SUMO wrestling with cell cycle progression Outline SUMO - - PowerPoint PPT Presentation

SUMO wrestling with cell cycle progression

• SUMO Introduction
• A critical role for SUMO in cell cycle progression
• SUMO regulates the Forkhead Box protein FoxM1
• SUMO regulates the Anaphase Promoting Complex

Outline

ubiquitin SUMO-1

Structural similarity between ubiquitin and SUMO

E3

SUMO

substrate

SUMO SUMO SUMO

Ubc9

SUMO

AMP

protease

SAE1 SAE2

SUMO

substrate SAE1-SAE2 ATP precursor E2 E1

SUMO-1 SUMO-2

SUMOylation cycle

SUMO-3

Bergink & Jentsch 2009 Nature

Comparing ubiquitin and SUMO systems

Cubenas-Potts et al., 2013 Dev. Cell

Molecular consequences of SUMOylation

30/09/2013 Proteasome Targe,ng Protein Targe,ng Complex Assembly Enzyme Regula,on

EYFP-SUMO-2

DNA Damage Response Transcription Ribosome Assembly Pre-mRNA Splicing Nuclear-Cytoplasmic Transport Cell Cycle Progression

SUMOylation regulates nuclear processes

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

SUMOylation consensus motif: ΨKxE Ψ = L, I, V, M, F

Target protein SUMO

ΨKxE

UBC9 interacts with SUMOylation consensus motifs

Bernier-Villamore et al. 2002 Cell

UBC9 RanGAP1

Proteins can non-covalently bind a SUMO moiety through a short conserved sequence termed a SUMO-Interaction Motif (SIM).

SUMO SIM

Sekiyama 2008 J. Biol. Chem. ZZ Zinc finger in HERC2 _ Danielsen et al. 2012 J. Cell Biol.

SUMO group modification in DNA repair

Psakhye & Jentsch 2012 Cell

anti-SUMO-2/3

acceptor site target K

SUMO

19 97 28 64 51 39 14 191

SUMO

Hendriks et al. 2014 & 2017 Nature Struct Mol Biol Hendriks et al. 2015 Nature Comm Schimmel et al. 2014 Molecular Cell Hendriks et al. 2016 Nature Rev Mol Cell Biol

SUMO target proteomics

Site-Specific SUMOylation Proteomics

substrate K

SUMO R FDGQPINETDTPAQLEMEDEDTIDVFQQQTGG XXXKXXXR/K chimeric peptide too large for mass spec R QQTGG XXXKXXXR/K 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

• 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

Outline

A critical role for SUMO in cell cycle progression

Mitosis G1 phase 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

• n SUMOylation

Kessler et al. 2012 Science

c-Myc driven tumors are dependent

• n SUMOylation

Kessler et al. 2012 Science

Prophase Prometaphase Metaphase Anaphase Telophase and cytokinesis Interphase

Day 0 Plate stable HeLa cell lines Day 1 Knockdown inducDon by doxycyclin Day 3 AddiDon of SiR-DNA and imaging NEB Chromosome alignment Chromosome segregaDon

SUMO in mitosis

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

Nacerddine…Dejean. 2005 Dev Cell

anaphase bridge UBE2I -/- UBE2I -/- UBE2I +/+ UBE2I +/+

Schimmel et al. 2014 Molecular Cell

Proteomic analysis of SUMOylation during cell cycle progression

input non-bound IP IP input IP IP input IP IP

14 19 28 39 51 64 97 191

input non-bound IP IP input non-bound IP IP input non-bound IP IP I II III I II III non-bound non-bound PonceauS an,-Flag

Purification of Flag-SUMO-2 conjugates

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

31

OverrepresentaDon of SUMO regulated proteins

Gene Ontology enrichment analysis of SUMO regulated proteins vs. background proteins

Summary of mass spectrometry screen

Schimmel et al. 2014 Molecular Cell

Confirmation of SUMOylation dynamics

Schimmel et al. 2014 Molecular Cell

• 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

Outline

34

FuncDonal analysis

Schimmel et al. 2014 Molecular Cell

STRING analysis reveals a highly interconnected protein network

• Specifically expressed in all proliferating cells
• Expression and transcriptional activity are tightly regulated during

the cell cycle:

• FoxM1 activity is essential for proper cell-cycle progression by

activating the expression of G2/M specific genes

Inhibition of FoxM1 activity Increase of FoxM1 expression Activation by phosphorylation

FoxM1: a Forkhead transcription factor

36

Laoukili et al. 2005 Nature Cell Biology

FoxM1 deficiency results in accumulation

• f tetraploid and polyploid cells

30/09/2013 37

FoxM1 SUMOylation during cell cycle progression

Schimmel et al. 2014 Molecular Cell

38

FoxM1 is extensively SUMOylated

His Pulldown His Pulldown His Pulldown His Pulldown

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 NRD FKH TAD

SUMOylation:

SUMO SUMO SUMO SUMO SUMO

NRD FKH TAD

SUMO SUMO SUMO SUMO SUMO Transciption Transciption Transciption

Schimmel et al. 2014 Molecular Cell

43

Increased polyploid population of cells expressing FoxM1 12KR

Schimmel et al. 2014 Molecular Cell

• 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

Outline

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

Chang et al. 2014 Nature Brenda Schulman lab David Barford lab

Classical function of APC/C during mitosis

Prometaphase Metaphase Anaphase APC/C

Securin

Ub Ub Ub Ub

Cyclin B1

Ub Ub Ub Ub

Separase ubiquiDnaDon + degradaDon release + acDvaDon cleavage of cohesins

APC/C subunit APC4 is SUMOylated

64 51 191 97 39 28 19 anD-SUMO 2/3

Eifler et al. 2018 Nature Comm

APC4 knockdown blocks cells in G2/M phase

shRNA 2 shRNA 1

G1 S G2/M

control virus control virus shRNA1 shRNA2 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

SUMO SUMO CDH1 Hsl1

APC3 APC4 SAE1/2

SUMO SUMO SUMO

UBC9 SAE1/2 UBC9

CDH1 Hsl1 Ub

UBA1 UbcH10

Ub Ub Ub Ub Ub

Y Y

ProtA

APC3 APC4

• 1. Purification of APC/C complex

Y

ProtA

APC3 APC4

• 2. In vitro SUMOylation

Y

ProtA

SUMO SUMO CDH1 Hsl1

APC3 APC4

• 3. Binding of substrate

and coactivator

SUMO SUMO CDH1 Hsl1

APC3 APC4

• 4. Elution from beads
• 5. In vitro ubiquitination of

substrate

Does APC4 SUMOylation affect APC/C?

Eifler et al. 2018 Nature Comm

APC1 APC2 APC3 APC4 APC5 APC6 APC7 APC8 191 97 64 51 39 28 14

Y

ProtA

APC3 APC4

• 1. Purification of APC/C complex

Does APC4 SUMOylation affect APC/C?

Eifler et al. 2018 Nature Comm

SAE1/2

SUMO SUMO SUMO

UBC9 APC4

• 2. In vitro SUMOylation

APC4 is the main SUMOylated APC/C subunit

Eifler et al. 2018 Nature Comm

SAE1/2 UBC9

CDH1 Hsl1

Y

ProtA

SUMO SUMO CDH1 Hsl1

APC3 APC4

• 3. Binding of substrate

and coactivator

• +

+ +

• +

+ + m WT m WT WT m WT m WT WT

• +
• +
• SUMO

+SUMO an,-Hsl1 an,-APC6 an,-Cdh1 Cdh1 Hsl1 E2+Ub 95 72 55

Does APC4 SUMOylation affect APC/C ?

Eifler et al. 2018 Nature Comm

0’ 2’ 10’ 30’ 0’ 2’ 10’ 30’

• SUMO

+SUMO an,-Hsl1 an,-APC11 98 62 14 6 +Ub:

SUMO SUMO CDH1 Hsl1

APC3 APC4

Ub Ub Ub Ub Ub

• 5. EluDon from beads
• 6. In vitro ubiquiDnaDon of substrate

Ub

UBA1 UbcH10

Y

In vitro acDvity of APC/C complex is enhanced a]er SUMOylaDon of APC4

Does APC4 SUMOylation affect APC/C ?

Eifler et al. 2018 Nature Comm

S S

In vitro SUMOyla,on Nega,ve control

S S

• Add cell lysate
• Bind to beads

and wash Elute with urea

S S

MS

Binding partners of SUMOylated APC/C

Eifler et al. 2018 Nature Comm

Recombinant APC/C complex

WT WT 2KR K772R K798R

• SUMO

+SUMO_K0 an,-APC4 10 µl assay unmodif. + 1 SUMO + 2 SUMOs 191 97 64 51

Recombinant APC/C: Brenda Schulman Eifler et al. 2018 Nature Comm

Binding partners of SUMOylated APC/C

Eifler et al. 2018 Nature Comm

Nature Reviews Cancer (2012) 12, 527-539

Motor domain Stalk Cargo Microtubule Unique non- motor domain

Kinesin KIF18B binds SUMOylated APC/C

KIF18B: In complex with KIF2C, constitutes the major microtubule plus-end depolymerizing activity in mitotic cells.

Ø APC4 SUMOylaDon increases the ubiquiDn E3 ligase acDvity of APC/C towards the novel human substrate KIF18B, but not towards securin.

KIF18B is a SUMO-dependent APC/C target

Eifler et al. 2018 Nature Comm

• SUMOylaDon of APC4 shi]s the preference of APC/C to a different

subset of targets, including KIF18B but not securin

– Via direct interacDon of these target with SUMOylated APC4 – Or through a conformaDonal change of the complex due to its SUMOylaDon

Ø Improper regulaDon of the APC/C during mitosis upon SUMO E1 knockdown might result in misregulaDon of this subset of targets, including KIF18B, resulDng in mitoDc delay and chromosome segregaDon defects

Fine-tuning APC/C activity by SUMO

Eifler et al.

A SIM motif in KIF18B binds SUMO in vitro

Eifler et al. 2018

• SUMO is critical for cell cycle progression
• SUMO regulates the Forkhead Box protein FoxM1
• SUMO regulates the Anaphase Promoting Complex

Summary

Daphne vd Elsland Sjaak Neefjes Jesper Olsen Matthias Mann Brenda Schulman Jan-Michael Peters René Medema Willeam Earnshaw

Acknowledgements