B. Webster R. Iyengar S. Liu S. Papadopol D. Concannon J. Wang Y. Auld D.S. Jenkins J.L. et al.Cell size homeostasis is maintained by CDK4-dependent activation of p38 MAPK.Dev. Cell. 2021; 56Abstract Full Text Full Text PDF Scopus (9) Google Scholar report that this inherent cell size homeostasis is maintained well even when protein synthesis is slowed by attenuating mTORC1 activity. To identify factors involved in the cell size control, they carried out a chemical screen on human RPE cells by using 248 inhibitors against 109 potential checkpoint regulators. Their screen identified two types of factors: “sensors” that maintain the correlation between G1 length and cell size, and “dials” that determine the target cell size for S phase entry. p38MAPK was one of the top sensors (Liu et al., 2018Liu S. Ginzberg M.B. Patel N. Hild M. Leung B. Li Z. Chen Y.-C. Chang N. Wang Y. Tan C. et al.Size uniformity of animal cells is actively maintained by a p38 MAPK-dependent regulation of G1-length.eLife. 2018; 7: e26947Crossref PubMed Scopus (33) Google Scholar) whose dysfunction significantly disrupts the correlation between cell size and G1 length. Even more intriguingly, they uncovered cyclin-dependent kinase 4 (CDK4) as one of the strongest “dials” that determine target cell size. Manipulating CDK4 activity to different levels, either by using low doses of the CDK4 inhibitor palbociclib or by increasing expression of the CDK4 activator, Cyclin D1, was found to shift cell sizes upward or downward respectively (Figure 1). The methods used to gauge cell sizes and division times in this study are truly cutting edge, and they provide an excellent example for the field. Because CDK4 plays an essential role in G1/S transition by phosphorylating Rb, one key question was whether other G1/S CDKs that regulate G1/S progression also affect target cell size. The authors examined this for CDK2 and CDK6. They found that although knockdown of these other CDKs elongated the cell cycle, it failed either to affect the correlation between birth size and G1 length or to change cell size itself. Their findings indicate a unique function of CDK4 in cell size control. Using epistasis tests, Tan et al. were also able to elucidate the relationship between CDK4 and p38MAPK. p38 is kept active through phosphorylation until cells reach target cell size. The authors quantified active p38 levels using either western blots or a p38 kinase translocation reporter (p38-KTR) at single cell resolution. When CDK4 function was impaired by palbociclib, the target cell size for S phase entry became larger, and p38 remained active at cell sizes that would exhibit inactive forms of p38 in the control cells. Logically, this places the “sensor” function of p38 downstream of and subsidiary to CDK4’s “dial” function (Figure 1). Finally, Tan et al. characterized the molecular changes underlying cell size control by CDK4. With reduced CDK4 activity, an increase in target cell size was accompanied by an increase in protein synthesis rates per cell and in bulk biomass, and by enlargement of subcellular components such as the nucleolus, mitochondria, and the endoplasmic reticulum. Changes in certain major growth-related signaling pathways and bioenergetic potential were found to be consistent with these cell size changes, such that the activity levels of mTORC1, ERK, c-Myc, hexokinase II, oxygen consumption, and ATP generation were all inversely correlated to CDK4 activity on a per cell basis (Figure 1). While these observations further emphasize the central role of CDK4 in cell size homeostasis, they present an intriguing paradox in light of many previous studies which showed that inhibition of CDK4 or Cyclin D actually suppresses growth, when measured in the context of cell clones, cell populations in culture, or organs, or (most often) during tumor progression (Satyanarayana and Kaldis, 2009Satyanarayana A. Kaldis P. Mammalian cell-cycle regulation: several Cdks, numerous cyclins and diverse compensatory mechanisms.Oncogene. 2009; 28: 2925-2939Crossref PubMed Scopus (531) Google Scholar). A few studies also show that increased Cyclin D/CDK4 has the potential to increase growth rates at both cellular and clonal levels in other organisms (Datar et al., 2000Datar S.A. Jacobs H.W. de la Cruz A.F.A. Lehner C.F. Edgar B.A. The Drosophila cyclin D-Cdk4 complex promotes cellular growth.EMBO J. 2000; 19: 4543-4554Crossref PubMed Scopus (227) Google Scholar; Rane et al., 1999Rane S.G. Dubus P. Mettus R.V. Galbreath E.J. Boden G. Reddy E.P. Barbacid M. Loss of Cdk4 expression causes insulin-deficient diabetes and Cdk4 activation results in β-islet cell hyperplasia.Nat. Genet. 1999; 22: 44-52Crossref PubMed Scopus (606) Google Scholar; Fantl et al., 1995Fantl V. Stamp G. Andrews A. Rosewell I. Dickson C. Mice lacking cyclin D1 are small and show defects in eye and mammary gland development.Genes Dev. 1995; 9: 2364-2372Crossref PubMed Scopus (601) Google Scholar). Although Tan et al., 2021Tan C. Ginzberg M.B. Webster R. Iyengar S. Liu S. Papadopol D. Concannon J. Wang Y. Auld D.S. Jenkins J.L. et al.Cell size homeostasis is maintained by CDK4-dependent activation of p38 MAPK.Dev. Cell. 2021; 56Abstract Full Text Full Text PDF Scopus (9) Google Scholar do not report such an effect, their measurements were focused on the sizes of individual cells rather than on the total biomass accumulated by cultures of cells over time, and indeed they did observe slower division after inhibiting CDK4, just as other researchers have. Future investigations of how CDK4 activity dictates the target cell size for S phase entry should be able to resolve these interesting paradoxes. Altogether, the work of Tan et al. provides exciting new insights that are leading us to a clearer picture of how cell size checkpoints function. The authors declare no competing interests. Cell size homeostasis is maintained by CDK4-dependent activation of p38 MAPKTan et al.Developmental CellMay 21, 2021In BriefTan et al. study homeostatic mechanisms that maintain animal cells at their appropriate target size. They find that p38 is part of a sensing mechanism that identifies inappropriately sized cells, while CDK4 is analogous to a thermostat dial that sets the target size set point referenced by p38. Full-Text PDF Open Archive