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IntroductionIntroduction

Cyclin dependent kinasaes (CDKs) are a family of serine/threonine kinases, which are responsible for cell cycle progression. They are regulated by several upstream pathways, such as the CDK activating kinase and interaction with Cyclin D. After association of CDK with cyclin, the active CDK/cyclin complex phosphorylates and herefore inactivates retinoblastoma protein family members. This leads to activation of E2F target genes. One of these genes is cyclin E which triggers G1 phase cell cycle progression.
CDKs and cyclins are particularly interesting because a dysfunction of their pathways are associated to numerous cancers. Among all genetic alterations of CDK/cyclin in cancer that one of CDK4 and cyclin D1 is most common. Appearance of breast cancer goes normally hand in hand with increased cyclin D1 levels, caused by genetic amplification or overexpression [1]. In liposarcomas the CDK4 gene is amplified [2]. Cyclin D1 translocations play also a role in mantle cell lymphoma and multiple myelomas [3]. Furthermore mutations of CDK4, which alter its structure in a manner that it becomes unable to bind regulative tumor supressor proteins result in defective CDK4 repression and therefore dysregulate the cell cycle. It becomes obvious that cyclin D1 CDK4 interaction is critical for the development of several cancers. Therefore CDKs, in particular CDK4 are a promising target of novel cancer drugs.

3D structure of CDK4 (cyan) with cyclin D1 (orange) in complex

Drag the structure with the mouse to rotate

CDK4 structureCDK4 structure

In order to gain structural information about CDKs, a crystallographic analysis of CDK in complex with cyclin D was performed. CDK4 and cyclin D1, were overexpressed in insect cells for crystallisation and slightly modified for better crystallization. CDK 4 possesses the typical of kinases, containing a 5-strandet ß-sheet and a N-terminal domain (residues 1-96). Within the N-terminal domain the helix alpha-C is packed against the ß-sheet. Furthermore an ATP binding site is located in between these domains, which might bind ATP after minimal conformational rearrangement of residues Asp-99, Asp-140, Lys-142, and Tyr-17. Residues 161-171 form a containing alpha-helices. Phosphorylation of this T-loop on residue Thr-172 or cyclin D binding may change its conformation in order to activate kinase activity of CDK4. Lambda-phosphatase incubation studies confirm the importance of phosphorylation of the T-loop for full kinase activity [4].


Cyclin D1 structureCyclin D1 structure

Cyclin D1 consists of a double glycin box domain fold, containing 11 alpha helices. Its secondary structure is generally equivalent to the structure of other cyclins. Furthermore, it contains 2 Rb binding sites comprising an LxCxE motive t its N-terminal site. Structural conservation of the RxL motive, responsible for peptide binding was observed on Cyclin D1 and Cyclin A. Peptide binding studies substantiate this observation [5].


Cyclin D1-CDK4 complexCyclin D1-CDK4 complex

CDK4 in complex with Cyclin D1 shows an engagement of the CDK4 alpha-helix with cyclin D1. Nevertheless, the helix does perform the conformational switch, normally known for CDK activation in other CDK/cyclin complexes [6]. Surprisingly, the CDK4/cyclin D1 structure reminds to the structures of inactive structures of non cyclin bound CDK2 and CDK7 [7]. Further stabilization of the CDK4 T-loop in the inactive confirmation is achieved by interactions of C- and N- terminal lobes of the kinase. These residues, containing an Asp158–Phe159–Gly160 are condensed into a helix, which is stabilized by interactions with the alpha-C-helix, ß4-strand, ß6-strand as well as the apex of the T-loop. The architecture of the T-Loop is similar to the one observed at CDK7 and CDK6. CDK4 kinase activation could be achieved due to movement of the alpha-C-helix. Although the C-alpha-lobe of CDK4 seems to be bound by Cyclin D1, the rotation of the C-lobe of CDK4 is not maximal. This reduces the buried surface area of of the CDK4/cyclin D1 interface in comparison of the buried surface of other CDK/cyclin complexes.

CDK4 in its biological contextCDK4 in its biological context

The cyclin-dependent kinase (CDK)4 forms the link between mitogenic and antimitogenic extracellular signals with the cell cycle and is located in the nucleus. As its name indicates, the assembly with a cyclin, in case of CDK4 a D-type cyclin, is prerequisite for the kinase activity of CDK4. Thus, in the active cyclin D-CDK4 complex, the D-type cyclin acts as a regulatory subunit that directs its complex partner to the retinoblastoma protein (pRB) substrate. pRB, the only physiologically important target of the cyclin D-CDK4 complex, is implicated in the coupling of the cell cycle clock with the transcription machinery. Thereby, it controls the passing of the restriction point in the G1 phase of the cell cycle. This G1 restriction point is the moment at which the cell commits whether it will proceed the cell cycle and proliferate by going towards the S phase or if potential DNA damage and metabolic disturbance have to be solved at first.
In its hypophosphorylated state, pRB binds to the transcription factor E2F and inhibits it from initiating transcription of a number of genes that are important for cell growth control [8][9]. In contrast, phosphorylation of pRB by CDK4 prevents pRB from binding to E2F, thereby allowing E2F to proceed with the activation of its regulated genes [10]. Thus, it can be resumed that kinase activity of cyclin D-CDK4 allows transcription initiation of cell growth genes by phosphorylation of the E2F-inhibitor pRB [11].


Regulation of CDK4Regulation of CDK4

The activation of CDK4 includes several different and independent regulatory steps allowing CDK4 to integrate the various signals implicated in cell growth and proliferation. This comprises the assembly of cyclin D-CDK4 complexes, the nuclear translocation of D-type cyclin-CDK4 complexes, activity regulation, and phosphorylation of CDK4.
At first, the most obvious regulation concerns the presence of both partners, CDK4 and the D-type cyclin, to be able to form a functional complex at all. This step also shows the inducibility of CDK4 by external growth signals. Extracellular mitogens induce the expression of the D-type cyclins required for cyclin D-CDK4 complex formation and thus, prevention of transcription factor inhibition by pRB. The concentration of the D-type cyclins has to pass an inhibitory threshold set by INK4 CDK4 inhibitory proteins that bind the isolated CDK4 thereby preventing the binding of cyclin. This already presents a possibility of negative regulation as growth inhibition signals may lead to an increasing accumulation of these inhibitory proteins. So, at this point, the key position of CDK4 for linking external signals with expression changes leading to cell cycle progress becomes clear.
After a functional complex is formed, activity of cyclin D-CDK4 can further be regulated on several levels. On one hand, this can take place by altering the stability of the complex by supplementary binding components. One example might be the Cip/Kip CDK inhibitors (p21, p27) which paradoxically have been shown to stabilize the complexes cyclin D3-CDK4 and cyclin D1-CDK4 [12][13]. Nevertheless, they are not essentially required for the formation of these complexes.
Furthermore, as the pRB substrate of cyclin D-CDK4 and the CDK-activating kinase (CAK) are nuclear proteins, the complex has to be imported into the nucleus. This is mediated by the CDK4 binding proteins p21 and p27, which, in contrast to the complex partners, possess an obvious nuclear localization signal. Therefore, p21 and p27 are required to determine this translocation of the cyclin D-CDK4 complex, thereby constituting another regulation possibility.
The Cip/Kip CDK inhibitors p21 and p27 can, as their names imply, inhibit the activity of the D-type cyclin-CDK4 complex. The underlying mechanisms are not understood yet, but it is supposed that the opposite effects of p21 and p27 on the CDK complex might depend on the relative stoichiometric ratio of CDK inhibitor and D-type cyclin [14] [15]. It can be retained that this inhibition represents another level of CDK4 activity regulation.
To be catalytically active, CDK4 of the complex has to be phosphorylated on a specific residue within its activation loop. This phosphorylation only takes place if CDK4 is present in form of the complex with cyclin D. The catalysing enzyme is the CDK-activating kinase (CAK) which is interestingly the cyclin H-CDK7 complex. However, as CDK7 has been found to be constitutively active [16], it does not seem as CAK would regulate CDK4 specifically in response to mitogenic stimulations. According to some observations, an increased binding of p27 to the cyclin D-CDK4 complex may prevent phosphorylation, but these results are in contradiction with more recent observations that exclude the prevention of the activating phosphorylation of CDK4 as activity inhibition of cyclin D-CDK4 by p27 [17].
To conclude, the activity of CDK4 can be regulated at several steps that are independently from each other. It is this complex process of regulation by integrating multiple signals and breaking them down on one point, the activity of CDK4 that allows the cell to respond in a simple manner – proliferation or not - to a variation of stimuli.

CDK4’s role in cancerCDK4’s role in cancer

A main characteristic of cancerous cells is the dysregulation of the cell cycle allowing uncontrolled cell growth and proliferation while safety mechanisms as senescence and apoptosis are inhibited. As the activation of the CDK4 pathway leads to transition from the G1 to the S phase of the cell-cycle via the inhibition of the retinoblastoma protein, this pathway plays an important role in cancerogenesis. In accordance with this idea, the CDK4 pathway was found to be altered in regulation in about 90% of studied melanomas [18]. In case of cancer variants where the dysregulation of the CDK4 signalling pathway is the main reason for melanoma development and not a secondary effect, drugs that target and inhibit CDK4 might be very promising. Therefore, further studies should be undertaken to develop new therapeutic agents fighting cancer at this central point of cell-cycle regulation. Furthermore recent research uncovered a transcriptional role of Cyclin D1. Knowing that Cyclin D1 levels in several cancers are altered this discovery might have a dramatic impact of the transcription of Cylcin D1 (and interaction partners) target genes [19]. In addition Cylcin D1 has a function in DNA repair in several cancers [20].


ReferencesReferences

  1. ↑ Arnold A, Papanikolaou A. Cyclin D1 in breast cancer pathogenesis. J Clin Oncol. 2005 Jun 20;23(18):4215-24. PMID:15961768 doi:http://dx.doi.org/10.1200/JCO.2005.05.064
  2. ↑ Binh MB, Sastre-Garau X, Guillou L, de Pinieux G, Terrier P, Lagace R, Aurias A, Hostein I, Coindre JM. MDM2 and CDK4 immunostainings are useful adjuncts in diagnosing well-differentiated and dedifferentiated liposarcoma subtypes: a comparative analysis of 559 soft tissue neoplasms with genetic data. Am J Surg Pathol. 2005 Oct;29(10):1340-7. PMID:16160477
  3. ↑ Amin HM, McDonnell TJ, Medeiros LJ, Rassidakis GZ, Leventaki V, O'Connor SL, Keating MJ, Lai R. Characterization of 4 mantle cell lymphoma cell lines. Arch Pathol Lab Med. 2003 Apr;127(4):424-31. PMID:12683869 doi:<0424:COMCLC>2.0.CO;2 http://dx.doi.org/10.1043/0003-9985(2003)127<0424:COMCLC>2.0.CO;2
  4. ↑ Day PJ, Cleasby A, Tickle IJ, O'Reilly M, Coyle JE, Holding FP, McMenamin RL, Yon J, Chopra R, Lengauer C, Jhoti H. Crystal structure of human CDK4 in complex with a D-type cyclin. Proc Natl Acad Sci U S A. 2009 Feb 23. PMID:19237565
  5. ↑ Day PJ, Cleasby A, Tickle IJ, O'Reilly M, Coyle JE, Holding FP, McMenamin RL, Yon J, Chopra R, Lengauer C, Jhoti H. Crystal structure of human CDK4 in complex with a D-type cyclin. Proc Natl Acad Sci U S A. 2009 Feb 23. PMID:19237565
  6. ↑ Jeffrey PD, Russo AA, Polyak K, Gibbs E, Hurwitz J, Massague J, Pavletich NP. Mechanism of CDK activation revealed by the structure of a cyclinA-CDK2 complex. Nature. 1995 Jul 27;376(6538):313-20. PMID:7630397 doi:http://dx.doi.org/10.1038/376313a0
  7. ↑ De Bondt HL, Rosenblatt J, Jancarik J, Jones HD, Morgan DO, Kim SH. Crystal structure of cyclin-dependent kinase 2. Nature. 1993 Jun 17;363(6430):595-602. PMID:8510751 doi:http://dx.doi.org/10.1038/363595a0
  8. ↑ La Thangue NB. DRTF1/E2F: an expanding family of heterodimeric transcription factors implicated in cell-cycle control. Trends Biochem Sci. 1994 Mar;19(3):108-14. PMID:8203017
  9. ↑ Nevins JR. E2F: a link between the Rb tumor suppressor protein and viral oncoproteins. Science. 1992 Oct 16;258(5081):424-9. PMID:1411535
  10. ↑ Chellappan SP, Hiebert S, Mudryj M, Horowitz JM, Nevins JR. The E2F transcription factor is a cellular target for the RB protein. Cell. 1991 Jun 14;65(6):1053-61. PMID:1828392
  11. ↑ Weinberg RA. The retinoblastoma protein and cell cycle control. Cell. 1995 May 5;81(3):323-30. PMID:7736585
  12. ↑ Bagui TK, Jackson RJ, Agrawal D, Pledger WJ. Analysis of cyclin D3-cdk4 complexes in fibroblasts expressing and lacking p27(kip1) and p21(cip1). Mol Cell Biol. 2000 Dec;20(23):8748-57. PMID:11073976
  13. ↑ Sugimoto M, Martin N, Wilks DP, Tamai K, Huot TJ, Pantoja C, Okumura K, Serrano M, Hara E. Activation of cyclin D1-kinase in murine fibroblasts lacking both p21(Cip1) and p27(Kip1). Oncogene. 2002 Nov 21;21(53):8067-74. PMID:12444543 doi:http://dx.doi.org/10.1038/sj.onc.1206019
  14. ↑ Blain SW, Montalvo E, Massague J. Differential interaction of the cyclin-dependent kinase (Cdk) inhibitor p27Kip1 with cyclin A-Cdk2 and cyclin D2-Cdk4. J Biol Chem. 1997 Oct 10;272(41):25863-72. PMID:9325318
  15. ↑ LaBaer J, Garrett MD, Stevenson LF, Slingerland JM, Sandhu C, Chou HS, Fattaey A, Harlow E. New functional activities for the p21 family of CDK inhibitors. Genes Dev. 1997 Apr 1;11(7):847-62. PMID:9106657
  16. ↑ Bockstaele L, Kooken H, Libert F, Paternot S, Dumont JE, de Launoit Y, Roger PP, Coulonval K. Regulated activating Thr172 phosphorylation of cyclin-dependent kinase 4(CDK4): its relationship with cyclins and CDK "inhibitors". Mol Cell Biol. 2006 Jul;26(13):5070-85. PMID:16782892 doi:10.1128/MCB.02006-05
  17. ↑ Bockstaele L, Coulonval K, Kooken H, Paternot S, Roger PP. Regulation of CDK4. Cell Div. 2006 Nov 8;1:25. PMID:17092340 doi:http://dx.doi.org/10.1186/1747-1028-1-25
  18. ↑ Sheppard KE, McArthur GA. The cell-cycle regulator CDK4: an emerging therapeutic target in melanoma. Clin Cancer Res. 2013 Oct 1;19(19):5320-8. doi: 10.1158/1078-0432.CCR-13-0259. PMID:24089445 doi:http://dx.doi.org/10.1158/1078-0432.CCR-13-0259
  19. ↑ Bienvenu F, Jirawatnotai S, Elias JE, Meyer CA, Mizeracka K, Marson A, Frampton GM, Cole MF, Odom DT, Odajima J, Geng Y, Zagozdzon A, Jecrois M, Young RA, Liu XS, Cepko CL, Gygi SP, Sicinski P. Transcriptional role of cyclin D1 in development revealed by a genetic-proteomic screen. Nature. 2010 Jan 21;463(7279):374-8. doi: 10.1038/nature08684. PMID:20090754 doi:http://dx.doi.org/10.1038/nature08684
  20. ↑ Jirawatnotai S, Hu Y, Michowski W, Elias JE, Becks L, Bienvenu F, Zagozdzon A, Goswami T, Wang YE, Clark AB, Kunkel TA, van Harn T, Xia B, Correll M, Quackenbush J, Livingston DM, Gygi SP, Sicinski P. A function for cyclin D1 in DNA repair uncovered by protein interactome analyses in human cancers. Nature. 2011 Jun 8;474(7350):230-4. doi: 10.1038/nature10155. PMID:21654808 doi:http://dx.doi.org/10.1038/nature10155

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