APC/CCdc20 targets E2F1 for degradation in prometaphase

The mechanisms that control E2F-1 activity are complex. We previously showed that Chk1 and Chk2 are required for E2F1 stabilization and p73 target gene induction following DNA damage. To gain further insight into the processes regulating E2F1 protein stability, we focused our investigation on the mechanisms responsible for regulating E2F1 turnover. Here we show that E2F1 is a substrate of the anaphase promoting complex or cyclosome (APC/C), a ubiquitin ligase that plays an important role in cell cycle progression. Ectopic expression of the APC/C activators Cdh1 and Cdc20 reduced the levels of co-expressed E2F-1 protein. Co-expression of DP1 with E2F1 blocked APC/C-induced E2F1 degradation, suggesting that the E2F1/DP1 heterodimer is protected from APC/C regulation. Following Cdc20 knockdown, E2F1 levels increased and remained stable in extracts over a time course, indicating that APC/CCdc20 is a primary regulator of E2F1 stability in vivo. Moreover, cell synchronization experiments showed that siRNA directed against Cdc20 induced an accumulation of E2F1 protein in prometaphase cells. These data suggest that APC/CCdc20 specifically targets E2F1 for degradation in early mitosis and reveal a novel mechanism for limiting free E2F1 levels in cells, failure of which may compromise cell survival and/or homeostasis.

[1]  E. Flemington,et al.  Regulation of E2F through ubiquitin-proteasome-dependent degradation: stabilization by the pRB tumor suppressor protein. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[2]  D. Livingston,et al.  The retinoblastoma gene product protects E2F-1 from degradation by the ubiquitin-proteasome pathway. , 1996, Genes & development.

[3]  W. Sellers,et al.  Transcription of the E2F-1 gene is rendered cell cycle dependent by E2F DNA-binding sites within its promoter , 1994, Molecular and cellular biology.

[4]  J. Nevins,et al.  Selective induction of E2F1 in response to DNA damage, mediated by ATM-dependent phosphorylation. , 2001, Genes & development.

[5]  W. Krek,et al.  Increased levels of E2F-1-dependent DNA binding activity after UV- or gamma-irradiation. , 1999, Nucleic acids research.

[6]  N. L. La Thangue,et al.  E2F and cell cycle control: a double-edged sword. , 2003, Archives of biochemistry and biophysics.

[7]  A. Shvarts,et al.  Degradation of E2F by the ubiquitin-proteasome pathway: regulation by retinoblastoma family proteins and adenovirus transforming proteins. , 1996, Genes & development.

[8]  Marc W. Kirschner,et al.  Autonomous regulation of the anaphase-promoting complex couples mitosis to S-phase entry , 2004, Nature.

[9]  C. Blattner,et al.  Transcription Factor E2F-1 Is Upregulated in Response to DNA Damage in a Manner Analogous to That of p53 , 1999, Molecular and Cellular Biology.

[10]  R. Spang,et al.  Role for E2F in Control of Both DNA Replication and Mitotic Functions as Revealed from DNA Microarray Analysis , 2001, Molecular and Cellular Biology.

[11]  David M. Livingston,et al.  p19ARF targets certain E2F species for degradation , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[12]  M. Kitagawa,et al.  Targeted disruption of Skp2 results in accumulation of cyclin E and p27Kip1, polyploidy and centrosome overduplication , 2000, The EMBO journal.

[13]  L. Johnston,et al.  Functional synergy between DP‐1 and E2F‐1 in the cell cycle‐regulating transcription factor DRTF1/E2F. , 1993, The EMBO journal.

[14]  C. Prives,et al.  p73 induction after DNA damage is regulated by checkpoint kinases Chk1 and Chk2. , 2004, Genes & development.

[15]  C. Wang,et al.  F-box protein Skp2: a novel transcriptional target of E2F , 2006, Oncogene.

[16]  Kim Nasmyth,et al.  Genes involved in sister chromatid separation are needed for b-type cyclin proteolysis in budding yeast , 1995, Cell.

[17]  M. Pagano,et al.  APC/C(Cdc20) controls the ubiquitin-mediated degradation of p21 in prometaphase. , 2007, Molecular cell.

[18]  V. Gorgoulis,et al.  Involvement of E2F transcription factor family in cancer. , 2005, European journal of cancer.

[19]  M. Scheffner,et al.  Interaction between ubiquitin–protein ligase SCFSKP2 and E2F-1 underlies the regulation of E2F-1 degradation , 1999, Nature Cell Biology.

[20]  Andrew M. Fry,et al.  Early mitotic degradation of Nek2A depends on Cdc20-independent interaction with the APC/C , 2006, Nature Cell Biology.

[21]  Wilhelm Krek,et al.  Negative regulation of the growth-promoting transcription factor E2F-1 by a stably bound cyclin A-dependent protein kinase , 1994, Cell.

[22]  D. Livingston,et al.  Binding to DNA and the retinoblastoma gene product promoted by complex formation of different E2F family members. , 1993, Science.

[23]  W. Krek,et al.  Increased levels of E2F-1-dependent DNA binding activity after UV- or γ-irradiation , 1999 .

[24]  Jeffrey M. Trimarchi,et al.  Transcription: Sibling rivalry in the E2F family , 2002, Nature Reviews Molecular Cell Biology.

[25]  J. Nevins,et al.  Toward an understanding of the functional complexity of the E2F and retinoblastoma families. , 1998, Cell growth & differentiation : the molecular biology journal of the American Association for Cancer Research.

[26]  A. Hershko,et al.  The cyclosome, a large complex containing cyclin-selective ubiquitin ligase activity, targets cyclins for destruction at the end of mitosis. , 1995, Molecular biology of the cell.

[27]  I. Screpanti,et al.  Differential regulation of E2F1 apoptotic target genes in response to DNA damage , 2003, Nature Cell Biology.

[28]  Sudhir Agrawal,et al.  Stabilization of E2F1 protein by MDM2 through the E2F1 ubiquitination pathway , 2005, Oncogene.

[29]  A. Gulino,et al.  Specific Role for p300/CREB-binding Protein-associated Factor Activity in E2F1 Stabilization in Response to DNA Damage* , 2004, Journal of Biological Chemistry.

[30]  M. Kirschner,et al.  A 20s complex containing CDC27 and CDC16 catalyzes the mitosis-specific conjugation of ubiquitin to cyclin B , 1995, Cell.

[31]  P. Farnham,et al.  Multiple DNA elements are required for the growth regulation of the mouse E2F1 promoter. , 1994, Genes & development.

[32]  A. Kentsis,et al.  The Mdm2 RING domain C‐terminus is required for supramolecular assembly and ubiquitin ligase activity , 2007, The EMBO journal.

[33]  Angelika Amon,et al.  The regulation of Cdc20 proteolysis reveals a role for the APC components Cdc23 and Cdc27 during S phase and early mitosis , 1998, Current Biology.

[34]  A. Blais,et al.  E2F-associated chromatin modifiers and cell cycle control. , 2007, Current opinion in cell biology.

[35]  Tim Hunt,et al.  Anaphase-Promoting Complex/Cyclosome–Dependent Proteolysis of Human Cyclin a Starts at the Beginning of Mitosis and Is Not Subject to the Spindle Assembly Checkpoint , 2001, The Journal of cell biology.

[36]  C. Stevens,et al.  Chk2 activates E2F-1 in response to DNA damage , 2003, Nature Cell Biology.

[37]  M. Giacca,et al.  Early mitotic degradation of the homeoprotein HOXC10 is potentially linked to cell cycle progression , 2003, The EMBO journal.

[38]  Yong Bok Park,et al.  Transcriptional repression of E2F gene by proteasome inhibitors in human osteosarcoma cells. , 2004, Biochemical and biophysical research communications.

[39]  S. Moreno,et al.  Cdh1/Hct1-APC Is Essential for the Survival of Postmitotic Neurons , 2005, The Journal of Neuroscience.

[40]  Y. Xiong,et al.  Phosphorylation- and Skp1-independent in vitro ubiquitination of E2F1 by multiple ROC-cullin ligases. , 2001, Cancer research.