In Vitro SUMOylation Assay to Study SUMO E3 Ligase Activity

Summary

Unlike ubiquitin ligases, few E3 SUMO ligases have been identified. This modified in vitro SUMOylation protocol is able to identify novel SUMO E3 ligases by an in vitro reconstitution assay.

Abstract

Small ubiquitin-like modifier (SUMO) modification is an important post-translational modification (PTM) that mediates signal transduction primarily through modulating protein-protein interactions. Similar to ubiquitin modification, SUMOylation is directed by a sequential enzyme cascade including E1-activating enzyme (SAE1/SAE2), E2-conjugation enzyme (Ubc9), and E3-ligase (i.e., PIAS family, RanBP2, and Pc2). However, different from ubiquitination, an E3 ligase is non-essential for the reaction but does provide precision and efficacy for SUMO conjugation. Proteins modified by SUMOylation can be identified by in vivo assay via immunoprecipitation with substrate-specific antibodies and immunoblotting with SUMO-specific antibodies. However, the demonstration of protein SUMO E3 ligase activity requires in vitro reconstitution of SUMOylation assays using purified enzymes, substrate, and SUMO proteins. Since in the in vitro reactions, usually SAE1/SAE2 and Ubc9, alone are sufficient for SUMO conjugation, enhancement of SUMOylation by a putative E3 ligase is not always easy to detect. Here, we describe a modified in vitro SUMOylation protocol that consistently identifies SUMO modification using an in vitro reconstituted system. A step-by-step protocol to purify catalytically active K-bZIP, a viral SUMO-2/3 E3 ligase, is also presented. The SUMOylation activities of the purified K-bZIP are shown on p53, a well-known target of SUMO. This protocol can not only be employed for elucidating novel SUMO E3 ligases, but also for revealing their SUMO paralog specificity.

Introduction

SUMO modification was initially identified as a reversible post-translational modification (PTM) that regulates protein stability¹. In addition to direct conjugation, SUMO can also be attached to a protein through non-covalent interaction by SUMO interaction motifs (SIMs)². Similar to the binding of tyrosyl-phosphorylation by molecules harboring Src homology 2 (SH2) or phosphotyrosine binding (PTB) domains^3,4, SUMO modification provides an additional interaction platform for selective recruitment of SIM-containing effector proteins^5,<....

Protocol

1. Preparation of Baculovirus Expression Constructs

1. Purify SUMO E3 ligase K-bZIP by cloning the cDNA of K-bZIP¹⁹ into a dual expression baculovirus vector (see Table of Materials) with an N-terminal epitope-tag. We have had success using an octapeptide tag (indicated throughout the protocol as vector-tag-K-bZIP).
   NOTE: The DNA template for K-bZIP polymerase chain reaction (PCR) cloning is cDNA reverse transcribed from RNA isolated from TREx F3H3-K-Rta BCBL-1 cells following doxycycline treatment²⁰.
   1. Transfer the full-length K-bZIP cDNA¹⁹ into the dual....

Results

According to the information provided by the manufacturer, the standard amount of E1 and E2 enzyme in the SUMOylation assay is 50 nM and 500 nM, respectively. The minimal amount of E2 conjugating enzyme Ubc9 that is able to SUMOylate p53 was first determined by an in vitro SUMOylation assay. As low as one-fifth of the amount of Ubc9 used in the standard in vitro SUMOylation assay protocol was able to efficiently SUMOylate p53 (Figure 1

Discussion

The in vitro SUMOylation protocol described here is routinely used to establish the SUMOylation status of identified Ubc9 substrates. The major limitation using the standard protocol to study the SUMOylation function of SUMO E3 ligase is the abundance of SUMO E1 activating and E2 conjugating enzymes Ubc9 that maximize the SUMO conjugation in in vitro systems. Considering this challenge, we believe that titration of the amount of SUMO E1 and E2 enzymes to the level that one can barely detect their activi.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
pFastBac	Invitrogen	10359-016	dual expression baculovirus vector
pCR2.1-TOPO vector	Invitrogen		PCR product vector
competent cell  E. coli DH5α	Yeastern Biotech	FYE678-80VL	competent E. coli cells A
E. coli DH10Bac	Invitrogen	10359-016	competent E. coli cells B
FugeneHD	Roche	04709705001	transfection reagent
Opti-MEM	Gibco	31985062	reduced serum media
T4 Ligase	NEW England BioLabs	M0202S
CpoI (RsrII)	Thermo Scientific	ER0741
Bluo-gal	Thermo Scientific	B1690	galactosidase substrate
IPTG	Sigma-Aldrich	I6758-1G
Grace’s Insect Medium	Gibco	11605094
Fetal Bovine Serum	Gibco	10082147
Gentamicin	Thermo Fisher	15750060
Ampicillin	Sigma-Aldrich	A9393-25G
Kanamycin	Sigma-Aldrich	K0254-20ML
gentamicin	Gibco	15710-064
tetracyclin	Sigma-Aldrich	87128-25G
LB Broth	Merk	1.10285.0500
HEPES	Sigma-Aldrich	H4034-100G
NaCl	Sigma-Aldrich	S9888-5KG
KCl	Merk	1.04936.1000
Na2HPO4	Sigma-Aldrich	S5136-500G
KH2PO4	J.T.Baker	3246-01
sodium dodecyl sulfate	Merk	1.13760.1000
β-mercaptoethanol	Bio-Rad	161-0710
TRIS (Base)	J.T.Baker	4109-06
Non-fat milk	Fonterra
glycerol	J.T.Baker	2136-01
Triton X-100	Amresco	0694-1L	detergent A
Tween 20	Amresco	0777-500ML	detergent B
Poloxamer 188 solution	Sigma-Aldrich	P5556-100ML	detergent C
Protease Inhibitor Cocktail Tablet	Roche	04 693 132 001
3x Flag peptide	Sigma	F4799
anti-FLAG m2 Magnetic beads	Sigma-Aldrich	M8823	antibody-tagged magnetic beads
SUMOlink SUMO-1 Kit	Active Motif	40120	standard SUMOylation protocol
SUMOlink SUMO-2/3 Kit	Active Motif	40220	standard SUMOylation protocol
QIAquick Gel Extraction Kit	QIAGEN	28704
QIAGEN Plasmid Mini Kit	QIAGEN	12123	plasmid extraction kit
Polypropylene tubes	Falcon	352059
Petri Dish	Falcon	351029
Cell lifter	Corning	CNG3008
Loading tip	Sorenson BioScience	28480
PVDF	PerkinElmer	NEF1002
Blotting filter paper	Bio-Rad	1703932
Mini slab gel apparatus (Bio-Rad Mini Protean II Cell)	Bio-Rad	1658001 EDU
Trans-Blot SD Semi-Dry Electrophoretic Transfer Cell	Bio-Rad	1703940
Pierce ECL Western Blotting	Thermo	32106	ECL reagent
suspension mixer	Digisystem laboratory instruments  Inc.	SM-3000
orbital shaker	Kansin instruments Co.	OS701
ImageQuant LAS 4000 biomolecular imager	GE Healthcare	28955810
Sf9	Thermo Scientific	B82501
anti-p53 antibody	Cell Signaling	#9282
anti-rabbit antibody	GE Healthcare	NA934-1ML