User:Iris To/Retinoblastoma Protein Regulation

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Retinoblastoma Protein

3n5u, resolution 3.20Å ()
Ligands: ,
Gene: PPP1A, PPP1CA (Homo sapiens)
Activity: Phosphoprotein phosphatase, with EC number 3.1.3.16


Resources: FirstGlance, OCA, RCSB, PDBsum
Coordinates: save as pdb, mmCIF, xml


The retinoblastoma protein (Rb) is a suppressor protein, also known as a tumor suppressor, involved in negative regulation of the cell cycle[1] due to its ability to bind to transcription factors like E2F[2]. It acts as a cell cycle checkpoint during the G1 phase, determining if the cell cycle should continue or stop. In its normal state, Rb is activated, which prevents the cell cycle to continue because it can recruit transcriptional co-repressors, blocking transcription[1]. Rb is deactivated after being phosphorylated by Cyclin-dependent kinases (Cdks)[3], and thus cannot associate factors that inhibit transcription factors that allow the cell cycle to continue. Normally, once the cell cycle is complete, Rb is activated once more by being dephosphorylated by phosphoprotein phosphatases (PP1)[4] to allow mitotic exit. Unregulated deactivation of Rb can lead to uncontrolled growth of cells, which is why studying this protein is important in cancer research.

Regulation of Rb

From previous studies, it has been determined that the phosphorylation state and activity of Rb are controlled by a balance of kinase and phosphatase activity, in which Cyclin/Cdks phosphorylate Rb from late G1 to mitosis and PP1 dephosphorylates Rb for mitotic exit[2]. An enzyme-docking site for PP1c (the catalytic subunit of PP1) was determined to overlap with the docking site for Cyclin/Cdks, more specifically the RxL Cyclin binding site[3] that brings Cyclin/Cdks to Rb, through a crystal structure; this crystal structure initiated studies on phosphatase and kinase competition for the docking site.

It has been found that PP1c directly inhibits phosphorylation of RbC from Cdk2-CycA[5]; it is not affected by phosphatase activity but by the presence of the KLRF docking site[6], which assists in binding the PP1 to Rb[2]. In addition, it has been observed that PP1c makes complexes with RbC when there are many phosphatases produced, thus overthrowing Cyclin/Cdk activity and inhibiting cell progression from the G1 phase. This competition is important in terms of cell signaling, which is affected by response to cellular stress, cell cycle exit, DNA damage, etc[2]. This is because the competition further controls cell overgrowth besides phosphorylation regulation. Studies determined a biochemical mechanism where directly competing kinases and phosphatase activity regulates Rb phosphorylation and activity, but there has not been an established study that defined a mechanism that controlled the outcome of the competition between each enzyme[2].

Relevance

While there are other characteristics of cancer cells, such as cell dispersal and apoptosis resistance, the foundation of cell proliferation is at the cell cycle[4]. Cyclin/Cdk complexes phosphorylate Rb to inactivate the Rb repressor function in the G1 phase, which if continually in that state, will cause unregulated cell proliferation. In order to regulate this, PP1 must dephosphorylate Rb and reactivate the suppressor function of Rb. PP1 and its dephosphorylation effects are targeted for anticancer drugs because it forms complexes with other proteins, which if released from the complex can lead to higher amounts of dephosporylation thus deactivating signal transductor proteins, transcription factors, etc. During anaphase and the G1 phase, it has been found that PP1 complexes have regulatory proteins, PNUTS, which during hypoxic conditions or presence of chemotherapeutics, would dissociate from PP1 to activate PP1 to Rb; this causes early dephosphorylation of Rb[5]. PP1 also complexes with enzyme, HDAC, where as a complex prevents PP1 from binding to Akt/protein kinase B, a signal transductor protein, and transcription factor cAMP response protein, CREB. Anticancer drugs target the removal of HDAC from PP1 in order to deactivate these proteins, preventing the continuation of cell proliferation[6]. With these findings, it is obvious that the inactivation and deactivation of Rb is crucial in the control of cell growth and proliferation. It is even more intriguing that the docking sites for PP1 and Cyclin/Cdk complexes overlap because the competition for the site determines the activity of Rb, which may or may not be good for the organism. Studying this competition in overlapping docking sites is important in cancer research because it can give a better understanding of how Rb can be manipulated if there is an error in regulation, which would lead to unwanted cell growth.


Structure of Rb-PP1c(PDB entry 3n5u)


3D Structures

1o9k with E2F
1h25 with Cdk2/Cyclin A


References

  1. Lee C, Chang JH, Lee HS, Cho Y. Structural basis for the recognition of the E2F transactivation domain by the retinoblastoma tumor suppressor. Genes Dev. 2002 Dec 15;16(24):3199-212. PMID:12502741 doi:10.1101/gad.1046102
  2. 2.0 2.1 2.2 2.3 2.4 2.5 Hirschi A, Cecchini M, Steinhardt RC, Schamber MR, Dick FA, Rubin SM. An overlapping kinase and phosphatase docking site regulates activity of the retinoblastoma protein. Nat Struct Mol Biol. 2010 Sep;17(9):1051-7. Epub 2010 Aug 8. PMID:20694007 doi:10.1038/nsmb.1868
  3. Lowe ED, Tews I, Cheng KY, Brown NR, Gul S, Noble ME, Gamblin SJ, Johnson LN. Specificity determinants of recruitment peptides bound to phospho-CDK2/cyclin A. Biochemistry. 2002 Dec 31;41(52):15625-34. PMID:12501191
  4. http://www.ias.ac.in/currsci/sep102001/515.pdf
  5. Kinkade, Rebecca. "Rb-Raf-1 Interaction as a Therapeutic Target for Proliferative Disorders." Diss. University of South Florida, 2008. Web.
  6. Airley, Rachel. Cancer Chemotherapy. Chichester, UK: Wiley-Blackwell, 2009. Print.
  7. Terrak, Mohammed, Kerff, Frederic, Langsetmo, Knut, Tao, Terence, & Dominguez, Roberto Structural basis of protein phosphatase 1 regulation. Nature, 429, 780–784 (17 June 2004); 10.1038/nature02582
  8. Wakula, P., Beullens, M., Ceulemans, H., Stalmans, W., and Bollen, M. (2003) J. Biol. Chem. 278, 18817–18823

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