Editing Cell cycle (section)

====Specific action of cyclin-CDK complexes====
[[Cyclin D]] is the first cyclin produced in the cells that enter the cell cycle, in response to extracellular signals (e.g. [[growth factor]]s). Cyclin D levels stay low in resting cells that are not proliferating. Additionally, [[Cyclin-dependent kinase 4|CDK4/6]] and [[Cyclin-dependent kinase 2|CDK2]] are also inactive because CDK4/6 are bound by [[INK4]] family members (e.g., p16), limiting kinase activity. Meanwhile, CDK2 complexes are inhibited by the CIP/KIP proteins such as p21 and p27,<ref>{{cite journal | vauthors = Goel S, DeCristo MJ, McAllister SS, Zhao JJ | title = CDK4/6 Inhibition in Cancer: Beyond Cell Cycle Arrest | journal = Trends in Cell Biology | volume = 28 | issue = 11 | pages = 911–925 | date = November 2018 | pmid = 30061045 | pmc = 6689321 | doi = 10.1016/j.tcb.2018.07.002 }}</ref> When it is time for a cell to enter the cell cycle, which is triggered by a mitogenic stimuli, levels of cyclin D increase. In response to this trigger, cyclin D binds to existing [[Cyclin-dependent kinase 4|CDK4]]/6, forming the active cyclin D-CDK4/6 complex. Cyclin D-CDK4/6 complexes in turn mono-phosphorylates the [[retinoblastoma]] susceptibility protein ([[Retinoblastoma protein|Rb]]) to pRb. The un-phosphorylated Rb tumour suppressor functions in inducing cell cycle exit and maintaining G0 arrest (senescence).<ref>{{cite journal | vauthors = Burkhart DL, Sage J | title = Cellular mechanisms of tumour suppression by the retinoblastoma gene | journal = Nature Reviews. Cancer | volume = 8 | issue = 9 | pages = 671–682 | date = September 2008 | pmid = 18650841 | pmc = 6996492 | doi = 10.1038/nrc2399 }}</ref>

In the last few decades, a model has been widely accepted whereby pRB proteins are inactivated by cyclin D-Cdk4/6-mediated phosphorylation. Rb has 14+ potential phosphorylation sites. Cyclin D-Cdk 4/6 progressively phosphorylates Rb to hyperphosphorylated state, which triggers dissociation of pRB–[[E2F]] complexes, thereby inducing G1/S cell cycle gene expression and progression into S phase.<ref>{{cite book | vauthors = Morgan DO |title=The cell cycle : principles of control |date=2007 |publisher=New Science Press |isbn=978-0-19-920610-0 |location=London |oclc=70173205 }}</ref>

Scientific observations from a study have shown that Rb is present in three types of isoforms: (1) un-phosphorylated Rb in G0 state; (2) mono-phosphorylated Rb, also referred to as "hypo-phosphorylated' or 'partially' phosphorylated Rb in early G1 state; and (3) inactive hyper-phosphorylated Rb in late G1 state.<ref>{{cite journal | vauthors = Paternot S, Bockstaele L, Bisteau X, Kooken H, Coulonval K, Roger PP | title = Rb inactivation in cell cycle and cancer: the puzzle of highly regulated activating phosphorylation of CDK4 versus constitutively active CDK-activating kinase | journal = Cell Cycle | volume = 9 | issue = 4 | pages = 689–699 | date = February 2010 | pmid = 20107323 | doi = 10.4161/cc.9.4.10611 | doi-access = free | url = https://dipot.ulb.ac.be/dspace/bitstream/2013/57637/1/17-PaternotCC9-4.pdf }}</ref><ref>{{cite journal | vauthors = Henley SA, Dick FA | title = The retinoblastoma family of proteins and their regulatory functions in the mammalian cell division cycle | journal = Cell Division | volume = 7 | issue = 1 | pages = 10 | date = March 2012 | pmid = 22417103 | pmc = 3325851 | doi = 10.1186/1747-1028-7-10 | doi-access = free }}</ref><ref name=":0">{{cite journal | vauthors = Narasimha AM, Kaulich M, Shapiro GS, Choi YJ, Sicinski P, Dowdy SF | title = Cyclin D activates the Rb tumor suppressor by mono-phosphorylation | journal = eLife | volume = 3 | pages = e02872 | date = June 2014 | pmid = 24876129 | pmc = 4076869 | doi = 10.7554/eLife.02872 | doi-access = free }}</ref> In early G1 cells, mono-phosphorylated Rb exists as 14 different isoforms, one of each has distinct [[E2F]] binding affinity.<ref name=":0" /> Rb has been found to associate with hundreds of different proteins<ref>{{cite book | vauthors = Morris EJ, Dyson NJ | title = Retinoblastoma protein partners | volume = 82 | pages = [https://archive.org/details/advancesincancer0000unse_w5o8/page/1 1–54] | date = 2001-01-01 | pmid = 11447760 | doi = 10.1016/s0065-230x(01)82001-7 | publisher = Academic Press | isbn = 9780120066827 | series = Advances in Cancer Research | url = https://archive.org/details/advancesincancer0000unse_w5o8/page/1 }}</ref> and the idea that different mono-phosphorylated Rb isoforms have different protein partners was very appealing.<ref name="pmid27401552">{{cite journal | vauthors = Dyson NJ | title = RB1: a prototype tumor suppressor and an enigma | journal = Genes & Development | volume = 30 | issue = 13 | pages = 1492–1502 | date = July 2016 | pmid = 27401552 | pmc = 4949322 | doi = 10.1101/gad.282145.116 }}</ref> A later report confirmed that mono-phosphorylation controls Rb's association with other proteins and generates functional distinct forms of Rb.<ref name="Sanidas">{{cite journal | vauthors = Sanidas I, Morris R, Fella KA, Rumde PH, Boukhali M, Tai EC, Ting DT, Lawrence MS, Haas W, Dyson NJ | display-authors = 6 | title = A Code of Mono-phosphorylation Modulates the Function of RB | journal = Molecular Cell | volume = 73 | issue = 5 | pages = 985–1000.e6 | date = March 2019 | pmid = 30711375 | pmc = 6424368 | doi = 10.1016/j.molcel.2019.01.004 }}</ref> All different mono-phosphorylated Rb isoforms inhibit E2F transcriptional program and are able to arrest cells in G1-phase. Different mono-phosphorylated forms of Rb have distinct transcriptional outputs that are extended beyond E2F regulation.<ref name="Sanidas" />

In general, the binding of pRb to E2F inhibits the E2F target gene expression of certain G1/S and S transition genes including [[Cyclin E|E-type cyclins]]. The partial phosphorylation of Rb de-represses the Rb-mediated suppression of E2F target gene expression, begins the expression of cyclin E. The molecular mechanism that causes the cell switched to cyclin E activation is currently not known, but as cyclin E levels rise, the active cyclin E-CDK2 complex is formed, bringing Rb to be inactivated by hyper-phosphorylation.<ref name=":0" /> Hyperphosphorylated Rb is completely dissociated from E2F, enabling further expression of a wide range of E2F target genes are required for driving cells to proceed into S phase [1]. It has been identified that cyclin D-Cdk4/6 binds to a C-terminal alpha-helix region of Rb that is only distinguishable to cyclin D rather than other cyclins, [[cyclin E]], [[Cyclin A|A]] and [[Cyclin B|B]].<ref name=":1">{{cite journal | vauthors = Topacio BR, Zatulovskiy E, Cristea S, Xie S, Tambo CS, Rubin SM, Sage J, Kõivomägi M, Skotheim JM | display-authors = 6 | title = Cyclin D-Cdk4,6 Drives Cell-Cycle Progression via the Retinoblastoma Protein's C-Terminal Helix | journal = Molecular Cell | volume = 74 | issue = 4 | pages = 758–770.e4 | date = May 2019 | pmid = 30982746 | pmc = 6800134 | doi = 10.1016/j.molcel.2019.03.020 }}</ref> This observation based on the structural analysis of Rb phosphorylation supports that Rb is phosphorylated in a different level through multiple Cyclin-Cdk complexes. This also makes feasible the current model of a simultaneous switch-like inactivation of all mono-phosphorylated Rb isoforms through one type of Rb hyper-phosphorylation mechanism. In addition, mutational analysis of the cyclin D- Cdk 4/6 specific Rb C-terminal helix shows that disruptions of cyclin D-Cdk 4/6 binding to Rb prevents Rb phosphorylation, arrests cells in G1, and bolsters Rb's functions in tumor suppressor.<ref name=":1" /> This cyclin-Cdk driven cell cycle transitional mechanism governs a cell committed to the cell cycle that allows cell proliferation. A cancerous cell growth often accompanies with deregulation of Cyclin D-Cdk 4/6 activity.

The hyperphosphorylated Rb dissociates from the E2F/DP1/Rb complex (which was bound to the [[E2F]] responsive genes, effectively "blocking" them from transcription), activating E2F. Activation of E2F results in transcription of various genes like [[cyclin E]], [[cyclin A]], [[DNA polymerase]], [[thymidine kinase]], etc. Cyclin E thus produced binds to [[Cyclin-dependent kinase 2|CDK2]], forming the cyclin E-CDK2 complex, which pushes the cell from G<sub>1</sub> to S phase (G<sub>1</sub>/S, which initiates the G<sub>2</sub>/M transition).<ref name="isbn0-12-324719-5">{{cite book | vauthors = Norbury C | veditors = Hardie DG, Hanks S | title = Protein kinase factsBook | publisher = Academic Press | location = Boston | year = 1995 | pages = [https://archive.org/details/proteinkinasefac0000unse/page/184 184] | chapter = Cdk2 protein kinase (vertebrates) | isbn = 978-0-12-324719-3 | chapter-url = https://archive.org/details/proteinkinasefac0000unse/page/184 }}</ref> [[Cyclin B]]-cdk1 complex activation causes breakdown of [[nuclear envelope]] and initiation of [[prophase]], and subsequently, its deactivation causes the cell to exit mitosis.<ref name="Robbins" /> A quantitative study of E2F transcriptional dynamics at the single-cell level by using engineered fluorescent reporter cells provided a quantitative framework for understanding the control logic of cell cycle entry, challenging the canonical textbook model. Genes that regulate the amplitude of E2F accumulation, such as Myc, determine the commitment in cell cycle and S phase entry. G1 cyclin-CDK activities are not the driver of cell cycle entry. Instead, they primarily tune the timing of E2F increase, thereby modulating the pace of cell cycle progression.<ref name="Dong, P. 2014" />