I-Rel: a novel rei-related protein that inhibits NF-KB transcriptional ...

temperature of 45°C was used. The PCR amplicons were cloned
into the HindUI-Xbal site of plasmid BL SK (Stratagene), and the
inserts of 50 clones were sequenced by the chain termination
method (Sanger et al. 1977) with T3 and T7 primers, using Sequenase
II (U.S. Biochemical).
The Hindlll--Xbal fragment obtained from the unique amplicon
was used as a probe to screen a Jurkat T-cell cDNA library
in X.ZAPII (Stratagene). From a total of 600,000 plaques screened
under stringent hybridization (42°C, 50% formamidc) and wash
conditions (65°C, O.lx SSC), seven positive plaques were obtained
through three rounds of purification. The BL SK phageniid
containing the cDNA (BL I-Rel res) was rescued as described
(Short et al. 1988) and sequenced by the chain termination
method (Sanger et al. 1977). Primers were synthesized to
permit overlapping sequencing of the cDNA in both directions.
Computer analysis and assembly of sequence information were
carried out using the GCG programs (University of Wisconsin,
Madison, WI).
Plasmid construction
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A pDS expression plasmid was used for expression of I-Rel in
Escherichia coh (Gentz et al. 1989). Expression is driven from a
bacteriophage T5 promoter under control of a lac operator.
Primers were synthesized corresponding either to the region
surrounding the initiator methionine (145-163 bp) or to amino
acid residue 123 at the beginning of the rel homology domain
(508-525 bp) to fuse I-Rel in-frame with the 6 histidine moiety
present in the pDS plasmid, and the I-Rel cDNA was amplified
using each of these primers for the 5' primers and a primer
corresponding to amino acid 425 (1399-1413 bp) as the 3'
primer. The amplified fragment was cloned into the BamHl-
Xbal sites of the vector pDS I-Rel(A3').
For in vitro transcription of I-Rel(A3'), the EcoRl-Xbal fragment
of pDS I-Rel(A3') was cloned into Bluescript SK (Stratagene)
to make BL I-Rel(A3'). For in vitro transcription of fulllength
I-Rel, the BamHl-Xbal fragment from BL I-Rel res was
exchanged with the BamHl-Xbal fragment of BL I-Rel(A3') to
make BL I-Rel. The expression vector CMV I-Rel was constructed
by subclonmg the Hindlll-Xbal fragment of BL I-Rel
between a CMV promoter-p-globin intron and an SV40 poly(A)
signal. The pCMVp50/p65 chimeric protein was generated by
fusing the sequence encoding amino acids 1-370 of p50 to the
sequence corresponding to amino acids 309-550 of p65 as described
(Ruben et al. 1992). The GAL4/1-Rel chimeric proteins
were constructed by first amplifying the fragment of I-Rel corresponding
to ammo acids 404—579, restricting the amplified
fragment with BamHl and Xbal, and cloning the fragment inframe
with the GAL4 sequence corresponding to amino acids
1-147 of the GAL4 DNA-binding domain in plasmid pSG424
(Sadowski and Ptashne 1989). Plasmid HKB-4CAT contains four
tandem copies of the KB sequence from the HIV-1 enhancer
(Leung and Nabel 1988). Plasmid IL-2R-421/-225 corresponding
to the IL-2/Ra-promoter has been described previously
(Ruben et al. 1988). Epitope-tagged I-Rel, p50, and p65 were
constructed by PCR using a 5' primer encoding the amino acid
sequence MYPYDVPDYA corresponding to the influenza HA
protein (Kolodziej and Young 1991), followed by cloning the
amplified product into Bluescript SK.
Cell culture and transfection
Jurkat T cells were maintained in RPMI-1640 medium containing
10% FCS and 50 |xg/ml of Gentamicin (GIBCO). For transient
transfection assays, 5 x 10"^ cells were transfected by the
DEAE-dextran procedure (Queen and Baltimore 1983). Cells
I-Rel inhibits NF-KB transcriptional activity
were transfected with 2 (xg of reporter plasmid and 1-4 |xg of the
CMV I-Rel expression vector. Cells were harvested 48 hr after
transfection, and CAT assays were performed as described previously
(Gorman et al. 1982). For PMA induction of Jurkat cells,
the cells were transfected with HKB-4CAT (1.5 ^g); 30 hr after
the transfection, PMA was added at 50 ng/ml. Cells were harvested
the following day. COS7 cells were maintained in
IMDM + 10% FCS supplemented with 4500 ixg/ml of glucose.
For transient transfection assays 2 x 10'' cells were plated on
35-mm plates, and a modified DEAE-dextran protocol (Dillon
et al. 1990) was used to transfect the cells the following day.
Cells were transfected with 1 [ig of the GAL4 UAS reporter
construct and 2 ixg of the GAL4/I-Rel chimeric expression vectors.
In vitro transcription and translation
and coimmunoprecipitation analysis
I-Rel, p50, or p65 cDNAs present in the Bluescript expression
vector was linearized with Xbal, and 1 |xg was used as template
for in vitro transcription with T7 RNA polymerase. The in
vitro-transcribed RNA was used to program a rabbit reticulocyte
translation lysate (Promega) that contained ['^^Slmethionine.
Protein products were analyzed on a 10% SDS-polyacrylamide
gel and fluorographed. For the coimmunoprecipitation
analysis, I |xl of RNA corresponding to protein containing the
HA tag was cotranslated with 1 fxl of RNA corresponding to
I-Rel, p50, or p65 lacking the tag sequence. For immunoprecipitation,
4 |xl of lysate was mixed with 150 |JL1 of immunoprecipitation
buffer [20 mM HEPES (pH 7.5), 250 mM NaCl, 4 mM
EDTA, and 0.1% NP-40]. Two microhters of anti-HA immune
sera (Babco) was added to the reaction followed by the addition
of protein G agarose beads (Pharmacia). The samples were incubated
at 4°C for 3 hr and washed four times with immunoprecipitation
buffer. Agarose beads were heated to 80°C for 10
min before gel loading. Protein products were analyzed on a
10% SDS-polyacrylamide gel and fluorographed.
Expression and purification of bacterial-expressed proteins
A pDS expression plasmid was used for expression of I-Rel, p50,
and p65 in Escherichia coli (Gentz et al. 1989). Briefly, expression
was driven from a bacteriophage T5 promoter under control
of a lac operator. Residues corresponding to the DNA-binding
domain of each protein (see above) were fused in-frame with
the 6 histidine moiety present m the pDS plasmid. Expression of
these proteins was induced in mid-logarithmic cultures of £.
coli by the addition of 1 mM IPTG. After 4 hr of incubation, cells
were pelleted and lysed in 6 M guanidine hydrochloride (pH 8.0).
The cleared lysate was adsorbed to a nickel chelate affinity
resin, and proteins were eluted with a pH step gradient of 6 M
guanidine-HCl. Purified protein was renatured slowly by dialyses
against H buffer [20 mM HEPES (pH 7.9), 0.2 mM EDTA, 1
mM DTT, 0.1% NP-40, and 0.5 mM PMSF] plus 300 mM KCl
containing 3, 1.5, 1, 0.5 M, and no guanidine-HCl, respectively.
For corenaturation experiments, proteins were diluted 1 : 10 in
6 M guanidine-HCl and mixed at a ratio of 1 : 1, 1 : 5, and 1 : 20
before dialysis.
For preparation of crude E. coli lysate containing I-Rel, p50,
and p65 proteins, mid-logarithmic cultures were induced with 1
mM IPTG for 2.5 hr, pelleted, and resuspended in H buffer without
NP-40. Cells were lysed by sonication with two 30-sec cycles,
and lysates were cleared by centrifugation and frozen at
- 70°C.
GENES & DEVELOPMENT 757