E2F and Signal Transduction Pathways

[Cell Cycle 4:3, 392-396; March 2005]; ©2005 Landes Bioscience

E2F and Signal Transduction Pathways Extra Views

ABSTRACT

*Correspondence to: Doron Ginsberg; Department of Molecular Cell Biology; The Weizmann Institute of Science; Rehovot 76100, Israel; Tel: 972.8.9342239; Fax: 972.8.9344125; Email: [email protected] Received 01/20/05; Accepted 01/24/05

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Genome-wide approaches recently undertaken to identify target genes of E2F transcription factors have helped to unveil and define the molecular basis for the involvement of E2Fs in a wide range of biological processes. These include differentiation, DNA replication, mitosis, the mitotic checkpoint, DNA damage checkpoints, DNA repair and apoptosis.1,2 Our recent observation that E2F positively affects the PI3-K/AKT signaling pathway through the transcriptional induction of the adaptor protein Gab23 implies that E2F also affects receptor-induced signaling. Here we discuss the possible involvement of E2F in additional signaling pathways and address the potential biological significance of the existence of such regulatory networks. The early signaling events that are triggered by growth factor receptor engagement and are necessary for immediate-early gene induction are rapid and transient. However, the initiation of S phase entry requires continuous exposure to mitogenic growth factors for several hours.4 Strikingly, in some settings, Ras, PI3-K and PKC exhibit a biphasic pattern of activity upon growth factor stimulation and their late activation constitutes one of the limiting signaling events required for the initiation of DNA synthesis.4 However, little is known about the mechanisms that regulate the late, delayed activation events following sustained growth factor stimulation. The question of how and when certain signaling cascades are regulated in cycling cells constantly exposed to growth factors has not been extensively addressed. The transcription-dependent control of the PI3-K/AKT signaling pathway by E2F3 suggests a possible mechanism for the late, delayed activity of PI3-K in stimulated cells. Several signaling pathways initiated by mitogenic stimuli ultimately converge to inactivate the function of the pRB family of pocket proteins leading to E2F activation.5 This represents the well-documented flow of information from the cell membrane to the nucleus. The effect of E2F on the expression of the adaptor Gab2 demonstrates a flow of information in the “reverse” direction, from a nuclear transcription factor to an upstream component of the PI3K/AKT pathway. Similarly, several other transcription factors have been shown to affect upstream components in signal transduction pathways. For example, p53 upregulates the expression of the PTEN phosphatase and the dual-specificity phosphatases PAC1 and DUSP5.6-8 An initial indication that E2F may transcriptionally regulate the activity of various signal transduction pathways came from the observation that many components of such pathways were found to be upregulated in screens that analyzed changes in gene expression in response to E2F.9-14 Potential E2F-regulated genes implicated in transducing extracellular signals include receptors, ligands, adaptor proteins and enzymes that function in a variety of signaling cascades (see Table 1).9-14 The identification of these genes as E2F-responsive targets currently relies mainly on microarray data and most of them have not been validated by additional assays. Nevertheless, future studies that demonstrate that E2F-mediated changes in expression of some of these genes affect signaling will help to establish a meaningful involvement of E2F in signal transduction. E2F-mediated transcriptional regulation of key players in signal transduction pathways may not, by itself, activate these pathways. However, it may sensitize cells and enable them to respond to sub-optimal stimuli that trigger these pathways. In this respect, it should be

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Previously published online as a Cell Cycle E-publication: http://www.landesbioscience.com/journals/cc/abstract.php?id=1556

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Department of Molecular Cell Biology; The Weizmann Institute of Science; Rehovot, Israel

The E2F family of transcription factors is most well known for its ability to regulate the expression of genes required for DNA replication and cell cycle progression. However, recent studies indicate that E2F can also regulate transcription of upstream components of signal transduction pathways. Here we discuss the effects of E2F activity on signal transduction pathways and its potential biological consequences.

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Marie Chaussepied Doron Ginsberg*

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We thank Jonathan Weitzman, Tzippi Hershko and Shirley Polager for critical reading of the manuscript. Work in the authors’ laboratory is supported by grants from the Israel Cancer Research Fund (ICRF) and the Israel Science Foundation. M.C. is supported by a post-doctoral fellowship from the Pasteur-Weizmann foundation. D.G. is an incumbent of the Recanati Career Development chair of Cancer research.

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ACKNOWLEDGEMENTS

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E2F, signal transduction, PI3-K/AKT

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KEY WORDS

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2005; Vol. 4 Issue 3

E2F and Signal Transduction Pathways

Table 1

Selected, putative E2F target genes involved in cell signaling Gene description

Reference

NOTCH3 MERTK TYRO3 EPHB6 EPHB4 FZD7 FZD8 FZD5 FZD1

fibroblast growth factor receptor 1 fibroblast growth factor receptor 2 fibroblast growth factor receptor 3 platelet-derived growth factor receptor, alpha polypeptide insulin-like growth factor 1 receptor platelet-derived growth factor receptor-like met proto-oncogene insulin receptor fms-related tyrosine kinase 4 bone morphogenetic protein receptor, type IA activin A receptor, type IIB activin A receptor, type IB activin A receptor, type I interleukin 15 receptor, alpha interleukin 1 receptor, type I interleukin 17 receptor B leukemia inhibitory factor receptor erythropoietin receptor tumor necrosis factor receptor superfamily, member 10b Notch gene homolog 1 (Drosophila) Notch homolog 3 c-mer proto-oncogene tyrosine kinase TYRO3 protein tyrosine kinase EPH receptor B6 EPH receptor B4 frizzled homolog 7 (drosophila) frizzled homolog 8 (drosophila) frizzled homolog 5 (drosophila) frizzled homolog 1 (drosophila)

10, 17 9, 27 9 9 9 9 10 10 9 10 9 10 9 9 10 10 9 9 10 11 9 9 9, 11 9, 10 9 9 10 9 10

ECGF1 VEGF VEGFC FGF9 FGF7 TGFAa BMP2a BMP4 BMP7 PBEF1 JAG2 JAG1 EFNA3 WNT10B

endothelial cell growth factor 1 (platelet-derived) vascular endothelial growth factor vascular endothelial growth factor C fibroblast growth factor 9 fibroblast growth factor 7 (keratinocyte growth factor) transforming growth factor, alpha bone morphogenetic protein 2 bone morphogenetic protein 4 bone morphogenetic protein 7 (osteogenic protein 1) pre-B-cell colony enhancing factor 1 jagged 2 jagged 1 (Alagille syndrome) ephrin-A3 wingless-type MMTV integration site family, member 10B

9 9, 11 9, 10 9 13 10 9, 10 9 9 10 9 9 9 10

PRKCD PRKCE PRKD3 PRKCZ PRKCI STK4

protein kinase C, delta protein kinase C, epsilon protein kinase D3 protein kinase C, zeta protein kinase C, iota serine/threonine kinase 4

10 10 9, 10 9 9 9

MRAS HRAS ARHN RHE RHOBTB2 RHOH RHOU RAP2A ARHGAP5

muscle RAS oncogene homolog v-Ha-ras Harvey rat sarcoma viral oncogene homolog ras homolog gene family, member N ras homolog gene family, member E Rho-related BTB domain containing 2 ras homolog gene family, member H ras homolog gene family, member U RAP2A, member of RAS oncogene family Rho GTPase activating protein 5

10 9 11 9 9 10 10 10 9

Gene symbol Receptors FGFR1a FGFR2a FGFR3a PDGFRA IGF1R PDGFRL METa INSR FLT4 BMPR1Aa ACVR2B ACVR1B ACVR1 IL15RA IL1R1 IL17RB LIFR EPOR TNFRSF10B

Ligands

Plasma Membrane to Cytoplasm Transduction Events Ser/Thr phosphorylation

Ras superfamilly

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Table 1

Selected, putative E2F target genes involved in cell signaling (continued)

Intracellular Signal Transduction Events Phosphorylation MAPK14a MAP4K2 MAP4K5 PRKACA CAMK2G DGKE MAP3K3 ARK5 STK24 DYRK1Aa DYRK2a TESK1 PRKRA ITPKA ITPK1

mitogen activated protein kinase 14 mitogen-activated protein kinase kinase kinase kinase 2 mitogen-activated protein kinase kinase kinase kinase 5 protein kinase, cAMP-dependent, catalytic, alpha calcium/calmodulin-dependent protein kinase (CaM kinase) II gamma diacylglycerol kinase, epsilon 64kDa mitogen activated protein kinase kinase kinase 3 AMP-activated protein kinase family member 5 serine/threonine kinase 24 (STE20 homolog, yeast) dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 1A dual-specificity tyrosine-(Y)-phosphorylation regulated kinase 2 testis-specific kinase 1 protein kinase, interferon-inducible double stranded RNA dependent activator inositol 1,4,5-trisphosphate 3-kinase A inositol 1,3,4-triphosphate 5/6 kinase

11 9 9 11 9 9 11 9 9, 10 10, 11 10 9 9 10 10

RASGRP1 RAPGEF2 TIAM1

RAS guanyl releasing protein 1 (calcium and DAG-regulated) Rap guanine nucleotide exchange factor (GEF) 2 T-cell lymphoma invasion and metastasis 1

9 9 10

DOK1 DVL3 AKAP1 TOB1 GRB7

docking protein 1, 62kDa (downstream of tyrosine kinase 1) dishevelled, dsh homolog 3 (Drosophila) A kinase (PRKA) anchor protein 1 transducer of ERBB2, 1 growth factor receptor-bound protein 7

9 9 9 9 11

AKAP12

A kinase (PRKA) anchor protein (gravin) 12

10

DUSP9 DUSP7 DUSP5 PPP2CA PPP3CA INPPL1 PTPN18

dual specificity phosphatase 9 dual specificity phosphatase 7 dual specificity phosphatase 5 protein phosphatase 2 (formerly 2A), catalytic subunit, alpha isoform protein phosphatase 3 (formerly 2B), catalytic subunit, alpha isoform (calcineurin A alpha) inositol polyphosphate phosphatase-like 1 protein tyrosine phosphatase, non-receptor type 18

9 9, 10 9 9 9 9 9

CBL

Cas-Br-M (murine) ecotropic retroviral transforming sequence

9

GDP/GTP exchange Protein-protein interaction

Dephosphorylation

Others

E2F target genes identified by microarray and ChIP-on chip analysis9-14 were functionally categorized using the DAVID (DAVID; http://www.david.niaid.nih.gov) annotation tool.26 A selected list of genes with Gene Ontology ascribed to Signal transducers and Catalytic activity is presented. aindicates genes for which induction by E2F has been validated by Northern analysis or RT-PCR.

noted that although elevated E2F activity caused AKT phosphorylation in serum-starved U-2OS cells, it was not sufficient to activate AKT in serum-starved human diploid fibroblasts (ref. 3 and MC and DG unpublished data). Unlike normal diploid fibroblasts, the human osteosarcoma U-2OS cell line exhibits autocrine receptor activation involving platelet-derived growth factor and insulin-like growth factor II.15,16 Thus, the differential response of U-2OS and WI38 cells may indicate that in U-2OS cells E2F potentiated, through a transcription-dependent mechanism, the transduction of existing signals. This implies that E2F activity may allow cells to respond more efficiently to growth factor stimulation. Indeed, Tashiro et al demonstrated that activation of the cyclin D/RB/E2F pathway sensitizes fibroblasts to bFGF and this is, most probably, attributed to transcriptional upregulation of FGF Receptor 1 by E2F.17 E2F activity is often deregulated in cancer cells.18 Cancer cells, whether in a primary or a metastatic lesion, must acquire the capacity to survive and proliferate in an initially unfavorable environment with limited availability of growth and survival factors.19 Thus, the effects of E2F on signaling pathways that control cell survival, cell 394

growth and cell proliferation may ultimately confer a selective advantage for the expansion of cells that exhibit deregulated E2F activity. Support for this notion comes from the observation that deregulated E2F expression has been shown to collaborate with activated Ras in the formation of papillomas in a transgenic mouse model.20 The molecular basis for this collaboration has not been elucidated but it is tempting to speculate that it is due to E2F-mediated sensitization to Ras-induced signals. In addition to this cell-autonomous activity, E2F may affect cell growth and survival in a non cell-autonomous fashion by altering the properties of the cellular and extracellular environment. As mentioned earlier, a number of potential E2F target genes identified by genome- wide approaches encode secreted or membrane-associated ligands (see Table 1)9-14 that may act in an autocrine and/or paracrine fashion. As the role and importance of the interaction between the tumor cells and the surrounding tissue in promoting tumor growth and progression has become the focus of increasing attention21 it will be interesting to understand whether and how deregulated E2F activity can contribute to shape a permissive and sup-

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portive microenvironment. Chronic inflammation A B predisposes to the development of cancer and both inflammatory and angiogenic responses accompany the transition from the premalignant to malignant stages.21 Interestingly, in E2F1-deficient mice the early steps in the wound healing process are significantly delayed, local inflammatory responses are reduced and the recruitment of inflammatory macrophages and neutrophils is also delayed.22 This maybe partially due to adhesion defects of E2F1 deficient cells but it may also represent signaling defects in these cells. The pRB family of pocket proteins negatively regulates E2F activity. The activation of various signal transduction pathways, including the PI3-K/AKT; RAL-GEFs/RAL; Raf/MEK/ERK and p38 pathways, inactivate the repressive function of pRB family members through a number of mechanisms that converge on their phosphorylation.5,23 Our findings3 suggest the existence of Figure 1. E2F regulates expression of components of signal transduction pathways. Receptor an amplification loop whereby E2F activity engagement by a cognate ligand promotes the recruitment and assembly of multimolecular potentiates PI3-K and AKT activation ultimately signaling complexes that initiate a cascade of biochemical events that ultimately effect gene leading to further E2F activation. Similar E2F- transcription. The triggering of a number of signaling pathways activates the E2F transcription mediated amplification loops may exist in addi- factor. Microarray analysis suggests that E2F also affect signal transduction by regulating the tional signal transduction pathways that contain transcription of components of signaling networks. E2F-upregulated gene products may function to control the activation of the same signaling pathway that led to E2F induction (A). Alternatively, an E2F-regulated component. Furthermore, such E2F target gene products may function in distinct signaling pathways (B). E2F-induced expression may provide a means of communication between two distinct signaling pathways. For example, one signal transduction pathway may lead to The duration and magnitude of the signals generated by receptor the activation of E2F that then transcriptionally upregulates the engagement are important in determining the biological output. A expression of a key component in a second signaling pathway thereby number of mechanisms act at various levels of the signaling cascade sensitizing the cell to this second pathway (Fig. 1). Of note, the to restrict the extent of activation. One such mechanism involves a existing DNA microarray data suggests that E2F coordinately regu- strict control of the phosphorylation status of proteins and lipids in lates sets of genes whose products participate in the same signaling the cell via a precise balance between kinase and phosphatase activipathways (see Table 1). ties. In this respect, the expression of several protein phosphatases, A recent DNA microarray analysis designed to study the relative including MAP/SAP kinases phosphatases and the lipid phosphatase contribution of release from pocket protein-mediated repression PTEN, has been shown to be subject to transcriptional regulation.6-8 versus transactivation by “free” E2F revealed that groups of E2F Negative regulators of receptor signaling may also be transcriptional target genes with common biological function were preferentially targets of E2F as suggested by the analysis of the existing microarray 9,10 Mechanisms likely exist to control the timely upregulated by either derepression or transactivation.9 A significant data (see Table 1). proportion of E2F target genes encoding growth factors and growth induction of positive versus negative regulators of receptor signaling. factor receptors, analyzed in this study, were upregulated both by The Nevins group has recently identified molecular mechanisms induction of E2F-regulated wild-type E2F1 and by an E2F1 mutant lacking the transactivation- that ensure the temporally restricted 24,25 It remains to be determined and pocket protein-binding domains (E2F1∆TA), suggesting that G1/S and G2/M specific genes. they are upregulated via derepression. Interestingly, Gab2 expression whether similar mechanisms operate in the E2F-signalling connection. was not induced by E2F1∆TA, however, expression of this mutant Although the study of E2F-mediated regulation of signal transduccaused an elevation in AKT phosphorylation (Chaussepied M, tion is in its early days, it opens new and exciting avenues of research Ginsberg D, unpublished data). These data indicate that E2F, most regarding the effects of deregulated E2F on tumor development. probably, regulates the expression of additional genes that function References in the PI3-K/AKT pathway. Their identity and mode of regulation 1. Bracken AP, Ciro M, Cocito A, Helin K. E2F target genes: Unraveling the biology. Trends remains to be determined. E2F1∆TA functions as a dominant Biochem Sci 2004; 29:409-17. negative mutant for both activation by E2F and repression by 2. DeGregori J. The genetics of the E2F family of transcription factors: Shared functions and unique roles. Biochim Biophys Acta 2002; 1602:131-50. E2F/RB complexes. Therefore, two nonexclusive interpretations 3. Chaussepied M, Ginsberg D. Transcriptional regulation of AKT activation by E2F. Mol could account for E2F1∆TA-induced AKT phosphorylation: it Cell 2004; 16:831-7. induces the derepression of gene(s) encoding positive regulators of 4. Jones SM, Kazlauskas A. Growth factor-dependent signaling and cell cycle progression. FEBS Lett 2001; 490:110-6. AKT activation; alternatively this mutant prevents the expression of 5. Sears RC, Nevins JR. Signaling networks that link cell proliferation and cell fate. J Biol a negative regulator of AKT phosphorylation. If E2F indeed regulates Chem 2002; 277:11617-20. both positive and negative regulators of AKT phosphorylation, this 6. Yin Y, Liu YX, Jin YJ, Hall EJ, Barrett JC. PAC1 phosphatase is a transcription target of may result in a transient induction of AKT phosphorylation by E2F. p53 in signalling apoptosis and growth suppression. Nature 2003; 422:527-31. www.landesbioscience.com

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7. Ueda K, Arakawa H, Nakamura Y. Dual-specificity phosphatase 5 (DUSP5) as a direct transcriptional target of tumor suppressor p53. Oncogene 2003; 22:5586-91. 8. Stambolic V, MacPherson D, Sas D, Lin Y, Snow B, Jang Y, Benchimol S, Mak TW. Regulation of PTEN transcription by p53. Mol Cell 2001; 8:317-25. 9. Young AP, Nagarajan R, Longmore GD. Mechanisms of transcriptional regulation by Rb-E2F segregate by biological pathway. Oncogene 2003; 22:7209-17. 10. Muller H, Bracken AP, Vernell R, Moroni MC, Christians F, Grassilli E, Prosperini E, Vigo E, Oliner JD, Helin K. E2Fs regulate the expression of genes involved in differentiation, development, proliferation, and apoptosis. Genes Dev 2001; 15:267-85. 11. Ma Y, Croxton R, Moorer Jr RL, Cress WD. Identification of novel E2F1-regulated genes by microarray. Arch Biochem Biophys 2002; 399:212-24. 12. Polager S, Kalma Y, Berkovich E, Ginsberg D. E2Fs upregulate expression of genes involved in DNA replication, DNA repair and mitosis. Oncogene 2002; 21:437-46. 13. Ren B, Cam H, Takahashi Y, Volkert T, Terragni J, Young RA, Dynlacht BD. E2F integrates cell cycle progression with DNA repair, replication, and G2/M checkpoints. Genes Dev 2002; 16:245-56. 14. Ishida S, Huang E, Zuzan H, Spang R, Leone G, West M, Nevins JR. Role for e2f in control of both DNA replication and mitotic functions as revealed from DNA microarray analysis. Mol Cell Biol 2001; 21:4684-99. 15. Betsholtz C, Westermark B, Ek B, Heldin CH. Coexpression of a PDGF-like growth factor and PDGF receptors in a human osteosarcoma cell line: Implications for autocrine receptor activation. Cell 1984; 39:447-57. 16. Raile K, Hoflich A, Kessler U, Yang Y, Pfuender M, Blum WF, Kolb H, Schwarz HP, Kiess W. Human osteosarcoma (U-2 OS) cells express both insulin-like growth factor-I (IGF-I) receptors and insulin-like growth factor-II/mannose-6-phosphate (IGF-II/M6P) receptors and synthesize IGF-II: Autocrine growth stimulation by IGF-II via the IGF-I receptor. J Cell Physiol 1994; 159:531-41. 17. Tashiro E, Maruki H, Minato Y, Doki Y, Weinstein IB, Imoto M. Overexpression of cyclin D1 contributes to malignancy by upregulation of fibroblast growth factor receptor 1 via the pRB/E2F pathway. Cancer Res 2003; 63:424-31. 18. Sherr CJ. Cancer cell cycles. Science 1996; 274:1672-7. 19. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000; 100:57-70. 20. Pierce AM, Fisher SM, Conti CJ, Johnson DG. Deregulated expression of E2F1 induces hyperplasia and cooperates with ras in skin tumor development. Oncogene 1998; 16:1267-76. 21. Mueller MM, Fusenig NE. Friends or foes - bipolar effects of the tumour stroma in cancer. Nat Rev Cancer 2004; 4:839-49. 22. D’Souza SJ, Vespa A, Murkherjee S, Maher A, Pajak A, Dagnino L. E2F-1 is essential for normal epidermal wound repair. J Biol Chem 2002; 277:10626-32. 23. Wang S, Nath N, Minden A, Chellappan S. Regulation of Rb and E2F by signal transduction cascades: Divergent effects of JNK1 and p38 kinases. Embo J 1999; 18:1559-70. 24. Giangrande PH, Zhu W, Schlisio S, Sun X, Mori S, Gaubatz S, Nevins JR. A role for E2F6 in distinguishing G1/S- and G2/M-specific transcription E2Fs link the control of G1/S and G2/M transcription. Genes Dev 2004; 18:294151. 25. Zhu W, Giangrande PH, Nevins JR. E2Fs link the control of G1/S and G2/M transcription. Embo J 2004; 23:4615-26. 26. Dennis Jr G, Sherman BT, Hosack DA, Yang J, Gao W, Lane HC, Lempicki RA. DAVID: Database for annotation, visualization, and integrated discovery. Genome Biol 2003; 4:3. 27. Tashiro E, Minato Y, Maruki H, Asagiri M, Imoto M. Regulation of FGF receptor-2 expression by transcription factor E2F-1. Oncogene 2003; 22:5630-5.

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