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

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

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

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

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

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

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

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

CDCA7 | Conservation AKT consensus site R X R X X T/S F/L CDCA7 T163 R P R R R T F >90% conserved human monkey dog mouse chicken frog zebrafish 24 49 69 78 112 190 261 363 AKT kinase 0.005% 1 T163 371 humCDCA7 zinc finger 261 361

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

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

CDCA7 is phosphorylated at t163 by AKT Inhibitors Vector CDCA7 Akt inh VIII IP: a-FLAG Blot: a-P-T163 Blot: a-FLAG

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

CDCA7 | Conservation How do we test for a nuclear localization signal? >90% conserved human monkey dog mouse chicken frog zebrafish 24 49 69 78 112 190 261 363 1 T163 371 humCDCA7 NLS? NLS? zinc finger 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

Where is cdca7 found? T163A CDCA7 a-Flag DAPI

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

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

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

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

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

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 Adapted from Hermeking, 2005 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

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

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

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

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

So how does cdca7 affect phenotype? Apoptosis proliferation

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

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

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

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