Distinct action of the retinoblastoma pathway on the DNA replication machinery defines specific roles for cyclin-dependent kinase complexes in prereplication complex assembly and S-phase progression
Authors: Jeanette G Cook
Jon M Lenihan
Emily Bosco
William Zagorski
Erik S Knudsen
Zhengdao Lan
Wesley A Braden
Michael F Reed
K Scott Luce
Journal: Molecular and Cellular Biology (2006)
Discipline: MOLECULAR BIOLOGY & GENETICS
Keywords: retinoblastoma; p16ink4a; tumor suppressors; human cancers; S phase
Affiliations: Department of Cell Biology, Vontz Center for Molecular Studies, 3125 Eden Avenue, Cincinnati, OH 45267-0521, USA., Department of Cell Biology and UC Cancer Center, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267, Department of Medicine, Johns Hopkins University, Baltimore, Maryland 21287, Department of Biochemistry and Biophysics, University of North Carolina, Chapel Hill, North Carolina 27599, Department of Surgery, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267

Abstract:  The retinoblastoma (RB) and p16ink4a tumor suppressors are believed to function in a linear pathway that is functionally inactivated in a large fraction of human cancers. Recent studies have shown that RB plays a critical role in regulating S phase as a means for suppressing aberrant proliferation and controlling genome stability. Here, we demonstrate a novel role for p16ink4a in replication control that is distinct from that of RB. Specifically, p16ink4a disrupts prereplication complex assembly by inhibiting mini-chromosome maintenance (MCM) protein loading in G1, while RB was found to disrupt replication in S phase through attenuation of PCNA function. This influence of p16ink4a on the prereplication complex was dependent on the presence of RB and the downregulation of cyclin-dependent kinase (CDK) activity. Strikingly, the inhibition of CDK2 activity was not sufficient to prevent the loading of MCM proteins onto chromatin, which supports a model wherein the composite action of multiple G1 CDK complexes regulates prereplication complex assembly. Additionally, p16ink4a attenuated the levels of the assembly factors Cdt1 and Cdc6. The enforced expression of these two licensing factors was sufficient to restore the assembly of the prereplication complex yet failed to promote S-phase progression due to the continued absence of PCNA function. Combined, these data reveal that RB and p16ink4a function through distinct pathways to inhibit the replication machinery and provide evidence that stepwise regulation of CDK activity interfaces with the replication machinery at two discrete execution points. 

Retracted: Yes

PubPeer comments:
 Figures 1F and 1G. Much more similar than you would expect. https: imgur.com 2dSIFPu , 
 Right set of panels figure 6A. Much more similar than you would expect. https: imgur.com Wu5A2wH , 
 Figure 1C. Much more similar than you would expect. https: imgur.com FSOxqRS , 
 Figures 1C and 1E. Much more similar than you would expect. https: imgur.com 4cU82WY , 
 Figures 8B and 2F much more similar than you would expect. https: imgur.com Z8FlC5u , 
 This is horrible....I have contacted the journal. , 
 Figure 6C. Much more similar than expected. https: imgur.com KELQ3Oy , 
 #3 Mol Cell Biol. 2006 Oct;26(20):7667-81 figure 1C. There appears to be a transduction with GFP in the left lane. Legend. (C) A2-4 cells cultured in the presence of DOX were transduced with GFP- or p16ink4a (p16)-encoding adenoviruses and harvested at 24 h postinfection. Left panel: cells were harvested, equal total protein amounts were resolved by SDS-PAGE, and the indicated proteins were detected by immunoblotting. Right panel: cells were harvested, fixed, stained with propidium iodide, and processed for flow cytometry. Histograms are from 10,000 events. Mol Cell Biol. 2004 Jun;24(12):5404-20. Legend. Figure 4D. (D, left panel) Asynchronously proliferating Rat-1 cells were infected with recombinant adenovirus expressing p16ink4a. Cells were harvested at 0 (lane 1) or 24 (lane 2) h postinfection. Equal total protein levels were resolved by SDS-PAGE, and the indicated proteins were detected by immunoblotting. The image is of the left panel. There does not seem to be any transduction with GFP. The right panel does refer to GFP. (Right panel) Rat-1 cells were infected with GFP- or p16ink4a-encoding adenoviruses and subjected to in situ extraction with CSK buffer at 24 h postinfection. Cells were subsequently fixed, and PCNA was detected by immunofluorescence. The values shown are means u00b1 standard deviation. At least 200 cells were counted per experiment. , 
 All your points are correct and sorry for not directly responding. Thank you for pointing out the myriad of issues with this paper! I have contacted MCB, I am not the Editor for the journal, but I am completely embarrassed to have this come from my laboratory...so I know what my preference is. Thank you, Erik , 
 2020 retraction. https: mcb.asm.org content 40 17 e00319-20 We retract this article due to image duplication and manipulation. We apologize to the scientific community and readers of Molecular and Cellular Biology and regret any inconvenience that this causes. ,