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The Evolutionarily Conserved C-terminal Domains in the Mammalian Retinoblastoma Tumor Suppressor Family Serve as Dual Regulators of Protein Stability and Transcriptional Potency*

  • Author Footnotes
    1 Supported in part by fellowships from the College of Natural Science at Michigan State University.
    Satyaki Sengupta
    Footnotes
    1 Supported in part by fellowships from the College of Natural Science at Michigan State University.
    Affiliations
    From the Department of Biochemistry and Molecular Biology

    Graduate Program in Physiology, and
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  • Raj Lingnurkar
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    From the Department of Biochemistry and Molecular Biology
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  • Timothy S. Carey
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    From the Department of Biochemistry and Molecular Biology
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  • Author Footnotes
    2 Supported in part by an undergraduate research award from the Lyman Briggs College at Michigan State University.
    Monica Pomaville
    Footnotes
    2 Supported in part by an undergraduate research award from the Lyman Briggs College at Michigan State University.
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    From the Department of Biochemistry and Molecular Biology
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  • Parimal Kar
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    From the Department of Biochemistry and Molecular Biology
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  • Michael Feig
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    From the Department of Biochemistry and Molecular Biology
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  • Catherine A. Wilson
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    Department of Animal Science, Michigan State University, East Lansing, Michigan 48824
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  • Jason G. Knott
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    From the Department of Biochemistry and Molecular Biology

    Department of Animal Science, Michigan State University, East Lansing, Michigan 48824
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  • David N. Arnosti
    Correspondence
    To whom correspondence may be addressed: Dept. of Biochemistry and Molecular Biology, Michigan State University, 603 Wilson Rd., East Lansing, MI 48824. Tel.: 517-353-3980; Fax: 517-353-9334
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    From the Department of Biochemistry and Molecular Biology
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  • R. William Henry
    Correspondence
    To whom correspondence may be addressed: Dept. of Biochemistry and Molecular Biology, Michigan State University, 603 Wilson Rd., East Lansing, MI 48824. Tel.: 517-353-3980; Fax: 517-353-9334.
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    From the Department of Biochemistry and Molecular Biology
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  • Author Footnotes
    * This work was supported, in whole or in part, by National Institutes of Health Grants R01GM095347 (to J. G. K.) and R01GM079098 (to D. N. A. and R. W. H.).
    1 Supported in part by fellowships from the College of Natural Science at Michigan State University.
    2 Supported in part by an undergraduate research award from the Lyman Briggs College at Michigan State University.
Open AccessPublished:April 22, 2015DOI:https://doi.org/10.1074/jbc.M114.599993
      The retinoblastoma (RB) tumor suppressor and related family of proteins play critical roles in development through their regulation of genes involved in cell fate. Multiple regulatory pathways impact RB function, including the ubiquitin-proteasome system with deregulated RB destruction frequently associated with pathogenesis. With the current study we explored the mechanisms connecting proteasome-mediated turnover of the RB family to the regulation of repressor activity. We find that steady state levels of all RB family members, RB, p107, and p130, were diminished during embryonic stem cell differentiation concomitant with their target gene acquisition. Proteasome-dependent turnover of the RB family is mediated by distinct and autonomously acting instability elements (IE) located in their C-terminal regulatory domains in a process that is sensitive to cyclin-dependent kinase (CDK4) perturbation. The IE regions include motifs that contribute to E2F-DP transcription factor interaction, and consistently, p107 and p130 repressor potency was reduced by IE deletion. The juxtaposition of degron sequences and E2F interaction motifs appears to be a conserved feature across the RB family, suggesting the potential for repressor ubiquitination and specific target gene regulation. These findings establish a mechanistic link between regulation of RB family repressor potency and the ubiquitin-proteasome system.
      Background: RB family protein abundance is dynamic and sensitive to growth conditions.
      Results: RB family turnover is mediated by C-terminal degrons in a process that is phosphorylation-sensitive.
      Conclusion: The RB family degrons are important regulatory domains linking stability and transcriptional potency.
      Significance: Dual control of stability and potency by RB family degrons may contribute to embryonic development.

      Introduction

      The retinoblastoma (RB)
      The abbreviations used are: RB
      retinoblastoma
      CDK
      cyclin-dependent kinase
      ES
      embryonic stem
      IE
      instability element
      LIF
      leukemia inhibitory factor
      TKO
      triple knock out
      RA
      retinoic acid.
      tumor suppressor regulates cell fate through its governance of distinct gene sets that promote cell division, differentiation, and apoptosis (
      • Burkhart D.L.
      • Sage J.
      Cellular mechanisms of tumour suppression by the retinoblastoma gene.
      ,
      • Dick F.A.
      • Rubin S.M.
      Molecular mechanisms underlying RB protein function.
      ). RB is related to two other family members, p107 and p130, that share substantial structural conservation along with some overlapping function in target gene regulation (
      • Hurford Jr., R.K.
      • Cobrinik D.
      • Lee M.H.
      • Dyson N.
      pRB and p107/p130 are required for the regulated expression of different sets of E2F responsive genes.
      • Black E.P.
      • Huang E.
      • Dressman H.
      • Rempel R.
      • Laakso N.
      • Asa S.L.
      • Ishida S.
      • West M.
      • Nevins J.R.
      Distinct gene expression phenotypes of cells lacking Rb and Rb family members.
      ,
      • Takahashi Y.
      • Rayman J.B.
      • Dynlacht B.D.
      Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression.
      • Chicas A.
      • Wang X.
      • Zhang C.
      • McCurrach M.
      • Zhao Z.
      • Mert O.
      • Dickins R.A.
      • Narita M.
      • Zhang M.
      • Lowe S.W.
      Dissecting the unique role of the retinoblastoma tumor suppressor during cellular senescence.
      ). Although not as tightly linked to tumor suppression as RB, tumor suppressive capacity has been assigned to these other family members in some contexts. In mouse studies, RB-deficient mice were prone to pituitary tumor formation (
      • Jacks T.
      • Fazeli A.
      • Schmitt E.M.
      • Bronson R.T.
      • Goodell M.A.
      • Weinberg R.A.
      Effects of an Rb mutation in the mouse.
      ), whereas mice deficient for RB and p107 or p130 developed retinoblastoma (
      • Ajioka I.
      • Martins R.A.
      • Bayazitov I.T.
      • Donovan S.
      • Johnson D.A.
      • Frase S.
      • Cicero S.A.
      • Boyd K.
      • Zakharenko S.S.
      • Dyer M.A.
      Differentiated horizontal interneurons clonally expand to form metastatic retinoblastoma in mice.
      ,
      • MacPherson D.
      • Conkrite K.
      • Tam M.
      • Mukai S.
      • Mu D.
      • Jacks T.
      Murine bilateral retinoblastoma exhibiting rapid-onset, metastatic progression and N-myc gene amplification.
      ), suggesting that p107 and p130 can influence tissue specific predisposition toward tumor development. Similarly, conditional loss of p130 in adult lung epithelial cells in a RB−/−/p53−/− null background enhanced development of small cell lung carcinoma (
      • Schaffer B.E.
      • Park K.S.
      • Yiu G.
      • Conklin J.F.
      • Lin C.
      • Burkhart D.L.
      • Karnezis A.N.
      • Sweet-Cordero E.A.
      • Sage J.
      Loss of p130 accelerates tumor development in a mouse model for human small-cell lung carcinoma.
      ). Thus, all three RB family members can act as tumor suppressors in specific contexts.
      In their roles as tumor suppressors, RB family members predominately function as transcriptional repressors of target genes through their antagonism of the E2F-DP family of transcription factors (
      • Dyson N.
      The regulation of E2F by pRB-family proteins.
      ,
      • Trimarchi J.M.
      • Lees J.A.
      Sibling rivalry in the E2F family.
      ). Recent evidence suggests that RB also plays a positive role in transcriptional activation of some pro-apoptotic response genes, again in a mechanism requiring E2F activity (
      • Ianari A.
      • Natale T.
      • Calo E.
      • Ferretti E.
      • Alesse E.
      • Screpanti I.
      • Haigis K.
      • Gulino A.
      • Lees J.A.
      Proapoptotic function of the retinoblastoma tumor suppressor protein.
      ), although RB may also induce apoptosis through a mechanism that is independent of transcription (
      • Hilgendorf K.I.
      • Leshchiner E.S.
      • Nedelcu S.
      • Maynard M.A.
      • Calo E.
      • Ianari A.
      • Walensky L.D.
      • Lees J.A.
      The retinoblastoma protein induces apoptosis directly at the mitochondria.
      ). In this scenario, RB-mediated tumor suppression is enabled through blockade of gene products necessary for cell growth with concomitant invocation of cell death pathways. As key regulators of cell fate, the RB family is tightly controlled by CDK-mediated phosphorylation in response to environmental conditions (
      • Buchkovich K.
      • Duffy L.A.
      • Harlow E.
      The retinoblastoma protein is phosphorylated during specific phases of the cell cycle.
      ,
      • Chen P.L.
      • Scully P.
      • Shew J.Y.
      • Wang J.Y.
      • Lee W.H.
      Phosphorylation of the retinoblastoma gene product is modulated during the cell cycle and cellular differentiation.
      • DeCaprio J.A.
      • Ludlow J.W.
      • Lynch D.
      • Furukawa Y.
      • Griffin J.
      • Piwnica-Worms H.
      • Huang C.M.
      • Livingston D.M.
      The product of the retinoblastoma susceptibility gene has properties of a cell cycle regulatory element.
      ). Hypo-phosphorylated RB interacts with E2F1-DP1 (
      • Chellappan S.P.
      • Hiebert S.
      • Mudryj M.
      • Horowitz J.M.
      • Nevins J.R.
      The E2F transcription factor is a cellular target for the RB protein.
      ), blocking activated transcription of cell cycle genes involved in DNA replication and S-phase progression (
      • Dyson N.
      The regulation of E2F by pRB-family proteins.
      ,
      • Trimarchi J.M.
      • Lees J.A.
      Sibling rivalry in the E2F family.
      ). In response to mitogenic signals, serial phosphorylation via cyclin D-CDK 4/6 and cyclin E-CDK2 renders RB family members inactive by disengaging their association with E2F complexes (
      • Hinds P.W.
      • Mittnacht S.
      • Dulic V.
      • Arnold A.
      • Reed S.I.
      • Weinberg R.A.
      Regulation of retinoblastoma protein functions by ectopic expression of human cyclins.
      • Lundberg A.S.
      • Weinberg R.A.
      Functional inactivation of the retinoblastoma protein requires sequential modification by at least two distinct cyclin-cdk complexes.
      ,
      • Narasimha A.M.
      • Kaulich M.
      • Shapiro G.S.
      • Choi Y.J.
      • Sicinski P.
      • Dowdy S.F.
      Cyclin D activates the Rb tumor suppressor by mono-phosphorylation.
      • Weinberg R.A.
      The retinoblastoma protein and cell cycle control.
      ). Cyclin-CDK activity is also critically regulated during early steps in normal embryonic development (
      • White J.
      • Dalton S.
      Cell cycle control of embryonic stem cells.
      ). Rapidly dividing pluripotent embryonic stem (ES) cells of the early developing embryo employ constitutive CDK-mediated inhibition of RB proteins as a mechanism to maintain rapid cell division during blastocyst formation (
      • Savatier P.
      • Huang S.
      • Szekely L.
      • Wiman K.G.
      • Samarut J.
      Contrasting patterns of retinoblastoma protein expression in mouse embryonic stem cells and embryonic fibroblasts.
      ,
      • Stead E.
      • White J.
      • Faast R.
      • Conn S.
      • Goldstone S.
      • Rathjen J.
      • Dhingra U.
      • Rathjen P.
      • Walker D.
      • Dalton S.
      Pluripotent cell division cycles are driven by ectopic Cdk2, cyclin A/E, and E2F activities.
      • White J.
      • Stead E.
      • Faast R.
      • Conn S.
      • Cartwright P.
      • Dalton S.
      Developmental activation of the Rb-E2F pathway and establishment of cell cycle-regulated cyclin-dependent kinase activity during embryonic stem cell differentiation.
      ). As ES cells differentiate, CDK activity plummets, allowing RB family proteins to regulate E2F activity in a cell cycle-dependent manner (
      • White J.
      • Stead E.
      • Faast R.
      • Conn S.
      • Cartwright P.
      • Dalton S.
      Developmental activation of the Rb-E2F pathway and establishment of cell cycle-regulated cyclin-dependent kinase activity during embryonic stem cell differentiation.
      ,
      • Sage J.
      The retinoblastoma tumor suppressor and stem cell biology.
      ). Despite this unifying model for cyclin-CDK regulation, there are significant differences in the coordination of RB family member activities and steady state levels. For example, RB and p107 are active in cycling cells, whereas p130 functions predominately in quiescent cells that have exited from the cell cycle. Experiments performed with staged cells showed that steady state p130 levels peak in G0, in contrast to RB and p107, which increase as cells progress through G1 (
      • Bhattacharya S.
      • Garriga J.
      • Calbó J.
      • Yong T.
      • Haines D.S.
      • Graña X.
      SKP2 associates with p130 and accelerates p130 ubiquitylation and degradation in human cells.
      ,
      • Tedesco D.
      • Lukas J.
      • Reed S.I.
      The pRb-related protein p130 is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCF(Skp2).
      ). Consistently, CDK4 activity has opposite effects on p107 and p130 steady state levels; inhibition of the enzyme leads to diminished levels of p107 and higher levels of p130 (
      • Rivadeneira D.B.
      • Mayhew C.N.
      • Thangavel C.
      • Sotillo E.
      • Reed C.A.
      • Graña X.
      • Knudsen E.S.
      Proliferative suppression by CDK4/6 inhibition: complex function of the retinoblastoma pathway in liver tissue and hepatoma cells.
      ). Thus, RB family member activity and stability clearly respond differently to cyclin-CDK signaling during cell cycle progression. However, the mechanisms that link regulation of RB family activity to their turnover are poorly understood.
      Previous studies from our laboratory showed that the Drosophila melanogaster RB homologue Rbf1 is subjected to proteasome-mediated turnover during embryonic development (
      • Acharya P.
      • Raj N.
      • Buckley M.S.
      • Zhang L.
      • Duperon S.
      • Williams G.
      • Henry R.W.
      • Arnosti D.N.
      Paradoxical instability-activity relationship defines a novel regulatory pathway for retinoblastoma proteins.
      ,
      • Ullah Z.
      • Buckley M.S.
      • Arnosti D.N.
      • Henry R.W.
      Retinoblastoma protein regulation by the COP9 signalosome.
      ). We further demonstrated that Rbf1 turnover is influenced by an “instability element” (IE) located within its C-terminal regulatory domain. Importantly, the IE region is also critical for full repressor potency for some cell cycle-regulated genes but not for non-canonical targets whose expression is not usually integrated with the cell cycle (
      • Acharya P.
      • Raj N.
      • Buckley M.S.
      • Zhang L.
      • Duperon S.
      • Williams G.
      • Henry R.W.
      • Arnosti D.N.
      Paradoxical instability-activity relationship defines a novel regulatory pathway for retinoblastoma proteins.
      ,
      • Raj N.
      • Zhang L.
      • Wei Y.
      • Arnosti D.N.
      • Henry R.W.
      Ubiquitination of retinoblastoma family protein 1 potentiates gene-specific repression function.
      ). Interestingly, Rbf1 ubiquitination also enhanced specific activity at select cell cycle target genes (
      • Raj N.
      • Zhang L.
      • Wei Y.
      • Arnosti D.N.
      • Henry R.W.
      Ubiquitination of retinoblastoma family protein 1 potentiates gene-specific repression function.
      ), suggesting that the potency of the repressor at specific genes and overall Rbf1 stability are coordinated. The IE region is well conserved within the mammalian p107 and p130 factors, and we hypothesized that the activity of mammalian RB family members may also be coordinated via integration of the cyclin-CDK signaling pathway with the ubiquitin-proteasome system. We demonstrate here that this regulatory mechanism is indeed shared among the human RB family proteins. The IE regions within the RB, p107, and p130 C-terminal domains negatively regulate repressor stability through a cyclin-CDK-responsive proteasome-dependent pathway and contribute to effective gene repression. These findings indicate that an evolutionarily conserved regulatory pathway links stability and potency for the mammalian RB family.

      Discussion

      The RB tumor suppressor family governs key steps in cellular proliferation through the transcriptional regulation of distinct genes associated with growth control (
      • Hurford Jr., R.K.
      • Cobrinik D.
      • Lee M.H.
      • Dyson N.
      pRB and p107/p130 are required for the regulated expression of different sets of E2F responsive genes.
      ,
      • Black E.P.
      • Huang E.
      • Dressman H.
      • Rempel R.
      • Laakso N.
      • Asa S.L.
      • Ishida S.
      • West M.
      • Nevins J.R.
      Distinct gene expression phenotypes of cells lacking Rb and Rb family members.
      ). Phosphorylation of RB proteins by cyclin-CDK complexes in mouse and human ES cells was previously demonstrated to inhibit RB/E2F interaction with consequent effects on gene expression and cell proliferation (
      • Stead E.
      • White J.
      • Faast R.
      • Conn S.
      • Goldstone S.
      • Rathjen J.
      • Dhingra U.
      • Rathjen P.
      • Walker D.
      • Dalton S.
      Pluripotent cell division cycles are driven by ectopic Cdk2, cyclin A/E, and E2F activities.
      ,
      • White J.
      • Stead E.
      • Faast R.
      • Conn S.
      • Cartwright P.
      • Dalton S.
      Developmental activation of the Rb-E2F pathway and establishment of cell cycle-regulated cyclin-dependent kinase activity during embryonic stem cell differentiation.
      ,
      • Wirt S.E.
      • Adler A.S.
      • Gebala V.
      • Weimann J.M.
      • Schaffer B.E.
      • Saddic L.A.
      • Viatour P.
      • Vogel H.
      • Chang H.Y.
      • Meissner A.
      • Sage J.
      G1 arrest and differentiation can occur independently of Rb family function.
      ,
      • Conklin J.F.
      • Baker J.
      • Sage J.
      The RB family is required for the self-renewal and survival of human embryonic stem cells.
      ). In this study we report that early steps during developmental regulation of the RB family in ES cells are additionally rendered through governance of subcellular localization. Specifically, RB and p107 accumulated in the nucleus during mouse ES cell differentiation mediated by concomitant LIF withdrawal and retinoic acid addition. This transition was concurrent with enhanced p107 and p130 association at select target genes. Promoter association by p107 and p130, but not RB in this developmental context, is similar to that observed in tissue culture experiments performed using T98G glioblastoma cells (
      • Takahashi Y.
      • Rayman J.B.
      • Dynlacht B.D.
      Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression.
      ). It is also interesting that p107 and p130 exhibited different behavior for promoter binding with some genes harboring only p107 and others harboring both p107 and p130. However, the functional significance of differential promoter association during ES differentiation remains to be determined. It should be noted that ES cells can undergo differentiation independently of RB family control (
      • Wirt S.E.
      • Adler A.S.
      • Gebala V.
      • Weimann J.M.
      • Schaffer B.E.
      • Saddic L.A.
      • Viatour P.
      • Vogel H.
      • Chang H.Y.
      • Meissner A.
      • Sage J.
      G1 arrest and differentiation can occur independently of Rb family function.
      ), and thus consequences for promoter-specific binding may depend upon additional cues, such as cell type and developmental context. These data point to two mechanisms governing activity of some RB family members during early development, one involving post-translational regulation by the cyclin-CDK system and another involving control of subcellular localization.
      Our data further indicate that RB family function in transcriptional repression is linked to increased repressor turnover. This connection was first suggested by observations that steady state levels of RB family members in ES cells were diminished during RA-induced differentiation and cell cycle attenuation. Our observations are also consistent with previous studies in mouse ES cells where total RB levels dropped in early G1 after release from nocodazole blockade (
      • Savatier P.
      • Huang S.
      • Szekely L.
      • Wiman K.G.
      • Samarut J.
      Contrasting patterns of retinoblastoma protein expression in mouse embryonic stem cells and embryonic fibroblasts.
      ). Taken together these observations suggest an intimate connection between cell cycle progression and RB protein levels in development. In response to RA, cellular mRNA levels encoding RB, p107, and p130 were either unaffected or were slightly increased, suggesting that changes in protein abundance during differentiation are influenced at a post transcriptional level, potentially involving regulated protein turnover. To understand the mechanism underlying RB family abundance, we tested the effect of proteasome inhibition on repressor levels in ES cells. However, these cells were extremely sensitive to MG132 treatment precluding direct assessment of RB family member half-lives. We, therefore, performed a biochemical structure-function study in a human osteosarcoma cell line that also maintains elevated CDK activity, analogously to that observed in ES cells. In this context, p107 and p130 turnover were indeed directed via a proteasome-mediated pathway that involved the evolutionarily conserved instability elements located within their C-terminal regulatory domains. The primary sequences of the mammalian p107 and p130 IE regions are most similar, and both of these are clearly related to the prototypical IE initially identified within Drosophila Rbf1 (
      • Acharya P.
      • Raj N.
      • Buckley M.S.
      • Zhang L.
      • Duperon S.
      • Williams G.
      • Henry R.W.
      • Arnosti D.N.
      Paradoxical instability-activity relationship defines a novel regulatory pathway for retinoblastoma proteins.
      ). However, RB differs substantially in primary sequence throughout the IE. Nonetheless, structural studies have indicated that pocket proteins maintain secondary and tertiary conservation throughout this region (
      • Rubin S.M.
      • Gall A.L.
      • Zheng N.
      • Pavletich N.P.
      Structure of the Rb C-terminal domain bound to E2F1-DP1: a mechanism for phosphorylation-induced E2F release.
      ). We show herein that the corresponding region within RB also functions as an autonomously acting degron, suggesting that regulation of repressor stability via these C-terminal degrons represents an important and evolutionarily conserved component of global RB family control.
      Mammalian RB family members are differentially expressed during cell cycle progression (
      • Bhattacharya S.
      • Garriga J.
      • Calbó J.
      • Yong T.
      • Haines D.S.
      • Graña X.
      SKP2 associates with p130 and accelerates p130 ubiquitylation and degradation in human cells.
      ,
      • Tedesco D.
      • Lukas J.
      • Reed S.I.
      The pRb-related protein p130 is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCF(Skp2).
      ,
      • Classon M.
      • Harlow E.
      The retinoblastoma tumour suppressor in development and cancer.
      ) with rapid degradation of hyperphosphorylated p130 correlated with G0 exit and cell cycle reentry. In contrast, p107 and RB levels tend to increase as cells progress toward S phase. Our studies showed that both wild type p107 and RB, but not mutant versions lacking the IE, are diminished by CDK4 inhibition and are consistent with a role of IE-mediated degron function in these cell cycle fluctuations. These findings are consistent with earlier observations wherein mouse ES cells ablated for the CDK2 inhibitor CDK2AP1 exhibited enhanced RB phosphorylation concomitant with increased RB abundance (
      • Kim Y.
      • Deshpande A.
      • Dai Y.
      • Kim J.J.
      • Lindgren A.
      • Conway A.
      • Clark A.T.
      • Wong D.T.
      Cyclin-dependent kinase 2-associating protein 1 commits murine embryonic stem cell differentiation through retinoblastoma protein regulation.
      ). Together, these studies demonstrate a clear linkage between the onset of CDK regulatory activity in ES cells and inversely correlated changes in RB family activity and abundance.
      Although the IE regions of RB, p107, and p130 share similar function in turnover control, the notable primary sequence divergence within these regions suggests that different E3 ligases participate in RB family turnover. We propose that these distinct IE regions provide regulatory flexibility for RB family responses to distinct cell signaling events. Such events may include differential responses to DNA damage or during regulated cell cycle progression. For example, the RBCNTer region can bind to the Mdm2 E3 ligase for targeted destruction of hypophosphorylated RB (
      • Sdek P.
      • Ying H.
      • Zheng H.
      • Margulis A.
      • Tang X.
      • Tian K.
      • Xiao Z.X.
      The central acidic domain of MDM2 is critical in inhibition of retinoblastoma-mediated suppression of E2F and cell growth.
      ,
      • Sdek P.
      • Ying H.
      • Chang D.L.
      • Qiu W.
      • Zheng H.
      • Touitou R.
      • Allday M.J.
      • Xiao Z.X.
      MDM2 promotes proteasome-dependent ubiquitin-independent degradation of retinoblastoma protein.
      ,
      • Uchida C.
      • Miwa S.
      • Kitagawa K.
      • Hattori T.
      • Isobe T.
      • Otani S.
      • Oda T.
      • Sugimura H.
      • Kamijo T.
      • Ookawa K.
      • Yasuda H.
      • Kitagawa M.
      Enhanced Mdm2 activity inhibits pRB function via ubiquitin-dependent degradation.
      ). As noted, corresponding regions in p107 and p130 are only minimally conserved with RB, and these proteins are refractory to Mdm2 expression (
      • Uchida C.
      • Miwa S.
      • Kitagawa K.
      • Hattori T.
      • Isobe T.
      • Otani S.
      • Oda T.
      • Sugimura H.
      • Kamijo T.
      • Ookawa K.
      • Yasuda H.
      • Kitagawa M.
      Enhanced Mdm2 activity inhibits pRB function via ubiquitin-dependent degradation.
      ). In contrast, proteolysis of p130, but not that of RB or p107, is dependent on the ubiquitin ligase activity of SCFSkp2 (
      • Bhattacharya S.
      • Garriga J.
      • Calbó J.
      • Yong T.
      • Haines D.S.
      • Graña X.
      SKP2 associates with p130 and accelerates p130 ubiquitylation and degradation in human cells.
      ,
      • Tedesco D.
      • Lukas J.
      • Reed S.I.
      The pRb-related protein p130 is regulated by phosphorylation-dependent proteolysis via the protein-ubiquitin ligase SCF(Skp2).
      ), which is minimally expressed during G0 but peaks during S phase (
      • Wei W.
      • Ayad N.G.
      • Wan Y.
      • Zhang G.J.
      • Kirschner M.W.
      • Kaelin Jr., W.G.
      Degradation of the SCF component Skp2 in cell-cycle phase G1 by the anaphase-promoting complex.
      ,
      • Wirbelauer C.
      • Sutterlüty H.
      • Blondel M.
      • Gstaiger M.
      • Peter M.
      • Reymond F.
      • Krek W.
      The F-box protein Skp2 is a ubiquitylation target of a Cul1-based core ubiquitin ligase complex: evidence for a role of Cul1 in the suppression of Skp2 expression in quiescent fibroblasts.
      • Zhang H.
      • Kobayashi R.
      • Galaktionov K.
      • Beach D.
      p19Skp1 and p45Skp2 are essential elements of the cyclin A-CDK2 S phase kinase.
      ), suggesting that E3 availability also plays a significant role in turnover of specific family members. Although SCFSkp2 and Mdm2 have been suggested as E3 ligases for cell cycle and DNA damage-associated degradation of RB family proteins, the involvement of these ligases in turnover during ES cell differentiation remains to be established. It is interesting that the RB C-terminal domain is also sufficient for F-box protein Skp2 association, but in this case the RB-Skp2 interaction is mainly implicated in the regulation of p27 turnover (
      • Ji P.
      • Jiang H.
      • Rekhtman K.
      • Bloom J.
      • Ichetovkin M.
      • Pagano M.
      • Zhu L.
      An Rb-Skp2-p27 pathway mediates acute cell cycle inhibition by Rb and is retained in a partial-penetrance Rb mutant.
      ), suggesting a role for the RB-IE in non-autonomous protein turnover. We consider it likely that multiple E3 ligases participate in RB family regulation through differential contacts with the IE regions of the different RB family members.
      Our study has additionally uncovered an intriguing aspect of mammalian RB regulation, namely that the sequences guiding repressor instability physically overlap with regions that are important for transcriptional repression. The inability of mutant p107 and p130 lacking the IE to fully engage in transcriptional repression is consistent with biochemical studies that have demonstrated a role for the IE in intermolecular contacts with the coiled coil-marked box regions of E2F1-DP1 complexes (Ref.
      • Rubin S.M.
      • Gall A.L.
      • Zheng N.
      • Pavletich N.P.
      Structure of the Rb C-terminal domain bound to E2F1-DP1: a mechanism for phosphorylation-induced E2F release.
      ; see also Fig. 7). In this regard our observations with the RB family of repressor proteins are similar to the intimate association of degrons within the activation domains of potent transactivator proteins, such as E2F1 and c-Myc (
      • Salghetti S.E.
      • Muratani M.
      • Wijnen H.
      • Futcher B.
      • Tansey W.P.
      Functional overlap of sequences that activate transcription and signal ubiquitin-mediated proteolysis.
      ), regulatory factors that control critical steps in cellular proliferation. A common theme emerges from these studies that key activators and repressors governing cell fate outcomes are inherently engineered. with limitations on their life span through turnover by the ubiquitin-proteasome system. Depending upon how the RB family interacts with distinct E2F-DP complexes, the interesting possibility arises that IE-E2F-DP engagement may reciprocally influence interactions with E3 ubiquitin ligases. In one model, E2F-DP complexes compete with E3 ligase for access to IE surfaces. In an alternative cooperative model, IE interactions with E2F complexes may portend engagement with E3 ubiquitin ligases. This latter model is supported, in part, by our data showing that the steady state levels of p107 and RB are diminished by cyclin D-CDK4 inhibition, a process that also licenses these pocket proteins for E2F-DP interactions and target gene engagement. Moreover, some cancer-associated p130 mutants tested in the current study showed increased steady state expression without significant effects on CCNA2 repression in vitro, suggesting that E3 binding and E2F-DP engagement are biochemically separable. Although ineffectual for perturbation of p130-mediated repression in this context, it remains possible that in vivo these mutations are associated with deregulation of other, as yet uncharacterized classes of target genes with significant effects on cellular physiology (
      • Chicas A.
      • Wang X.
      • Zhang C.
      • McCurrach M.
      • Zhao Z.
      • Mert O.
      • Dickins R.A.
      • Narita M.
      • Zhang M.
      • Lowe S.W.
      Dissecting the unique role of the retinoblastoma tumor suppressor during cellular senescence.
      ,
      • Cam H.
      • Balciunaite E.
      • Blais A.
      • Spektor A.
      • Scarpulla R.C.
      • Young R.
      • Kluger Y.
      • Dynlacht B.D.
      A common set of gene regulatory networks links metabolism and growth inhibition.
      ). We note that in the developing Drosophila embryo, Rbf1 associates with many genes involved in cell signaling and metabolism (
      • Acharya P.
      • Negre N.
      • Johnston J.
      • Wei Y.
      • White K.P.
      • Henry R.W.
      • Arnosti D.N.
      Evidence for autoregulation and cell signaling pathway regulation from genome-wide binding of the Drosophila retinoblastoma protein.
      ), and similar categories of genes may likewise become deregulated during human cancer progression in cells lacking IE function. In flies, expression of Rbf1 lacking the IE enhanced DNA replication in vitro (
      • Raj N.
      • Zhang L.
      • Wei Y.
      • Arnosti D.N.
      • Henry R.W.
      Rbf1 degron dysfunction enhances cellular DNA replication.
      ) and drove increased organ size when expressed in a tissue-specific manner during development (
      • Elenbaas J.S.
      • Mouawad R.
      • Henry R.W.
      • Arnosti D.N.
      • Payankaulam S.
      Role of Drosophila retinoblastoma protein instability element in cell growth and proliferation.
      ), suggesting a critical role for IE function in developmental and proliferative pathways.

      ACKNOWLEDGMENTS

      We are grateful to Julien Sage and Jenny Hsu for providing the wild type J1-ES cells, triple knock out ES cells, and p107−/− mouse embryonic fibroblasts. We also thank Erik Knudsen and Fred Dick for kindly sharing plasmids and Seth Rubin for providing a mouse monoclonal p130 antibody. We also acknowledge the assistance of Melinda Frame at the Michigan State University Center for Advanced Microscopy for help with confocal imaging.

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