Rb Pocket Research Articles

Retinoblastoma mutation to predict poor outcomes in non-small cell lung cancer (NSCLC).

9060 Background: Genomic profiling of tumor DNA has revealed the diversity in NSCLC. The retinoblastoma gene ( RB1) encodes for RB pocket protein that plays an important role in cell cycle progression by interacting with various transcriptional factors. Here we determine the frequency and prognostic significance of RB1mutation in NSCLC and compare it to that in small cell lung cancer (SCLC). Methods: This IRB-approved retrospective review on NSCLC patients included Stage III and IV patients with genomic and clinical data. Primary outcome was median overall survival (OS) and secondary outcome was progression-free survival (PFS). OS and PFS were calculated by the Kaplan-Meier method and compared between mutant and wild-type (wt) RB1using the Log-Rank test. The effect of RB1mutation status on OS and PFS was further evaluated using the multivariable Cox model, controlling the effects of age, sex, stage, smoking history and chemotherapy. All tests are two-sided and p-values ≤ 0.05 were considered statistically significant. Results: We identified RB1 mutation in 8.2% of NSCLC patients (16 of 195 patients). With a median follow-up of 15.1 months, the median OS for wt RB1was 28.3 months and for mutant RB1 was 8.3 months (HR = 2.59, p-value = 0.002). The median PFS for wt RB1 was 21.8 months vs 6.4 months for mutant RB1 (HR = 2.85, p-value = 0.0002). RB1 mutation was associated with worse OS ( p= 0.017, HR = 2.17) and PFS ( p= 0.005, HR = 2.37) in multivariate analyses after adjusting for traditional risk factors like, age, sex, stage, smoking history and chemotherapy. Interestingly, a previously described benign deletion (A16-A18) in RB1 protein was identified in 5 of the 16 RB1 mutant patients and was associated with far worse outcomes compared to other RB1 mutations. In contrast to NSCLC, RB1 mutation was identified in 75% of 64 SCLC patients. Furthermore, wt RB1 was associated with significantly shorter OS ( p= 0.002), PFS ( p= 0.004) and chemotherapy refractoriness ( p= 0.033) in SCLC. Conclusions: RB1 mutation is present in a minority of patients with advanced NSCLC and is associated with poor prognosis. In contrast, RB1 mutation is present in the majority of SCLC patients and is associated with a favorable prognosis.

RB: An essential player in adult neurogenesis.

The fundamental mechanisms underlying adult neurogenesis remain to be fully clarified. Members of the cell cycle machinery have demonstrated key roles in regulating adult neural stem cell (NSC) quiescence and the size of the adult-born neuronal population. The retinoblastoma protein, Rb, is known to possess CNS-specific requirements that are independent from its classical role as a tumor suppressor. The recent study by Vandenbosch etal. has clarified distinct requirements for Rb during adult neurogenesis, in the restriction of proliferation, as well as long-term adult-born neuronal survival. However, Rb is no longer believed to be the main cell cycle regulator maintaining the quiescence of adult NSCs. Future studies must consider Rb as part of a larger network of regulatory effectors, including the other members of the Rb family, p107 and p130. This will help elucidate the contribution of Rb and other pocket proteins in the context of adult neurogenesis, and define its crucial role in regulating the size and fate of the neurogenic niche.

Structural Conservation and E2F Binding Specificity within the Retinoblastoma Pocket Protein Family

The human pocket proteins retinoblastoma (Rb), p107, and p130 are critical negative regulators of the cell cycle and contribute to tumor suppression. While strong structural conservation within the pocket protein family provides for some functional redundancy, important differences have been observed and may underlie the reason that Rb is a uniquely potent tumor suppressor. It has been proposed that distinct pocket protein activities are mediated by their different E2F transcription factor binding partners. In humans, Rb binds E2F1–E2F5, whereas p107 and p130 almost exclusively associate with E2F4 and E2F5. To identify the molecular determinants of this specificity, we compared the crystal structures of Rb and p107 pocket domains and identified several key residues that contribute to E2F selectivity in the pocket family. Mutation of these residues in p107 to match the analogous residue in Rb results in an increase in affinity for E2F1 and E2F2 and an increase in the ability of p107 to inhibit E2F2 transactivation. Additionally, we investigated how phosphorylation by Cyclin-dependent kinase on distinct residues regulates p107 affinity for the E2F4 transactivation domain. We found that phosphorylation of residues S650 and S975 weakens the E2F4 transactivation domain binding. Our data reveal molecular features of pocket proteins that are responsible for their similarities and differences in function and regulation.

Merkel cell polyomavirus T antigens promote cell proliferation and inflammatory cytokine gene expression.

Merkel cell polyomavirus (MCV) is clonally integrated in over 80 % of Merkel cell carcinomas and mediates tumour development through the expression of viral oncoproteins, the large T (LT) and small T antigens (sT). Viral integration is associated with signature mutations in the T-antigen locus that result in deletions of C-terminal replicative functions of the LT antigen. Despite these truncations, the LT LXCXE retinoblastoma (Rb) pocket protein family binding domain is retained, and the entire sT isoform is maintained intact. To investigate the ability of MCV oncoproteins to regulate host gene expression, we performed microarray analysis on cells stably expressing tumour-derived LT, tumour-derived LT along with sT, and tumour-derived LT with a mutated Rb interaction domain. Gene expression alterations in the presence of tumour-derived LT could be classified into three main groups: genes that are involved in the cell cycle (specifically the G1/S transition), genes involved in DNA replication and genes involved in cellular movement. The LXCXE mutant LT largely reversed gene expression alterations detected with the WT tumour-derived LT, while co-expression of sT did not significantly affect these patterns of gene expression. LXCXE-dependent upregulation of cyclin E and CDK2 correlated with increased proliferation in tumour-derived LT-expressing cells. Tumour-derived LT and tumour-derived LT plus sT increased expression of multiple cytokines and chemokines, which resulted in elevated levels of secreted IL-8. We concluded that, in human fibroblasts, the LXCXE motif of tumour-derived LT enhances cellular proliferation and upregulates cell cycle and immune signalling gene transcription.

Phosphorylation-induced Conformational Changes in the Retinoblastoma Protein Inhibit E2F Transactivation Domain Binding

Inactivation of the retinoblastoma protein (Rb) through phosphorylation is an important step in promoting cell cycle progression, and hyperphosphorylated Rb is commonly found in tumors. Rb phosphorylation prevents its association with the E2F transcription factor; however, the molecular basis for complex inhibition has not been established. We identify here the key phosphorylation events and conformational changes that occur in Rb to inhibit the specific association between the E2F transactivation domain (E2F(TD)) and the Rb pocket domain. Calorimetry assays demonstrate that phosphorylation of Rb reduces the affinity of E2F(TD) binding approximately 250-fold and that phosphorylation at Ser(608)/Ser(612) and Thr(356)/Thr(373) is necessary and sufficient for this effect. An NMR assay identifies phosphorylation-driven conformational changes in Rb that directly inhibit E2F(TD) binding. We find that phosphorylation at Ser(608)/Ser(612) promotes an intramolecular association between a conserved sequence in the flexible pocket linker and the pocket domain of Rb that occludes the E2F(TD) binding site. We also find that phosphorylation of Thr(356)/Thr(373) inhibits E2F(TD) binding in a manner that requires the Rb N-terminal domain. Taken together, our results suggest two distinct mechanisms for how phosphorylation of Rb modulates association between E2F(TD) and the Rb pocket and describe for the first time a function for the structured N-terminal domain in Rb inactivation.

Abstract 3216: CREG1, an epigenetically regulated gene, cooperates with p16INK4a in cellular senescence

Abstract By passing cellular senescence, an irreversible growth arrest of cells that is activated in normal cells upon shortening of telomere and other cellular stress, to become immortal is one of the prerequisites for carcinogenesis. Using spontaneously immortal Li-Fraumeni Syndrome (LFS) fibroblasts, we found that CREG1 (Cellular Repressor of E1A-stimulated Genes1) is one of the genes whose expression fit the criteria of senescence-associated genes, decrease expression during immortalization and increase in senescence. Moreover, we found that epigenetic mechanism regulates CREG1 expression. CREG1 is a secreted glycoprotein that was shown to bind RB-family pocket proteins and inhibits E2F transactivation activity. Therefore, we hypothesize that CREG1 plays a role in cellular senescence involving p16INK4a/Rb pathway. Ectopic expression of CREG1 in the immortal LFS cell lines decreases cell proliferation but does not directly induce senescence. We confirmed this in osteosarcoma and fibrosarcoma cancer cell lines commonly seen in Li-Fraumeni Syndrome. Because CREG1 was shown to interact with RB pocket proteins in vitro, we tested that growth suppression by CREG1 may depend on the phosphorylation status of Rb. We found that p16INK4a is also downregulated in immortal cells and that coexpression of CREG1 and p16INK4a, and inhibitor of CDK and Rb phosphorylation, has a greater effect than CREG1 and p16INK4a alone to reduce cell growth, induce cell cycle arrest and cellular senescence in immortal LFS, osteosarcoma and fibrosarcoma cell lines. Moreover, cooperation of CREG1 and p16INK4a inhibits the expression of cyclin A and cyclin B by inhibiting promoter activity thereby decreasing mRNA and protein levels; these proteins are required for S-phase entry and G2/M transition. Whether CREG1 directly involved in transcriptional repression of cyclin A and B mediated by p16INK4a will be confirmed by ChIP assay. In conclusion, we are the first to find that CREG1 enhances p16INK4a-induced senescence by transcriptional repression of cell cycle mediated genes. Analysis of how CREG1 and its cooperation with p16INK4a regulate cell cycle and cellular senescence may provide better understanding of cellular immortalization, an early step in human tumorigenesis. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3216.

Molecular Mechanisms for Phosphorylation Driven Dissociation of Rb-E2F Complexes

The Retinoblastoma Protein (Rb) functions as a negative regulator of cell growth in part by physically sequestering and repressing the transactivation activity of E2F. It is well established that phosphorylation of Rb by Cyclin Dependent Kinases disrupts binding between Rb and E2F, however it is unknown which of the 15 CDK consensus phosphorylation sites on Rb are required to disrupt the interaction between the pocket domain of Rb and the transactivation domain of E2F (E2FTD). In this work, we use calorimetric assays to reveal that phosphorylation at S608/S612 and T356/T373 are together sufficient to reduce the affinity of E2FTD for Rb pocket 250-fold, the same as fully-phosphorylated Rb. Nuclear Magnetic Resonance is used to identify the phosphorylation-dependent conformational changes that directly inhibit E2FTD binding. Specifically, we have found that phosphorylation of S608/S612 promotes intramolecular binding between the flexible pocket linker and the pocket domain of Rb, while phosphorylation at T356/T373 enhances binding between the N-terminal domain and pocket domain of Rb. Taken together, our results reveal two novel mechanisms for how phosphorylation of Rb modulates binding between E2FTD and Rb pocket, and we describe for the first time a function for the N-terminal domain in the inactivation of Rb.

Inhibition of RNA Polymerase III Transcription by BRCA1

RNA polymerase III (RNA pol III) transcribes structural RNAs involved in RNA processing (U6 snRNA) and translation (tRNA), thereby regulating the growth rate of cells. Proper initiation by RNA pol III requires the transcription factor TFIIIB. Gene-external U6 snRNA transcription requires TFIIIB consisting of Bdp1, TBP, and Brf2. Transcription from the gene-internal tRNA promoter requires TFIIIB composed of Bdp1, TBP, and Brf1. TFIIIB is a target of tumor suppressors, including PTEN, ARF, p53, and RB, and RB-related pocket proteins. Breast cancer susceptibility gene 1 (BRCA1) tumor suppressor plays a role in DNA repair, cell cycle regulation, apoptosis, genome integrity, and ubiquitination. BRCA1 has a conserved amino-terminal RING domain, an activation domain 1 (AD1), and an acidic carboxyl-terminal domain (BRCA1 C-terminal region). In Saccharomyces cerevisiae, TFIIB interacts with the BRCA1 C-terminal region domain of Fcp1p, an RNA polymerase II phosphatase. The TFIIIB subunits Brf1 and Brf2 are structurally similar to TFIIB. Hence, we hypothesize that RNA pol III may be regulated by BRCA1 via the TFIIB family members Brf1 and Brf2. Here we report that: (1) BRCA1 inhibits both VAI (tRNA) and U6 snRNA RNA pol III transcription; (2) the AD1 of BRCA1 is responsible for inhibition of U6 snRNA transcription, whereas the RING domain and AD1 of BRCA1 are required for VAI transcription inhibition; and (3) overexpression of Brf1 and Brf2 alleviates inhibition of U6 snRNA and VAI transcription by BRCA1. Taken together, these data suggest that BRCA1 is a general repressor of RNA pol III transcription.

Roscovitine Prevents Alloreactive T Cell Expansion and TNF-a-Mediated Proinflammatory Gene Expression and Protects against GvHD.

Graft versus host disease (GvHD) is induced by donor T cells stimulated by recipient's alloantigens and remains a frequent and severe complication of allogeneic stem cell transplantation (SCT). Cell cycle progression in response to antigenic stimulation plays a critical role in determining the fate of activated T lymphocytes. Cyclin dependent kinase (Cdk)-2 is a key regulator of the cell cycle that promotes phosphorylation of Rb and related pocket proteins thereby reversing their ability to sequester E2F transcription factors and allowing transcription of E2F target genes that facilitate G1/S transition and S-phase progression. Cdk2 also phosphorylates Smad2 and Smad3 and this event reduces Smad3 transcriptional activity and antiproliferative function. We have determined that induction of T cell tolerance results in impaired cdk2 activity leading to reduced level of Smad3 phosphorylation on cdk-specific sites and increased Smad3 antiproliferative function due to upregulation of p15. Since cdk2 is involved in multiple steps of T cell activation, we hypothesized that pharmacologic inhibition of cdk2 might provide an effective strategy to control expansion of alloreactive T cells and might have clinical application for the control of GvHD. To test this hypothesis we used (R)-roscovitine (CYC202), a potent inhibitor of cdk2-cyclin E, which in higher concentrations also inhibits other cdk-cyclin complexes including cdk7, cdk9 and cdk5. Due to its anti-proliferative effects (R)-roscovitine is currently in clinical trials as anticancer drug. To evaluate the effects of (R)-roscovitine on alloreactive T cell responses in vitro we stimulated C57BL/6 T cells with MHC disparate Balb/c splenocytes. Addition of (R)-roscovitine in these cultures resulted in blockade of alloreactive T cell proliferation and IL-2 production. Biochemical analysis revealed that (R)-roscovitine prevented downregulation of p27, phosphorylation of Rb, and synthesis of cyclin A, suggesting that the cell cycle was blocked at G1/S phase. Recently it was determined that (R)-roscovitine is a potent inhibitor of TNF-α-mediated activation of NF-κB pathway in cancer cells. Because induction of NF-κB target genes by TNF-α is essential for regulation of inflammatory immune responses we investigated whether (R)- roscovitine might have a similar effect on TNF-α-mediated NF-κB activation in T cells. Addition of (R)-roscovitine in T cell cultures incubated with TNF-α inhibited TNF-ainduced IkB kinase (IKK) kinase activity resulting in defective IkBa phosphorylation, degradation and re-synthesis. These events lead to defective phosphorylation of p65 on Ser 536, a bona fide IKK site that is required for TNF-a-mediated NF-kB transcription, indicating that roscovitine is an inhibitor of the canonical IKK signaling in T cells. Based on these findings, we examined whether (R)-roscovitine might repress TNF-α induced activation of the NF-κB target genes MCP-1 and RANTES that play a direct role in the pathobiology of GvHD. Incubation of T cells with TNF-α induced both MCP- 1 and RANTES gene and protein expression and these events was suppressed by (R)- roscovitine. Thus, (R)-roscovitine reverses both TCR-mediated clonal expansion and TNF-α mediated inflammatory responses of T cells. Because these properties are highly desirable in therapeutic interventions to prevent GvHD, we employed a mouse model of GvHD to examine the effects of (R)-roscovitine in vivo. Recipient (C57BL/6 x DBA/2) F1 mice were lethally irradiated and were subsequently infused with bone marrow cells and splenocytes, as source of allogeneic T cells, from parental C57BL/6 donors. With this regimen, recipients succumbed to severe acute GvHD with a median survival time of 28 days. Administration of (R)-roscovitine at the time of allogeneic bone marrow transplantation and on a daily basis thereafter for a total 21 days increased median survival of allograft recipients to 68 days (p=0.003). These results indicate that (R)-roscovitine has direct effects on regulating alloreactive T cell immune responses and may provide a novel therapeutic approach for control of GvHD.

Retinoblastoma protein and the leukemia-associated PLZF transcription factor interact to repress target gene promoters

Translocations of the retinoic acid receptor-alpha (RARalpha) locus with the promyelocytic leukemia zinc-finger (PLZF) or PML genes lead to expression of oncogenic PLZF-RARalpha or PML-RARalpha fusion proteins, respectively. These fusion oncoproteins constitutively repress RARalpha target genes, in large part through aberrant recruitment of multiprotein co-repressor complexes. PML and PML-RARalpha have previously been shown to associate with the retinoblastoma (Rb) tumour suppressor protein in its hypophosphorylated state. Here, we demonstrate that PLZF also interacts with Rb in vitro and in vivo. The interaction between PLZF and Rb is mediated through the Rb pocket and the region of PLZF that lies between its transcriptional repression (poxvirus and zinc-finger, POZ) and DNA-binding (zinc-finger) domains. In addition, Rb can simultaneously interact with PLZF and the E2F1 S phase-inducing transcription factor, suggesting that these proteins can exist in the same multiprotein complex. In contrast to the interaction of Rb with PML or E2F1, the PLZF-Rb interaction is not dependent on hypophosphorylation of Rb. These data are supported by chromatin immunoprecipitation analysis, which indicates that PLZF associates with the promoter region of CDC6, a known E2F/Rb target gene. Co-expression of PLZF and Rb results in enhancement of transcriptional repression of PLZF and E2F/Rb target genes, indicating functional co-operation between the two proteins. Both PLZF and Rb have been shown to function in stem cells and taken together these data suggest that interactions between PLZF and Rb could be important in stem cell biology.

Retinoblastoma Protein and the Leukemia-Associated PLZF Transcription Factor Interact To Repress Target Gene Promoters.

Translocations of the retinoic acid receptor alpha (RARA) locus with the PLZF or PML genes lead to expression of oncogenic PLZF-RARα or PML-RARα fusion proteins, respectively. These fusion oncoproteins constitutively repress RARα target genes, in large part through aberrant recruitment of multiprotein co-repressor complexes. PML and PML-RARα have previously been shown to associate with the retinoblastoma (Rb) tumour suppressor protein in its hypophosphorylated state. Here we demonstrate that PLZF also interacts with Rb in vitro and in vivo. The interaction between PLZF and Rb is mediated through the Rb pocket and the region of PLZF that lies between its transcriptional repression (POZ) and DNA binding (zinc-finger) domains. Additionally, Rb can simultaneously interact with PLZF and the E2F1 S phase-inducing transcription factor, suggesting that these proteins can exist in the same multiprotein complex. In contrast to the interaction between PML or E2F1 with Rb, the PLZF-Rb interaction is not dependent on hypophosphorylation of Rb. The interaction between PLZF and Rb is further underlined by chromatin immunoprecipitation analysis of PLZF binding to genomic DNA, which shows that PLZF associates with genes controlling cell proliferation known to be regulated by Rb and E2F (for example cdc6). Co-expression of PLZF and Rb results in enhancement of transcriptional repression of PLZF and E2F target genes, indicating functional co-operation between the two proteins. Both PLZF and Rb have been shown to have roles in stem cell biology and, taken together, these data provide a plausible scenario in which interactions between PLZF and Rb function in stem cell commitment or maintenance and self-renewal. The oncogenic PLZF-RARα fusion also interacts with Rb, suggesting that deregulation of Rb function may be a factor in the molecular pathogenesis of PLZF-RARα associated acute promyelocytic leukemia.

Roscovitine Prevents TCR-Mediated T Cell Clonal Expansion In-Vitro and Protects Against GvHD In-Vivo.

Cell cycle re-entry of quiescent T lymphocytes is required for generation of productive T cell responses. Cyclin-dependent kinases (cdk), particularly cdk2, have an essential role in cell cycle re-entry. Cdk2 promotes phosphorylation of Rb and related pocket proteins thereby reversing their ability to sequester E2F transcription factors. Besides Rb, cdk2 phosphorylates Smad2 and Smad3. Smad3 inhibits cell cycle progression from G1 to S phase, and impaired phosphorylation on the cdk-mediated sites renders it more effective in executing this function. In contrast, cdk-mediated phosphorylation of Smad3 reduces Smad3 transcriptional activity and antiproliferative function. Recently, we determined that induction of T cell tolerance resulted in impaired cdk2 activity, leading to reduced levels of Smad3 phosphorylation on cdk-specific sites and increased Smad3 antiproliferative function due to upregulation of p15. We hypothesized that pharmacologic inhibition of cdk2 during antigen-mediated T cell stimulation might provide an effective strategy to control T cell expansion and induce tolerance. (R)-roscovitine (CYC202) is a potent inhibitor of cdk2-cyclin E, which in higher concentrations also inhibits other cdk-cyclin complexes including cdk7, cdk9 and cdk5. It is currently in clinical trials as anticancer drug and recently was shown to induce long-lasting arrest of murine polycystic kidney disease. We examined the effect of roscovitine on T cell responses in vitro and in vivo. We stimulated C57BL/6 T cells with anti-CD3-plus-anti-CD28 mAbs, DO11.10 TCR-transgenic T cells with OVA peptide or C57BL/6 T cells with MHC disparate Balb/c splenocytes. Addition of roscovitine in these cultures resulted in blockade of cell proliferation without induction of apoptosis. Biochemical analysis revealed that roscovitine prevented phosphorylation of cdk2, downregulation of p27, phosphorylation of Rb and synthesis of cyclin A, suggesting an effective G1/S cell cycle block. To determine whether roscovitine could also inhibit clonal expansion of activated T cells in vivo, we employed a mouse model of GvHD. Recipient (C57BL/6 x DBA/2) F1 mice were lethally irradiated and were subsequently infused with bone marrow cells and splenocytes, as source of allogeneic T cells, from parental C57BL/6 donors. Roscovitine or vehicle-control was given at the time of allogeneic BMT and on a trice-weekly basis thereafter for a total of three weeks. Administration of roscovitine protected against acute GvHD resulting in a median survival of 49 days in the roscovitine-treated group compared to 24 days in the control group (p=0.005), and significantly less weight loss. Importantly, roscovitine treatment had no adverse effects on engraftment, resulting in full donor chimerism in the treated mice. To examine whether tolerance had been induced by in vivo treatment with roscovitine, we examined in vitro rechallenge responses. While control C57BL/6 T cells exhibited robust responses when stimulated with (C57BL/6 x DBA/2) F1 splenocytes, responses of T cells isolated from roscovitine-treated recipients against (C57BL/6 x DBA/2) F1 splenocytes were abrogated. These results indicate that roscovitine has direct effects on preventing TCR-mediated clonal expansion in vitro and in vivo and may provide a novel therapeutic approach for control of GvHD.

Crystal Structure of the Retinoblastoma Protein N Domain Provides Insight into Tumor Suppression, Ligand Interaction, and Holoprotein Architecture

The retinoblastoma susceptibility protein, Rb, has a key role in regulating cell-cycle progression via interactions involving the central "pocket" and C-terminal regions. While the N-terminal domain of Rb is dispensable for this function, it is nonetheless strongly conserved and harbors missense mutations found in hereditary retinoblastoma, indicating that disruption of its function is oncogenic. The crystal structure of the Rb N-terminal domain (RbN), reveals a globular entity formed by two rigidly connected cyclin-like folds. The similarity of RbN to the A and B boxes of the Rb pocket domain suggests that Rb evolved through domain duplication. Structural and functional analysis provides insight into oncogenicity of mutations in RbN and identifies a unique phosphorylation-regulated site of protein interaction. Additionally, this analysis suggests a coherent conformation for the Rb holoprotein in which RbN and pocket domains directly interact, and which can be modulated through ligand binding and possibly Rb phosphorylation.

Setting the Stage for S Phase

In a recent issue of Molecular Cell, Tyagi et al. (2007) show that E2F1, a positive regulator of S phase entry, recruits cofactor HCF-1 and associated hSet1/MLL histone H3 lysine 4 methyltransferase complex, facilitating the activation of genes required for proliferation.

A Novel p27kip1-Smad3 Regulated Pathway Is Involved in Induction of T Cell Tolerance.

Induction and maintenance of peripheral tolerance is essential for homeostasis of the immune system. In vivo studies demonstrate the significance of tolerance induction in preventing autoimmunity, graft rejection and GVHD. Upregulation of the cyclin-dependent kinase inhhibitor, p27, correlates with induction of T cell tolerance in vitro and in vivo. p27 interacts with cdk2, cdc2, grb2, and Rho family GTPases. Extensive studies support an essential role of cdks, particularly cdk2, in cell cycle re-entry. Cdk2 promotes cell cycle progression in part by phosphorylating Rb and related pocket proteins thereby reversing their ability to sequester E2F transcription factors. Recent work indicates that cdk2 phosphorylates Smad2 and Smad3. Smad3 inhibits progression from G1 to S phase, and impaired phosphorylation on the cdk-specific sites renders it more effective in executing this function. In contrast, cdk-mediated phosphorylation of Smad3 reduces Smad3 transcriptional activity and antiproliferative function. In spite the strong correlation between p27 expression level and T cell tolerance, it remains unclear whether p27 has a causative role in induction of tolerance. Here, we examined the role of p27 during induction of tolerance of naïve T cells in vivo, using RAG2 deficient, DO11.10 TCR-transgenic T cells that lack the cyclin-cdk-binding domain of p27 (p27Δ) thereby disrupting only the interactions of p27 with cyclin-cdk complexes. We adoptively transferred CD4+ T cells from RAG2−/−DO11.10 TCR-transgenic mice (DO11.10) or RAG2−/−DO11.10 TCR-transgenic p27Δ mice (DO11.10/p27Δ) into syngeneic wild-type recipients and compared the development of immune responses to immunogenic or tolerizing stimulus in vivo. Following exposure to immunogenic or tolerizing stimulus, DO11.10 and DO11.10/p27Δ CD4+ T cells underwent equal numbers of divisions in vivo, and both cell types exhibited reduced number of divisions in response to tolerizing stimulus. Strikingly, only wild-type DO11.10 TCR-transgenic T cells were tolerized as determined by impaired cyclin E activation, proliferation, and IL-2 production upon antigen-specific rechallenge. Compared to primed wild-type DO11.10 cells, tolerized wild-type DO11.10 cells exhibited impaired cdk2 and cdc2 activity, reduced levels of Smad3 phosphorylation on cdk-specific sites, and increased Smad3-transactivation leading to upregulation of the cdk4/6-specific cdk inhibitor p15. In contrast, after either priming or tolerizing stimulus, DO11.10/p27Δ cells exhibited comparable cdk2 and cdc2 activity, cdk-mediated phosphorylation of Smad3, low-level Smad3 transactivation, and no upregulation of p15. Furthermore, knockdown of Smad3 by expression of Smad3 shRNA in wild-type DO11.10 T cells recapitulated the functional and molecular findings observed in DO11.10/p27Δ cells, preventing induction of tolerance and upregulation of p15, and resulting in production of IL-2 and cell cycle progression. In contrast, expression of Smad3 mutant resistant to cdk-mediated phosphorylation in DO11.10/p27Δ cells recapitulated the molecular and functional effects of tolerance and resulted in inhibition of IL-2 production, upregulation of p15 and blockade of cell cycle progression. These results show that p27 plays a causative role in the induction of tolerance of naïve T cells and Smad3 is a critical component of a pathway downstream of p27 regulating the induction of tolerance in vivo.

Structure of the Rb C-Terminal Domain Bound to E2F1-DP1: A Mechanism for Phosphorylation-Induced E2F Release

The retinoblastoma (Rb) protein negatively regulates the G1-S transition by binding to the E2F transcription factors, until cyclin-dependent kinases phosphorylate Rb, causing E2F release. The Rb pocket domain is necessary for E2F binding, but the Rb C-terminal domain (RbC) is also required for growth suppression. Here we demonstrate a high-affinity interaction between RbC and E2F-DP heterodimers shared by all Rb and E2F family members. The crystal structure of an RbC-E2F1-DP1 complex reveals an intertwined heterodimer in which the marked box domains of both E2F1 and DP1 contact RbC. We also demonstrate that phosphorylation of RbC at serines 788 and 795 destabilizes one set of RbC-E2F-DP interactions directly, while phosphorylation at threonines 821 and 826 induces an intramolecular interaction between RbC and the Rb pocket that destabilizes the remaining interactions indirectly. Our findings explain the requirement of RbC for high-affinity E2F binding and growth suppression and establish a mechanism for the regulation of Rb-E2F association by phosphorylation.

Retinoblastoma and the Related Pocket Protein p107 Act as Coactivators of NeuroD1 to Enhance Gene Transcription

Gene inactivation studies have suggested that the product of the retinoblastoma gene, Rb, is particularly limiting in pituitary pro-opiomelanocortin (POMC)-expressing cell lineages. Indeed, in Rb knock-out mice, these cells develop tumors with high frequency. To understand the implication of limiting Rb expression in these cells, we investigated the action of Rb and its related pocket proteins, p107 and p130, on POMC gene transcription. This led to the identification of the neurogenic basic helix-loop-helix transcription factor, NeuroD1, as a target of Rb action. Rb and to a lesser extent p107, but not p130, enhance NeuroD1-dependent transcription, and this activity appears to depend on direct protein interactions between the Rb pocket and the helix-loop-helix domain of NeuroD1. In vivo, NeuroD is found in a complex that includes Rb and also the orphan nuclear receptor NGFI-B, which mediates corticotropin-releasing hormone activation of POMC transcription. The formation of a similar complex in vitro requires the presence of Rb as a bridge between NeuroD and NGFI-B. In POMC-expressing AtT-20 cells, Rb and p107 are present on the POMC promoter and inhibition of their expression through small interfering RNA decreases POMC mRNA levels. The action of Rb and its related proteins on POMC transcription may contribute to the establishment and/or maintenance of the differentiation phenotype.

Hierarchical Requirement of SWI/SNF in Retinoblastoma Tumor Suppressor-mediated Repression of Plk1

Plk1 (Polo-like kinase 1) is a critical regulator of cell cycle progression that harbors oncogenic activity and exhibits aberrant expression in multiple tumors. However, the mechanism through which Plk1 expression is regulated has not been extensively studied. Here we demonstrate that Plk1 is a target of the retinoblastoma tumor suppressor (RB) pathway. Activation of RB and related pocket proteins p107/p130 mediate attenuation of Plk1. Conversely, RB loss deregulates the control of Plk1 expression. RB pathway activation resulted in the repression of Plk1 promoter activity, and this action was dependent on the SWI/SNF chromatin remodeling complex. Although SWI/SNF subunits are lost during tumorigenesis and cooperate with RB for transcriptional repression, the mechanism through which SWI/SNF impinges on RB action is unresolved. Therefore, we delineated the requirement of SWI/SNF for three critical facets of Plk1 promoter regulation: transcription factor binding, corepressor binding, and histone modification. We find that E2F4 and pocket protein association with the Plk1 promoter is independent of SWI/SNF. However, these analyses revealed that SWI/SNF is required for histone deacetylation of the Plk1 promoter. The importance of SWI/SNF-dependent histone deacetylation of the Plk1 promoter was evident, because blockade of this event restored Plk1 expression in the presence of active RB. In summary, these data demonstrate that Plk1 is a target of the RB pathway. Moreover, these findings demonstrate a hierarchical role for SWI/SNF in the control of promoter activity through histone modification.

The role of cyclin D2 and p21/waf1 in human T-cell leukemia virus type 1 infected cells

BackgroundThe human T-cell leukemia virus type 1 (HTLV-1) Tax protein indirectly influences transcriptional activation, signal transduction, cell cycle control, and apoptosis. The function of Tax primarily relies on protein-protein interactions. We have previously shown that Tax upregulates the cell cycle checkpoint proteins p21/waf1 and cyclin D2. Here we describe the consequences of upregulating these G1/S checkpoint regulators in HTLV-1 infected cells.ResultsTo further decipher any physical and functional interactions between cyclin D2 and p21/waf1, we used a series of biochemical assays from HTLV-1 infected and uninfected cells. Immunoprecipitations from HTLV-1 infected cells showed p21/waf1 in a stable complex with cyclin D2/cdk4. This complex is active as it phosphorylates the Rb protein in kinase assays. Confocal fluorescent microscopy indicated that p21/waf1 and cyclin D2 colocalize in HTLV-1 infected, but not in uninfected cells. Furthermore, in vitro kinase assays using purified proteins demonstrated that the addition of p21/waf1 to cyclin D2/cdk4 increased the kinase activity of cdk4.ConclusionThese data suggest that the p21/cyclin D2/cdk4 complex is not an inhibitory complex and that p21/waf1 could potentially function as an assembly factor for the cyclin D2/cdk4 complex in HTLV-1 infected cells. A by-product of this assembly with cyclin D2/cdk4 is the sequestration of p21/waf1 away from the cyclin E/cdk2 complex, allowing this active cyclin-cdk complex to phosphorylate Rb pocket proteins efficiently and push cells through the G1/S checkpoint. These two distinct functional and physical activities of p21/waf1 suggest that RNA tumor viruses manipulate the G1/S checkpoint by deregulating cyclin and cdk complexes.

Structural basis for the recognition of the E2F transactivation domain by the retinoblastoma tumor suppressor.

Repression of E2F transcription activity by the retinoblastoma (Rb) tumor suppressor through its interaction with the transactivation domain of the E2F transcription factor is one of the central features of G1/S arrest in the mammalian cell cycle. Deregulation of the Rb-E2F interaction results in hyperproliferation, lack of differentiation, and apoptosis, and can lead to cancer. The 2.2-A crystal structure of the Rb pocket complexed with an 18-residue transactivation-domain peptide of E2F-2 reveals that the boomerang-shaped peptide binds to the highly conserved interface between the A-box and the B-box of the Rb pocket in a bipartite manner. The N-terminal segment of the E2F-2 peptide in an extended beta-strand-like structure interacts with helices from the conserved groove at the A-B interface, whereas the C-terminal segment, which contains one 3(10) helix, binds to a groove mainly formed by A-box helices. The flexibility in the middle of the E2F-2 peptide is essential for the tight association of E2F to the Rb pocket. The binding of Rb to the E2F-2 peptide conceals several conserved residues that are crucial for transcription activation of E2F. We provide the structural basis for the Rb-mediated repression of E2F transcription activity without the requirement of histone-modifying enzymes.