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CDCA7 A case study in cellular regulation

CDCA7 A case study in cellular regulation. January 23 rd , 2014 Tim Gabor | Dr. Michael Scheid| York University. Lecture key words. Cell cycle Transcription factors Phosphorylation Heterodimerization Immunohistochemistry Immunprecipitation Growth factors Apoptosis Colony formation

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CDCA7 A case study in cellular regulation

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  1. CDCA7 A case study in cellular regulation January 23rd, 2014 Tim Gabor | Dr. Michael Scheid| York University

  2. Lecture key words • Cell cycle • Transcription factors • Phosphorylation • Heterodimerization • Immunohistochemistry • Immunprecipitation • Growth factors • Apoptosis • Colony formation • Nuclear localization • Consensus sequences • Motifs

  3. CDCA7 | a case study in cellular regulation • Cell cycle control is the endgame of cellular regulation - critical balance between proliferation and apoptosis  CANCER

  4. CDCA7 | a case study in cellular regulation • Cell cycle control is the endgame of cellular regulation - critical balance between proliferation and apoptosis  CANCER • Modes: • -phosphorylation, • -subcellular localization - heterodimerization

  5. CDCA7 | What is known • Myc and E2F target gene with peak expression at G1-S • Novel member of cell division cycle-associated gene family • Frequently overexpressed in human tumors • JPO2 binds Myc and promotes Myc dependent transformation • JPO2 and CDCA7 share cysteine rich C-term which may bind DNA • Not known if CDCA7 interacts with Myc

  6. Myc | Just the facts • Discovered in Burkitt’s lymphoma patients • Member of bHLH-LZ family of transcription factors • Requires heterodimerization with Max to transactivate • Regulates the expression of ~10-15% of genes • Role in development, cell division, cell growth, metabolism, angiogenesis • Early response gene induced by growth factors, levels peak at G0-G1 • Driving force of cell cycle and malignant transformation • Active in 70% of human cancers • ~100,000 cancer deaths per year in the US due to changes in Myc

  7. Myc PI3K Myc AKT AKT AKT P P P P P P P P P P P P rictor TOR Growth Factors PIP2 PIP3 Receptor Tyrosine Kinase 14-3-3 Cytoplasm PDK1 14-3-3 CDCA7 14-3-3 14-3-3 CDCA7 14-3-3 14-3-3 CDCA7 Pro-apoptotic Genes ? Transcription Nucleus

  8. CDCA7 | a case study in cellular regulation • Cell cycle control is the endgame of cellular regulation - critical balance between proliferation and apoptosis  CANCER • Modes: • -phosphorylation • -subcellular localization -heterodimerization

  9. CDCA7 | Conservation AKT consensus site RXRXXT/SF/L CDCA7 T163 RPRRRTF >90% conserved human monkey dog mouse chicken frog zebrafish 112 24 49 69 78 190 261 363 AKT kinase 0.005% 371 T163 1 humCDCA7 zinc finger 261 361

  10. CDCA7 is phosphorylated at t163 • Many ways to prove phosphorylation • Custom made antibody against phospho-T163 WT CDCA7+ CIP T163A + CIP Vector T163A CDCA7 T163A Radioactivity and mutational analysis Phosphotase a-FLAG a-P-T163

  11. CDCA7 is phosphorylated at t163 Treatments w/ growth factors T= 0’ T= 20’ T= 30’ Immunohistochemistry T= 40’ T= 50’ T= 60’ Vector 360 15 45 120 5 0 PDGF (min) a-P-T163 a-FLAG Merge 3.9 3.6 4.7 4.0 2.2 1.0 Ratio P-T163/ Total CDCA7

  12. CDCA7 is phosphorylated at t163 by AKT Inhibitors IP: a-FLAG Blot: a-P-T163 CDCA7 Aktinh VIII Vector IP: a-FLAG Blot: a-FLAG

  13. CDCA7 | a case study in cellular regulation • Cell cycle control is the endgame of cellular regulation - critical balance between proliferation and apoptosis  CANCER • Modes: • -phosphorylation • -subcellular localization -heterodimerization

  14. CDCA7 | Conservation >90% conserved human monkey dog mouse chicken frog zebrafish 112 24 49 69 78 190 261 363 371 T163 1 humCDCA7 zinc finger NLS? NLS? 261 361 157-186 RRPRRRTFPGVASRRNPERRARPLTRSRSR How do we test for a nuclear localization signal? Isolate region in question and test its ability to target an innocuous protein to the nucleus

  15. Where is cdca7 found? a-Flag DAPI CDCA7 T163A CDCA7

  16. CDCA7 contains an NLS • Passive diffusion into nucleus <45 KDa SV40 SV40 KE R171E 157-188 R171/176E R176E 157-188 CDCA7 157-167 CDCA7 157-188 (T163A) 167-188 CDCA7 R184E R176/184E 157-RRPRRRTFPGVASRRNPERRARPLTRSRSRIL-188 Supplementary Figure 2

  17. Phosphorylation alters localization Nuclei Merge a-CDCA7 Unstimulated PDGF PDGF + LY Supplementary Figure 3

  18. CDCA7 | a case study in cellular regulation • Cell cycle control is the endgame of cellular regulation - critical balance between proliferation and apoptosis  CANCER • Modes: • -phosphorylation • -subcellular localization -heterodimerization

  19. CDCA7 | Conservation >90% conserved human monkey dog mouse chicken frog zebrafish 112 24 49 69 78 190 261 363 371 T163 1 humCDCA7 zinc finger NLS? NLS? 261 361 157-186 RRPRRRTFPGVASRRNPERRARPLTRSRSR 14-3-3 consensus binding site R-[S/F/Y]-X-pS/T -X -P cdcA7 T163 RRR T F P Mekk2 T283 G R K T F P

  20. 14-3-3 | Just the facts • Large family of highly conserved, small, acidic polypeptides of 28-33 kDa • Seven different isoforms in humans, 14-3-3σ directly implicated in cancer • Binds to protein ligands at defined phospho-serine/threonine motif RSXpS/TXP • Over 200 known ligands • 14-3-3 regulates process relevant to cancer biology: cell-cycle progression, apoptosis and mitogenic signaling

  21. 14-3-3 | Modes of influence • 14-3-3 exists as a dimer and offers two binding sites for phospho-S/T motifs • Can function as adaptor protein for: • a) two proteins that would otherwise not associate • b) one protein with two 14-3-3 motifs = high affinity • Affects change by: • Alteration of enzymatic activity – maintains RAF1 in inactive state • Alteration of DNA-binding activity – increases p53 DNA-binding after DNA damage • Sequestration - BAD, FKHRL1, HDAC5 and CDC25C are localized to cytoplasm • Altering protein-protein interactions - reduced affinity of CDC25A to CDC2 • Adaptor protein functions – bridging of RAF1 to BCR • Sequestration • Altering protein-protein interactions Adapted from Hermeking, 2005

  22. CDCA7 binds14-3-3 and is phospho dependent Western blots S/F/Y 14-3-3 consensus site - - R X pT X P Wildtype P159A R158A P165A R160A R162A T163A F164A R161A Vector Blot: a-FLAG a-P-T163 a-14-3-3

  23. 14-3-3 alters CDCA7 localization Is 14-3-3 masking the NLS within the T163 region? a-Flag DAPI CDCA7 T163A CDCA7 R161A CDCA7 R161A/ T163A CDCA7

  24. CDCA7 | What is known • Myc and E2F target gene with peak expression at G1-S • Novel member of cell division cycle-associated gene family • Frequently overexpressed in human tumors • JPO2 binds Myc and promotes Myc dependent transformation • JPO2 and CDCA7 share cysteine rich C-term which may bind DNA • Not known if CDCA7 interacts with Myc

  25. CDCA7 binds the transcription factor Myc Co-immunoprecipitation D(230-370) CDCA7 D(170-370) CDCA7 D(260-370) CDCA7 D(153-370) CDCA7 D(112-137) CDCA7 D(1-234) CDCA7 D(1-146) CDCA7 D(1-172) CDCA7 D(1-202) CDCA7 T163A CDCA7 CDCA7 WT CDCA7 + WT + T163A + D(112-137) + D(1-146) - D(1-172) - D(1-202) His-Myc Pulldown Blot: a-FLAG - D(1-234) + D(260-370) + D(230-370) + D(170-370) - D(153-370) Input Blot: a-FLAG

  26. So how does cdca7 affect phenotype? Apoptosis proliferation

  27. 14-3-3/CDCA7 binding influence Myc-induced transformation Colony formation assay

  28. 14-3-3/CDCA7 binding influence Myc-induced apoptosis Rat1 Trypan blue exclusion Myc-Rat1 Sh1-Myc-Rat1

  29. Myc PI3K Myc AKT AKT AKT P P P P P P P P P P P P rictor TOR Growth Factors PIP2 PIP3 Receptor Tyrosine Kinase 14-3-3 Cytoplasm PDK1 14-3-3 CDCA7 14-3-3 14-3-3 CDCA7 14-3-3 14-3-3 CDCA7 Pro-apoptotic Genes ? Transcription Nucleus

  30. summary • CDCA7 is a novel target of AKT required for Myc-dependent apoptosis • Phosphorylation of T163 inhibits CDCA7/Myc apoptosis by: • Promoting 14-3-3 binding • Disruption of Myc binding • Shuttling to the cytoplasm • Potential for medical intervention in Myc tumors where AKT is dysregulated

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