3191801

From CellBiology

Larissa's Individual Project

Overview

Scientists for eons have been studying the processes of cellular structure and development in order to understand the mechanisms of replication to facilitate possible medical treatments. in 1983 it was discovered that entry into the Mitotic phase of the cell cycle was reliant on new protein synthesis reactions. [1][2]

Through the study of sea urchins and clam embryo's two proteins with very distinctive features were identified, that is they showed patterns of degradation and accumulation. These patterns were periodic in nature whereby accumulation of proteins occurred throughout interphase rapidly degrading at the end of mitosis, signifying a control for the entry and exit into the mitotic phase of the cell cycle.[3][4].These proteins were named cyclins. Overtime suggestions that cyclins played a key role in inducing mitosis through their timed expression and effective interactions with their associated cyclin dependent kinases(CDKs) in the cell cycle were proved true. [5] It can be noted that in higher eukaryotes the control of the cell cycle is not only through a multitude of cyclins but also the proteins kinases attached to them(CDKs). This is due to the specificity of the cyclin/cdk complexes that form through different stages of the cycle. [6]. CDK2 and CDK4 for example are found primarily to associate with cyclins D and E to facilitate the progression of the cycle from the G1 phase to the S phase of the cell cycle, however CDK2 is also associated with cyclin A throughout the S phase of the cycle. Based on my research on the S-phase of the cell cycle, I have decided to use Cyclin E/cdk2 as my focal point for my individual project.

CYCLIN E/CDK2

Introduction

Cyclin E is found in a network of molecular proteins controlling the cell cycle. It is important for cell cycle progression and is frequently overexpressed in cancer cells.Cyclin E is associated with CDK2 and functions specifically to regulate the transition between the G1 phase and Synthesis phase of the cycle.There is extensive evidence to indicate that the activation of cyclin E/Cdk2 leads to the initiation of DNA replication. Activity of the cyclin has been found to be maximal during G1 phase.

Structure

Figure 1: 3D resprentation of a cyclinE/cdk2 complex. URL:http://www.ncbi.nlm.nih.gov/pubmed/15660127


The structure of cyclin E is very similar to the structure of cyclin A .Two five- hexagonal domains with additional N- and C- termini attached from the central point around which cyclin E is based. [7]. As mentioned there are 2 domains, the first is named the N-terminal cyclin box fold, and makes the N-terminal helix accompanied by 5 helices. The central portion of the third Helix is surrounded by four helixes and forms an organised hydrophobic core. Figure 1 illustrates the helical boundaries of the protein and its association with cdk2.





Actions of Cyclin E /cdk2 during the Cycle

The main action of cyclin E/cdk2 complex is to initiate cellular synthesis by enabling a transition from G1 phase of the cell cycle to S-phase of the cycle. The Cyclin E/cdk2 complex is formed when Cyclin E associates with cdk2 during early G1 phase. This complex is considered to be a central regulator of the G1/S transition as it initiates multiple cell cycle events, including DNA replication, centrosome duplication, and activation of the E2F transcriptional program.[8]Through phosphorylation of a limited number of targets including pRb, the cyclin E/cdk2 complex activity allows for the transition to S phase of the cycle.[9] On entry into S phase, cyclin E is abruptly destroyed by the proteasome to which it is targeted by umbiquin, and is therefore rendered inactive. Cyclin A is expressed in response to this and binds to the cyclin E/cdk2 complex.Association with cdk2 drives cells through S phase.


Expression of Cyclin E /cdk2

With reference to figure 2, it can be seen that CyclinE/cdk2 complex is expressed during the first half of the cell cycle. Beginning during early G1 phase, peaking at the start of the s-phase and then terminating expression between mid to late s-phase.The peak at the start of the S-phase signifies the phosphorylation of the complex with key substrates to initiate DNA synthesis.[10]

Expression of cyclin E and its binding with cdk2 will be suppressed in 2 situations. the first being a control mechanism in times of error during replication or to prevent a second cyclin from occurring before the completion of the first. in this instance levels of cyclin E/cdk2 will be low. the second situation is one where expression can be suppressed by Cyclin- dependent kinase inhibitors, which inhibit the action of cyclin E thereby rendering the cell static. The inhibitors not only suppress further progression in the s-phase of the cycle but may also affect the structure and function of the complex when a external substance such a oncogenic viral protein interact with the inhibitor. [11][12]
Figure 2: Expression of cyclin complexes during cell cycle.http://en.wikipedia.org/wiki/Cyclin_E

Regulation

Cyclin E levels are regulated throughout the cell cycle. This is monitored both transcriptional and proteolytic mechanism.[13]Due to the nature of the protein being one that is highly unstable and readily degradable- the primary mechanism for regulation of cyclin E levels is through transcription via umbiquitin-dependent pathways. [14] [15].Activity of the cyclin is regulated by phosphorylative processes.

Current Research

Resent studies on the interaction between various substances with cyclin E/cdk2 in the presence of CKIs have depicted possible explanations to the process of neoplasia, as well as various prognosis's for common cancers. [16]. Expression of Clyclin E in neoplasia has become a more pronounced and research has shown that though the cyclin functions to allow for normal cellular proliferation and development, over expression leads to an unstable acceleration of the G1 phase. Acceleration in the G1 phase and accumulation of cyclin E is reflective of gene amplification -often leading to mutation and developing cancer.[17] Knowledge of neoplasms and the process of neoplasia is largely due to investigations on cyclin E. Current research with cyclin E/cdk2 targets refining prognosis's and developing possible treatments for common cancer patients. [18]

Conclusion

The complex formed between cyclin E and cdk2 protein provides a control mechanism in the cell cycle by monitoring the progression from G1 phase to the s phase, thereby initiating DNA synthesis. Though the mechanism for specificity of this complex is unknown, cyclin E/cdk2 has initiated further research in cellular replication and cellular control, which has lead to increased interest and investigation in research involving neoplasia and cancer, in the hope that there will one day be a break through in medicative control as a result of better understanding of this complex compound.


Links

  • The cell cycle and checkpoint controls: towards understanding of genome maintenance mechanisms. [[10]]
  • Kinetic Model of cyclin E/ cdk2 developmental timer. [[11]]
  • Cell cycle: virtual laboratory [[12]]

References

  1. H.Lodish ,A.Berk ,L.Zipursky ,P. Matsudaira ,D. Baltimore ,J. D.W. H. Freeman and CompanyMolecular cell biology,2000
  2. Copper, G. M., & Hausman, R. E. (2006). The Cell: A Molecular Approach, 4th ed. 2006 ASM Press and Sinauer Associates, Inc.
  3. H.Lodish ,A.Berk ,L.Zipursky ,P. Matsudaira ,D. Baltimore ,J. D.W. H. Freeman and CompanyMolecular cell biology,2000
  4. J. Méndez,Cell Proliferation without Cyclin E-CDK2.Cell, Volume 114, Issue 4, Pages 398-399
  5. R.Honda1, E.Lowe1, E.Dubinina1, V.Skamnaki, A.Cook, N. Brown and L. Johnson. The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent rolesThe EMBO Journal (2005) 24, 452–463. [[1]]
  6. H.Lodish ,A.Berk ,L.Zipursky ,P. Matsudaira ,D. Baltimore ,J. D.W. H. Freeman and CompanyMolecular cell biology,2000
  7. R.Honda1, E.Lowe1, E.Dubinina1, V.Skamnaki, A.Cook, N. Brown and L. Johnson. The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent rolesThe EMBO Journal (2005) 24, 452–463. [[2]]
  8. Donellan and R Chetty. Cyclin E in human cancer. The FASEB Journal.(1999) Vol13, p773-780. [[3]]
  9. A Koff, A Giordano, D Desai, K Yamashita, JW Harper, S Elledge, T Nishimoto, DO Morgan, BR Franza, and JM Roberts. Formation and activation of a cyclin E-cdk2 complex during the G1 phase of the human cell cycle.Science, Vol 257, Issue 5077, 1689-1694
  10. L.Cam, J. Polanowska, E.Fabbrizio, M.Olivier, A.Philips, E .Eaton, M.Classon, Y.Geng, and C.Sardet. Timing of cyclin E gene expression depends on the regulated Association of a bipartite repressor element with a novel E2F complex.EMBO Journal April (1999)Vol 18(7)pp1878-1890[[4]]
  11. R. Donellan and R Chetty. Cyclin E in human cancer. The FASEB Journal.(1999) Vol13, p773-780. [[5]]
  12. X.Ye, Y.Wei, G.Nalepa, and J.HarperThe Cyclin E/Cdk2 Substrate p220NPAT Is Required for S-Phase Entry, Histone Gene Expression, and Cajal Body Maintenance in Human Somatic Cells.Molecular and Cellular biology. December 2003.Vol 23 p8586-8600
  13. Mark Jackman1*, Yumiko Kubota1, Nicole Den Elzen1, Anja Hagting1, and Jonathon Pines1Cyclin A and Cyclin E-Cdk complexes shuttle between the nucleus and the cytoplasm:2001[[6]]
  14. B.Urman, R.Sheaff, K.Thress, M.Groudine, and J.Roberts, (1996) Turnover of cyclin E by the ubiquitin-proteasome pathway is regulated by Cdk2 binding and cyclin phosphorylation, Genes Dev 10, 1979-90.
  15. W. Brensnahan, I Boldogh, T. Ma, T. Albrencht, E. Thompson.( October1996) Cyclin E/cdk2 activity is controlled by different mechanisms in the G0 and G1 phases of the cell cycle. Cell growth and differenciation.Vol(7) pg 1283-1290. [[7]]
  16. N.Delk ,K.Hunt, K.Keyomarsi,(2009). Altered Subcellular Localization of Tumor-Specific Cyclin E Isoforms Affects Cyclin-Dependent Kinase 2 Complex Formation and Proteasomal Regulation. Cancer Research. 69: 2817-2825
  17. R. Donellan and R Chetty. Cyclin E in human cancer. The FASEB Journal.(1999) Vol13, p773-780. [[8]]
  18. R.Honda1, E.Lowe1, E.Dubinina1, V.Skamnaki, A.Cook, N. Brown and L. Johnson. The structure of cyclin E1/CDK2: implications for CDK2 activation and CDK2-independent rolesThe EMBO Journal (2005) 24, 452–463. [[9]]



Lecture Feedback

Lecture 4 - Nucleus

I found the nuclear lamins in the cytoskeleton of the nucleus to be very interesting. Their interaction with the nuclear envelope in terms of attachment of DNA to the nuclear envelope and its ability to affect the assembly state of the envelope due to its phosphorylation state in particular, provided a better understanding of the internal nuclear structures and relations and interaction of nuclear bodies therein.

Lecture 5 - Exocytosis

The lecture on exocytosis was not a difficult lecture to understand in that it was well set out, however the concept of secretory vesicles required a little more attention due to its relation with membrane turnover.

Lecture 7 - Mitochondria

  • respiration
  • growth
  • cell division and growth-mitosis and meiosis
  • cell signalling
  • most cellular procedures require energy with is generated by the mitochondria they are the cellular powerplants in an organism

Lecture 8 -Cellular junction

Cellular junctions are areas where cell adhesion takes place the following are examples of cellular proteins that are adhesive molecules which are located on the cellular surface and bind the the extecellular matrix of a cell. 1. L-CAM = liver cell adhesion 2. I-CAM = intracellular adhesion molecule 3. Ng-CAM = neuron-glia adhesion molecule 4. V-CAM = vascular cell adhesion molecule


lecture 10- intermediate filaments

Stratum spinosum is the layer between the basal and granulosa layer. they contain adhesive structures known as Desmosomes which are located in epitheliod cells-ie. simple and stratified squamous epithelia


===Lab 6 ===-

Lecture 14- Confocal Microscopy

  • laser
  • spinning disc

Lecture 15 - Cell Cycle

S-phase- is an abbreviated term for the "synthesis"-phase