INVESTIGATING THE ROLE AND REGULATION OF THE SPECIFIC ANDGENERAL PRB-E2F1 INTERACTIONS

2008 ◽  
Vol 31 (4) ◽  
pp. 6
Author(s):  
Matthew J Cecchini ◽  
Frederick A Dick
Keyword(s):  
Tumor Suppressor ◽  
Binding Site ◽  
Family Members ◽  
Retinoblastoma Tumor Suppressor Protein ◽  
Retinoblastoma Tumor Suppressor ◽  
C Terminus ◽  
Proliferation And Apoptosis ◽  
Retinoblastoma Tumor ◽  
And Control

Background/Purpose. The retinoblastoma tumor suppressor protein (pRB) plays a central role in proliferative control and is a frequent target for inactivation in cancer. The G1-Sphase transition of the cell cycle is regulated by pRB, which is capable of interacting with E2F family members and inhibiting the transcription of genesrequired to progress into S-phase. E2F1 is unique from other E2F family members as it can induce both apoptosis and proliferation. To control these contrasting functions of E2F1, a second E2F1 binding site exists in the C-terminus of pRB that can control apoptosis separately from proliferation. This anti-apoptotic function of pRBcan in some circumstances promote tumorigenesis, which leads to the question; is pRB a tumor suppressor or an oncogene? Methods. To investigate this, a gene-targeted mouse model is being engineered to selectively disrupt the ability of pRB to control proliferation through the general E2F binding site while still retaining the ability tocontrol apoptosis through the specific E2F1 site. Results. A series of novel mutants were engineered to selectively disrupt the binding of E2Fs at the general site, and prevent pRB from controlling proliferation. The mutants retain the ability to bind E2F1 and control apoptosis through thespecific binding site, which is not disrupted. Conclusions. By separating the ability of pRB to control proliferation and apoptosis under in vivo conditions, a better understanding into the significance of these two functions in development and tumorigenesis can be gained.

scholarly journals The Corepressor mSin3a Interacts with the Proline-Rich Domain of p53 and Protects p53 from Proteasome-Mediated Degradation

2001 ◽  
Vol 21 (12) ◽  
pp. 3974-3985 ◽  
Author(s):  
Jack T. Zilfou ◽  
William H. Hoffman ◽  
Michael Sank ◽  
Donna L. George ◽  
Maureen Murphy
Keyword(s):  
Tumor Suppressor ◽  
Binding Domain ◽  
Protein Stabilization ◽  
Independent Manner ◽  
Retinoblastoma Tumor Suppressor Protein ◽  
Rich Domain ◽  
Retinoblastoma Tumor Suppressor ◽  
Retinoblastoma Tumor ◽  
Sin3 Complex

ABSTRACT While the transactivation function of the tumor suppressor p53 is well understood, less is known about the transrepression functions of this protein. We have previously shown that p53 interacts with the corepressor protein mSin3a (hereafter designated Sin3) in vivo and that this interaction is critical for the ability of p53 to repress gene expression. In the present study, we demonstrate that expression of Sin3 results in posttranslational stabilization of both exogenous and endogenous p53, due to an inhibition of proteasome-mediated degradation of this protein. Stabilization of p53 by Sin3 requires the Sin3-binding domain, determined here to map to the proline-rich region of p53, from amino acids 61 to 75. The correlation between Sin3 binding and stabilization supports the hypothesis that this domain of p53 may normally be subject to a destabilizing influence. The finding that a synthetic mutant of p53 lacking the Sin3-binding domain has an increased half-life in cells, compared to wild-type p53, supports this premise. Interestingly, unlike retinoblastoma tumor suppressor protein, MDMX, and p14ARF, Sin3 stabilizes p53 in an MDM2-independent manner. The ability of Sin3 to stabilize p53 is consistent with the model whereby these two proteins must exist on a promoter for extended periods, in order for repression to be an effective mechanism of gene regulation. This model is consistent with our data indicating that, unlike the p300-p53 complex, the p53-Sin3 complex is immunologically detectable for prolonged periods following exposure of cells to agents of DNA damage.

scholarly journals The Mus musculus papillomavirus type 1 E7 protein binds to the retinoblastoma tumor suppressor - implications for viral pathogenesis

2021 ◽  
Author(s):  
Tao Wei ◽  
Miranda Grace ◽  
Aayushi Uberoi ◽  
James C Romero-Masters ◽  
Denis Lee ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Mus Musculus ◽  
Model Organisms ◽  
Retinoblastoma Tumor Suppressor ◽  
C Terminus ◽  
E6 And E7 ◽  
Retinoblastoma Tumor ◽  
E7 Proteins

The species specificity of papillomaviruses has been a significant roadblock for performing in vivo pathogenesis studies in common model organisms. The Mus musculus papillomavirus type 1 (MmuPV1) causes cutaneous papillomas that can progress to squamous cell carcinomas in laboratory mice. The papillomavirus E6 and E7 genes encode proteins that establish and maintain a cellular milieu that allows for viral genome synthesis and viral progeny synthesis in growth-arrested, terminally differentiated keratinocytes. The E6 and E7 proteins provide this activity by binding to and functionally reprogramming key cellular regulatory proteins. The MmuPV1 E7 protein lacks the canonical LXCXE motif that mediates the binding of multiple viral oncoproteins to the cellular retinoblastoma tumor suppressor protein, RB1. Our proteomic experiments, however, revealed that MmuPV1 E7 still interacts specifically with RB1. We show that MmuPV1 E7 interacts through its C-terminus with the C-terminal domain of RB1. Binding of MmuPV1 E7 to RB1 did not cause significant activation of E2F-regulated cellular genes. MmuPV1 E7 expression was shown to be essential for papilloma formation. Experimental infection of mice with MmuPV1 virus expressing an E7 mutant that is defective for binding to RB1 caused delayed onset, lower incidence, and smaller sizes of papillomas. Our results demonstrate that the MmuPV1 E7 gene is essential and that targeting non-canonical activities of RB1, which are independent of RB1's ability to modulate the expression of E2F-regulated genes, contribute to papillomavirus-mediated pathogenesis.

scholarly journals Retinoblastoma Protein Contains a C-terminal Motif That Targets It for Phosphorylation by Cyclin-cdk Complexes

1999 ◽  
Vol 19 (2) ◽  
pp. 1068-1080 ◽  
Author(s):  
Peter D. Adams ◽  
Xiaotong Li ◽  
William R. Sellers ◽  
Kayla B. Baker ◽  
Xiaohong Leng ◽  
...  
Keyword(s):  
Site Directed Mutagenesis ◽  
Binding Motif ◽  
Retinoblastoma Tumor Suppressor Protein ◽  
Retinoblastoma Tumor Suppressor ◽  
C Terminus ◽  
Sequence Elements ◽  
Stable Association ◽  
Retinoblastoma Tumor

ABSTRACT Stable association of certain proteins, such as E2F1 and p21, with cyclin-cdk2 complexes is dependent upon a conserved cyclin-cdk2 binding motif that contains the core sequence ZRXL, where Z and X are usually basic. In vitro phosphorylation of the retinoblastoma tumor suppressor protein, pRB, by cyclin A-cdk2 and cyclin E-cdk2 was inhibited by a short peptide spanning the cyclin-cdk2 binding motif present in E2F1. Examination of the pRB C terminus revealed that it contained sequence elements related to ZRXL. Site-directed mutagenesis of one of these sequences, beginning at residue 870, impaired the phosphorylation of pRB in vitro. A synthetic peptide spanning this sequence also inhibited the phosphorylation of pRB in vitro. pRB C-terminal truncation mutants lacking this sequence were hypophosphorylated in vitro and in vivo despite the presence of intact cyclin-cdk phosphoacceptor sites. Phosphorylation of such mutants was restored by fusion to the ZRXL-like motif derived from pRB or to the ZRXL motifs from E2F1 or p21. Phospho-site-specific antibodies revealed that certain phosphoacceptor sites strictly required a C-terminal ZRXL motif whereas at least one site did not. Furthermore, this residual phosphorylation was sufficient to inactivate pRB in vivo, implying that there are additional mechanisms for directing cyclin-cdk complexes to pRB. Thus, the C terminus of pRB contains a cyclin-cdk interaction motif of the type found in E2F1 and p21 that enables it to be recognized and phosphorylated by cyclin-cdk complexes.

scholarly journals Peptidic degron in EID1 is recognized by an SCF E3 ligase complex containing the orphan F-box protein FBXO21

2015 ◽  
Vol 112 (50) ◽  
pp. 15372-15377 ◽  
Author(s):  
Cuiyan Zhang ◽  
Xiaotong Li ◽  
Guillaume Adelmant ◽  
Jessica Dobbins ◽  
Christoph Geisen ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Family Members ◽  
Binding Motif ◽  
Retinoblastoma Tumor Suppressor ◽  
Ubiquitin Ligase Complex ◽  
Retinoblastoma Tumor ◽  
F Box Protein ◽  
Necessary And Sufficient

EP300-interacting inhibitor of differentiation 1 (EID1) belongs to a protein family implicated in the control of transcription, differentiation, DNA repair, and chromosomal maintenance. EID1 has a very short half-life, especially in G0 cells. We discovered that EID1 contains a peptidic, modular degron that is necessary and sufficient for its polyubiquitylation and proteasomal degradation. We found that this degron is recognized by an Skp1, Cullin, and F-box (SCF)-containing ubiquitin ligase complex that uses the F-box Only Protein 21 (FBXO21) as its substrate recognition subunit. SCFFBXO21 polyubiquitylates EID1 both in vitro and in vivo and is required for the efficient degradation of EID1 in both cycling and quiescent cells. The EID1 degron partially overlaps with its retinoblastoma tumor suppressor protein-binding domain and is congruent with a previously defined melanoma-associated antigen-binding motif shared by EID family members, suggesting that binding to retinoblastoma tumor suppressor and melanoma-associated antigen family proteins could affect the polyubiquitylation and turnover of EID family members in cells.

scholarly journals In vivo association of E2F and DP family proteins.

1995 ◽  
Vol 15 (5) ◽  
pp. 2536-2546 ◽  
Author(s):  
C L Wu ◽  
L R Zukerberg ◽  
C Ngwu ◽  
E Harlow ◽  
J A Lees
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activity ◽  
Family Members ◽  
Tumor Suppressor Protein ◽  
Retinoblastoma Tumor Suppressor Protein ◽  
Retinoblastoma Tumor Suppressor ◽  
Expression Of Genes ◽  
Transcription Factor E2f ◽  
Retinoblastoma Tumor

The mammalian transcription factor E2F plays an important role in regulating the expression of genes that are required for passage through the cell cycle. This transcriptional activity is inhibited by association with the retinoblastoma tumor suppressor protein (pRB) or its relatives p107 and p103. The first cDNA from the E2F family to be cloned was designated E2F-1, and multiple E2F family members have now been identified. They bind to DNA as heterodimers, interacting with proteins known as DP. Here we demonstrate that DP is also a family of polypeptides with at least two members (hDP-1 and hDP-2). Both hDP-1 and hDP-2 bind to all E2F family members in vivo, and each complex is capable of activating transcription. However, the various E2F/DP complexes display strong differences in the ability to bind to either pRB or p107 in vivo, and the specificity of pRB or p107 binding is mediated by the E2F subunit.

scholarly journals Differential Regulation of Retinoblastoma Tumor Suppressor Protein by G1 Cyclin-Dependent Kinase Complexes In Vivo

2001 ◽  
Vol 21 (14) ◽  
pp. 4773-4784 ◽  
Author(s):  
Sergei A. Ezhevsky ◽  
Alan Ho ◽  
Michelle Becker-Hapak ◽  
Penny K. Davis ◽  
Steven F. Dowdy
Keyword(s):  
Cyclin E ◽  
Active Form ◽  
Dominant Negative ◽  
Tumor Suppressor Protein ◽  
Cyclin D ◽  
Cyclin Dependent Kinase ◽  
Retinoblastoma Tumor Suppressor Protein ◽  
Retinoblastoma Tumor Suppressor ◽  
Retinoblastoma Tumor

ABSTRACT The retinoblastoma tumor suppressor protein (pRB) negatively regulates early-G1 cell cycle progression, in part, by sequestering E2F transcription factors and repressing E2F-responsive genes. Although pRB is phosphorylated on up to 16 cyclin-dependent kinase (Cdk) sites by multiple G1 cyclin-Cdk complexes, the active form(s) of pRB in vivo remains unknown. pRB is present as an unphosphorylated protein in G0 quiescent cells and becomes hypophosphorylated (∼2 mol of PO4 to 1 mol of pRB) in early G1 and hyperphosphorylated (∼10 mol of PO4 to 1 mol of pRB) in late G1 phase. Here, we report that hypophosphorylated pRB, present in early G1, represents the biologically active form of pRB in vivo that is assembled with E2Fs and E1A but that both unphosphorylated pRB in G0 and hyperphosphorylated pRB in late G1 fail to become assembled with E2Fs and E1A. Furthermore, using transducible dominant-negative TAT fusion proteins that differentially target cyclin D-Cdk4 or cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes, namely, TAT-p16 and TAT–dominant-negative Cdk2, respectively, we found that, in vivo, cyclin D-Cdk4/6 complexes hypophosphorylate pRB in early G1 and that cyclin E-Cdk2 complexes inactivate pRB by hyperphosphorylation in late G1. Moreover, we found that cycling human tumor cells expressing deregulated cyclin D-Cdk4/6 complexes, due to deletion of the p16 INK4a gene, contained hypophosphorylated pRB that was bound to E2Fs in early G1and that E2F-responsive genes, including those for dihydrofolate reductase and cyclin E, were transcriptionally repressed. Thus, we conclude that, physiologically, pRB is differentially regulated by G1 cyclin-Cdk complexes.

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