How does rb function

Mooney, L. Jia, R. Raptis, and A. Yoshio, T. Morita, Y. Kimura, M. Tsujii, N. Hayashi, and K. Muller, K. Vousden, and J. Powell, D. Piwnica-Worms, and H. Araki, T. Ebata, A.

Guo, K. Tobiume, S. Wolf, and K. Xia and H. Gadea, M. Anguille, and P. Lefort, A. Mandinova, P. Ostano et al. Gao, L. Wang, and Y. Lapasset, C. Croft, D. Crighton, M. Samuel et al. Ongusaha, H.

Boswell et al. Hall, W. Lu, J. Godfrey et al. Bennett and A. View at: Google Scholar V. Bennett and J. View at: Google Scholar P.

Stabach, I. Ranieri et al. Harris, A. Daeden, and G. Lecuit and A. Ladoux, W. Nelson, J. Yan, and R. Berx and F. Siemens, R. Jackstadt, S. Chang, C. Chao, W. Xia et al. Kim, A. Veronese, F. Pichiorri et al.

Wang, W. Wang, Y. Chang et al. Bae, S. An et al. Muller and K. Muller, P. Caswell, B. Doyle et al. Arjonen, R. Kaukonen, E. Mattila et al. Iwanicki, H. Chen, C. Iavarone et al. Lee, J. Ahn, T. Kim, J. Lee, and J. Li and C. Zhang, W. Yan, and X. Kogan-Sakin, Y. Tabach, Y. Buganim et al. View at: Google Scholar G.

Bossi, F. Marampon, R. Maor-Aloni et al. Engel, E. Welsh, M. Emmons, P. Santiago-Cardona, and W. Barczyk, S. Carracedo, and D. Sikkema, A. Strikwerda, M. Sharma et al. Filipenko, S. Attwell, C. Roskelley, and S. Fielding, I. Dobreva, and S. Fielding, S. Lim, K. Montgomery, I. Kwon, S. Godinho, N. Chandhok et al. Araki, K. Kawauchi, H. Hirata, M. Yamamoto, and Y. Mammoto, S. Huang, K. Oh, and D. Gunduz, V. Vazquez-Rivera et al.

Fehon, A. McClatchey, and A. Tomita, A. Van Bokhoven, G. Van Leenders et al. Nakajima, A. Patino-Garcia, S. Bruheim et al. View at: Google Scholar D.

Tamura, T. It is also true that the mutations seen in human cancers do not extend to all members of the families of proteins operating in the pathway. For instance, although mutation of the Rb gene is frequent, mutations in other members of the Rb family, which includes the p and p genes, are not common events in human cancers. Why this might be the case is not clear, although it does appear that Rb plays a more widespread role in the control of E2F activity than the other Rb family members Nevertheless, although mutations involving p have not been described, one study has identified loss of function mutations within the p gene in small cell lung carcinoma cell lines Perhaps most striking is the general lack of mutations or chromosomal alterations in members of the E2F family.

Although homozygous deletion of E2F1 in the mouse does lead to increased tumor formation in several tissues 37 , possibly linked to a role for E2F1 as a signal for apoptosis, a link between E2F1 and human cancer has not been established. Likewise, mutations or alteration that would lead to loss of expression or deregulated expression of other E2F proteins have not been clearly established in human tumors.

Thus, even though E2F3 appears to play a particularly important role in the control of cellular proliferation 13 , 55 and E2F4 appears to play a widespread role in determining cellular differentiation 56 , direct alteration of these genes does not appear to contribute significantly to the development of human cancer. Alterations in the E2F4 gene involving changes in the trinucleotide repeat element have been observed, but there is no evidence as yet that these alterations have an impact on E2F4 function or that they contribute to the cancer phenotype.

The potential explanations for the apparent lack of direct alterations of E2F genes in cancer are numerous but perhaps the most likely reason is the overlap in function that is evident within the family, as well as the potential disruptive role of deregulated expression, which might not allow a productive event. As this understanding continues to grow, one hopes that insights will offer new clues to therapeutic approaches to treat cancer.

In this regard, strategies focused on the control of E2F proteins hold particular promise, since activation of E2F activity is the ultimate consequence of deregulation of the Rb pathway, irrespective of the nature of the mutation. In this sense, targeting E2Fs could be a more generic approach. The problem, of course, is that the activation of E2Fs is part of the normal process of cell growth and thus any attempt to control E2F activity will have a negative impact on normal tissue that has a proliferating component.

In that sense, therapeutics focused on E2F activities would not be particularly different from drugs that inhibit DNA replication activities. Moreover, as discussed above, most cancers do not involve direct alterations of the E2F genes and thus, the elevated levels of E2F activities present in tumors represent normal proteins.

The more attractive possibility is to take advantage of small alterations in the normal balance of events—those changes that are not typical of the normal cell.

Although logical, these approaches are nevertheless unlikely to be effective as efficient mechanisms to stop or kill tumors. Rather, the development of small molecules that have specificity for key components of the pathway, which may play particularly important roles in deregulated events of cancer, is more likely to be successful.

More specifically, Hartwell et al. Figure 2. Figure 3. Natl Acad. USA , 68 , — Nature , , — Cell , 65 , — Cell , 64 , — USA , 92 , — Cell , 79 , — Cell Growth Differ. Genes Dev. Cell , 98 , — USA , 88 , — USA , 91 , — Nature Genet. Cell , 85 , — Cell , 83 , — Although the complete structure of Rb has yet to be solved, several groups have recently solved the core structure as an intra-strand pseudo-dimer of dimers with the Cdk sites present on loops between structured regions or on the C-terminus abutting the B-box Burke et al.

Based on the binding preferences of individual Rb mono-phosphorylated isoforms to specific E2F transcription factors, our data suggest that the generation of 14 mono-phosphorylated Rb isoforms may serve as a mechanism to post-translationally functionally diversify Rb in early G 1 phase from a single un-phosphorylated isoform in G 0.

In addition to E2F transcription factors, Rb has also been shown to bind to over additional cellular proteins Morris and Dyson, , leaving open the potential for differential binding preferences of mono-phosphorylated Rb to specific cellular targets. Excluding hyper-phosphorylated Rb, we were surprised at the complete absence of any multi-phosphorylated Rb isoforms.

While this will require extensive structural analyses beyond the scope of our study, we speculate that the substrate recognition of Rb by cyclin D's N-terminal LxCxE motif weak binding to Rb's pocket domain Dowdy et al. This also allows for a simultaneous switch-like inactivation of all 14 mono-phosphorylated Rb isoforms by one processive hyper-phosphorylation mechanism.

Our study addresses several critical problems arising from numerous biochemical analyses of Rb phosphorylation going back more than 20 years. By constitutively mono-phosphorylating Rb, the nascent neoplastic cell avoids cell cycle exit and differentiation mediated by un-phosphorylated Rb, and also maintains a high level of metabolism. This notion is entirely consistent with the observed subtle and highly tolerated cancer predisposing mutations of p16 deletion and cyclin D overexpression in mouse models that avoid activation of oncogene-induced apoptosis Burkhart and Sage, The net effect is a subtle, but irreversible, oncogenic step forward.

Once the metabolic threshold has been exceeded, the sensor activates cyclin E:Cdk2 resulting in Rb inactivation by hyper-phosphorylation, induction of E2F target gene transcription and progression across the Restriction Point into late G 1 phase Haberichter et al. We are currently investigating the mechanics of this putative mechanism and the identity of the metabolic sensor. We predict that additional oncogenic and metabolic pathways ultimately converge on and activate cyclin E:Cdk2 complexes to inactivate Rb by hyper-phosphorylation at the Restriction Point and drive cells into late G 1 phase.

Louis, MO or exposure to 20 Grays of ionizing radiation. U2OS-p16 cells Jiang et al. Co-immunoprecipitations were performed as described Ezhevsky et al. Immunoblotting was performed as described Ezhevsky et al. Immunoprecipitation-kinase assays were performed as described Ezhevsky et al.

Mean values of triplicate samples were normalized to betamicroglobulin. Whole-genome microarray analysis was performed as described Eguchi et al. Heat maps were created with Cluster 3. An edited version of the letter sent to the authors after peer review is shown, indicating the substantive concerns or comments; minor concerns are not usually shown. Reviewers have the opportunity to discuss the decision before the letter is sent see review process.

Similarly, the author response typically shows only responses to the major concerns raised by the reviewers. Your article has been favorably evaluated by Tony Hunter Senior editor , a Reviewing editor, and 3 reviewers. The Reviewing editor and the other reviewers discussed their comments before we reached this decision, and the Reviewing editor has assembled the following comments to help you prepare a revised submission.

Narasimha et al. Finally they present data that suggests that un-phosphorylated pRb drives cell cycle exit G0 entry or prevents G1 entry. The experiments have been performed with care, but some of the conclusions drawn are not warranted by the data.

Given the extensive scope of the current study, the reviewers believe that the remaining issues can be adequately addressed by changes to the text of the manuscript without the need for further experimentation. The inability to phosphorylate pRb in response to DNA damage results in activation of E2F-dependent transcription and genomic instability.

They state that line the appearance of tetraploid cells indicates that the cells 'failed to activate the DNA damage checkpoint'. This is neither tested nor likely. The reviewers recommend that the text of the manuscript should be revised to note these points. Can the authors elaborate on this point? Although it is beyond the scope of the paper to test alternative models for E2F-dependent transcriptional activation, the reviewers believe that this topic should be included in the discussion section of the manuscript.

We completely concur that the DNA damage response is intact. We have clarified the text accordingly. This specific comment was meant to reinforce that the rather subtle differences observed between E2F2 and E2F3 binding to specific mono-phosphorylated Rb isoforms was consistently observed in three independent experiments, whereas E2F1 and E2F4 showed much larger binding differences. Once the metabolic threshold has been exceeded, the sensor activates cyclin E:Cdk2 resulting in Rb inactivation by hyper-phosphorylation, induction of E2F target gene transcription and progression across the Restriction Point into late G 1 phase.

We have expanded the Discussion section to include our thoughts on cyclin E:Cdk2 activation. We also assert that there is no progressive multi-phosphorylating, hypo-phosphorylation of Rb.

We believe that both the depth of analyses and the magnitude of impact on understanding G 1 cell cycle progression warrants publication in eLife. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Iowa for constructs, antibodies and cell lines. This article is distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use and redistribution provided that the original author and source are credited.

Cancer Res 56 : — Genome 3 : Cancer Res 60 : 8— Cobrinik D. Oncogene 24 : — Genes dev 7 : — Condorelli G, Giordano A. J Cell Biochem 67 : — Mol Cell 8 : — Oncogene 22 : — Oncogene , review in this issue. J Biol Chem : — J Cell Sci : — Dimova DK, Dyson N. Oncogene 44 : 30— Trends Biol Sci 16 : — EMBO J 22 : — Cell 73 : — Science : — Dyson N.

Curr Opin Cell Biol 14 : — Genes Dev 12 : — Oncogene 21 : — Gallo G, Giordano A. J Cell Phys 2 : — EMBO J 23 : — Mol Cell 6 : — Cell : 79— Mol Cell Biol 13 : — Mol Cell Biol 16 : — Hitomi M, Stacey DW. Curr Biol 19 : — Cancer Res 1 : — Hunter T, Pines J. Cell 79 : — Gene : 37— Oncogene 49 : — EMBO J 14 : — Front Biosci 3 : D—D Mol Cell Biol 24 : — Oncogene 11 : — Kim YT, Zhao M.

Yonsei Med J 31 : — Knudson AG. Mol Cell Biol 19 : — Hum Pathol 33 : — Nature : — Cancer Cell 2 : — Genes Dev 16 : — Mol Cell Biol 2 : — Curr Opin Genet Dev 14 : 55— Eur J Cancer 35 : —