SUMO wrestling with cell cycle progression
Outline • SUMO Introduction • A critical role for SUMO in cell cycle progression • SUMO regulates the Forkhead Box protein FoxM1 • SUMO regulates the Anaphase Promoting Complex
Structural similarity between ubiquitin and SUMO SUMO-1 ubiquitin
SUMOylation cycle substrate protease SUMO SUMO precursor substrate SUMO SAE1-SAE2 E3 ATP AMP SUMO Ubc9 SUMO E2 E1 SAE1 SAE2 SUMO SUMO-1 SUMO-2 SUMO-3
Comparing ubiquitin and SUMO systems Bergink & Jentsch 2009 Nature
Molecular consequences of SUMOylation Protein Complex Assembly Targe,ng Proteasome Enzyme Regula,on Targe,ng 30/09/2013 Cubenas-Potts et al., 2013 Dev. Cell
SUMOylation regulates nuclear processes Cell Cycle Progression Transcription EYFP-SUMO-2 DNA Damage Response Ribosome Assembly Pre-mRNA Splicing Nuclear-Cytoplasmic Transport
SUMO-2 is essential for embryonic development Wang et al. 2014 EMBO Reports
SENP1-deficient mouse embryos are aenemic Cheng et al. 2007 Cell
SUMOylation consensus sites SUMO Ψ KxE Target protein SUMOylation consensus motif: Ψ KxE Ψ = L, I, V, M, F
UBC9 interacts with SUMOylation consensus motifs UBC9 RanGAP1 Bernier-Villamore et al. 2002 Cell
Proteins can non-covalently bind a SUMO moiety through a short conserved sequence termed a SUMO-Interaction Motif (SIM). SUMO SIM ZZ Zinc finger in HERC2 _ Danielsen et al. 2012 J. Cell Biol. Sekiyama 2008 J. Biol. Chem.
SUMO group modification in DNA repair Psakhye & Jentsch 2012 Cell
SUMO target proteomics acceptor site SUMO 191 K 97 target 64 51 39 28 19 SUMO 14 Hendriks et al. 2014 & 2017 Nature Struct Mol Biol Hendriks et al. 2015 Nature Comm anti-SUMO-2/3 Schimmel et al. 2014 Molecular Cell Hendriks et al. 2016 Nature Rev Mol Cell Biol
Site-Specific SUMOylation Proteomics SUMO K chimeric peptide XXX K XXXR/K substrate too large for mass spec R FDGQPINETDTPAQLEMEDEDTIDVFQQQTGG XXX K XXXR/K R QQTGG suitable for mass spec
The SUMO Consensus Motif Hendriks et al. 2014 Nature Struct Mol Biol
SUMOylation dynamics Hendriks et al. 2016 Nature Rev Mol Cell Biol
Outline • Global overview of SUMOylation system • A critical role for SUMO in cell cycle progression • SUMO regulates the Forkhead Box protein FoxM1 • SUMO regulates the Anaphase Promoting Complex
A critical role for SUMO in cell cycle progression G1 phase Mitosis S phase G2 phase
Knockdown of the SUMOylation machinery reduces cell proliferation Colony forma,on assay (day 10) Schimmel et al. 2014 Molecular Cell
c-Myc driven tumors are dependent on SUMOylation Kessler et al. 2012 Science
c-Myc driven tumors are dependent on SUMOylation Kessler et al. 2012 Science
SUMO in mitosis NEB Interphase Prophase Prometaphase Metaphase Chromosome Anaphase alignment Telophase and cytokinesis Chromosome segregaDon Day 0 Day 1 Day 3 Plate stable Knockdown inducDon AddiDon of SiR-DNA HeLa cell lines by doxycyclin and imaging
Mitotic delay upon SUMO E1 knockdown Ø Knockdown of the SUMO E1 subunits leads to a delay in mitotic progression and chromosome segregation defects Eifler et al. 2018
SUMO machinery knockdown
The SUMO Pathway Is Essential for Chromosomal Integrity in Mice UBE2I -/- UBE2I -/- UBE2I +/+ UBE2I +/+ anaphase bridge Nacerddine … Dejean. 2005 Dev Cell
Proteomic analysis of SUMOylation during cell cycle progression Schimmel et al. 2014 Molecular Cell
Purification of Flag-SUMO-2 conjugates I II I II III III non-bound non-bound non-bound non-bound non-bound non-bound input input input input input input IP IP IP IP IP IP IP IP IP IP IP IP 191 97 64 51 39 28 19 14 PonceauS an,-Flag Schimmel et al. 2014 Molecular Cell
Summary of mass spectrometry screen Schimmel et al. 2014 Molecular Cell
Summary of mass spectrometry screen 328 SUMOylated proteins are regulated in a cell cycle dependent manner Schimmel et al. 2014 Molecular Cell
Summary of mass spectrometry screen Gene Ontology enrichment analysis of SUMO regulated proteins vs. background proteins OverrepresentaDon of SUMO regulated proteins 31 Schimmel et al. 2014 Molecular Cell
Confirmation of SUMOylation dynamics Schimmel et al. 2014 Molecular Cell
Outline • Global overview of SUMOylation system • A critical role for SUMO in cell cycle progression • SUMO regulates the Forkhead Box protein FoxM1 • SUMO regulates the Anaphase Promoting Complex
STRING analysis reveals a highly interconnected protein network FuncDonal analysis 34 Schimmel et al. 2014 Molecular Cell
FoxM1: a Forkhead transcription factor • Specifically expressed in all proliferating cells • Expression and transcriptional activity are tightly regulated during the cell cycle: Inhibition of FoxM1 activity Increase of FoxM1 expression Activation by phosphorylation • FoxM1 activity is essential for proper cell-cycle progression by activating the expression of G2/M specific genes
FoxM1 deficiency results in accumulation of tetraploid and polyploid cells Laoukili et al. 2005 Nature Cell Biology 36
FoxM1 SUMOylation during cell cycle progression 30/09/2013 Schimmel et al. 2014 Molecular Cell 37
FoxM1 is extensively SUMOylated His Pulldown His Pulldown His Pulldown His Pulldown 38 Schimmel et al. 2014 Molecular Cell
SUMOylation is required for FoxM1 activity Schimmel et al. 2014 Molecular Cell
Autorepression of FoxM1 NRD FKH TAD NRD FKH TAD Transciption Laoukili J. et al. (2008) Mol. Cell. Biol. 28:3076-87
Autorepression of FoxM1 NRD FKH TAD NRD FKH TAD Transciption Schimmel et al. 2014 Molecular Cell
Model: SUMOylation relieves FoxM1 autorepression NRD FKH TAD NRD FKH TAD Transciption SUMOylation: Transciption NRD FKH TAD SUMO SUMO SUMO SUMO SUMO Transciption NRD FKH TAD SUMO SUMO SUMO SUMO SUMO Schimmel et al. 2014 Molecular Cell
Increased polyploid population of cells expressing FoxM1 12KR Schimmel et al. 2014 Molecular Cell 43
Outline • Global overview of SUMOylation system • A critical role for SUMO in cell cycle progression • SUMO regulates the Forkhead Box protein FoxM1 • SUMO regulates the Anaphase Promoting Complex Karolin Eifler-Olivi
Mitotic delay upon SUMO E1 knockdown Ø Knockdown of the SUMO E1 subunits leads to a delay in mitotic progression and chromosome segregation defects Eifler et al. 2018
The Anaphase Promoting Complex Brenda Schulman lab David Barford lab Chang et al. 2014 Nature
Classical function of APC/C during mitosis APC/C ubiquiDnaDon + degradaDon Ub Ub Ub Ub Ub Ub Ub Ub Securin Cyclin B1 release + acDvaDon Separase cleavage of cohesins Prometaphase Metaphase Anaphase
APC/C subunit APC4 is SUMOylated 191 97 64 51 39 28 19 anD-SUMO 2/3 Eifler et al. 2018 Nature Comm
APC4 knockdown blocks cells in G2/M phase control virus shRNA 1 shRNA 2 G1 S G2/M shRNA1 shRNA2 control virus Eifler et al. 2018 Nature Comm
Mutation of K772 and K798 in APC4 abolishes APC4 SUMOylation Eifler et al. 2018 Nature Comm
Functional relevance of APC/C SUMOylation Eifler et al. 2018 Nature Comm
Functional relevance of APC/C SUMOylation Eifler et al. 2018 Nature Comm
Does APC4 SUMOylation affect APC/C? ProtA SAE1/2 ProtA Y CDH1 SUMO Hsl1 Y APC3 UBC9 APC3 APC4 SAE1/2 ProtA APC4 SUMO SUMO Y 1. Purification of APC/C complex 2. In vitro SUMOylation UBC9 APC3 3. Binding of substrate Hsl1 CDH1 and coactivator UBA1 Ub APC3 Ub Ub Ub Ub Ub APC3 APC4 UbcH10 SUMO SUMO Hsl1 Hsl1 CDH1 CDH1 APC4 APC4 SUMO SUMO SUMO SUMO Eifler et al. 2018 5. In vitro ubiquitination of 4. Elution from beads Y Nature Comm substrate
Does APC4 SUMOylation affect APC/C? APC1 191 APC2 97 ProtA APC3 APC4 Y APC5 APC3 APC6 64 APC7 APC8 APC4 51 1. Purification of APC/C complex 39 28 14 Eifler et al. 2018 Nature Comm
APC4 is the main SUMOylated APC/C subunit SAE1/2 SUMO UBC9 APC4 SUMO SUMO 2. In vitro SUMOylation Eifler et al. 2018 Nature Comm
Does APC4 SUMOylation affect APC/C ? Cdh1 - - + + + - - + + + CDH1 Hsl1 m WT m WT WT m WT m WT WT Hsl1 E2+Ub - - - - + - - - - + an,-Hsl1 95 an,-APC6 72 SAE1/2 ProtA an,-Cdh1 Y 55 UBC9 APC3 -SUMO +SUMO Hsl1 CDH1 APC4 SUMO SUMO 3. Binding of substrate and coactivator Eifler et al. 2018 Nature Comm
Does APC4 SUMOylation affect APC/C ? -SUMO +SUMO UBA1 Ub +Ub: 0’ 2’ 10’ 30’ 0’ 2’ 10’ 30’ UbcH10 98 62 Ub Ub Ub Ub Ub an,-Hsl1 APC3 14 Hsl1 CDH1 6 Y an,-APC11 APC4 SUMO SUMO 5. EluDon from beads In vitro acDvity of APC/C complex is 6. In vitro ubiquiDnaDon of substrate enhanced a]er SUMOylaDon of APC4 Eifler et al. 2018 Nature Comm
Binding partners of SUMOylated APC/C S S In vitro SUMOyla,on S S S S -Add cell lysate Elute with -Bind to beads urea MS and wash Nega,ve control Eifler et al. 2018 Nature Comm
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