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ISSN
0582-9879 Acta Biochim et Biophysica Sinica 2004, 36(1): 11-15 CN 31-1300/Q
Protein Product Encoded by a Human Novel Gene E9730 Enhances
AP-1 Activity through Interacting with Jab1
Zhao-Qing WANG, Han-Dong WEI, and Fu-Chu HE*
( Department of System Biology, Beijing Institute of Radiation
Medicine, Chinese Human National Center at Beijing, Beijing 100850, China)
Abstract A novel human gene, named E9730 (a clone number
of fetal liver cDNA library), has beenidentified from more than 14,000 expressed
sequence tags (ESTs) based on our large scale sequencing of human fetal liver
cDNA libraries. Although sequencing of this novel human gene indicates that
it is a leucine zipper protein, the function of E9730 and its homongous
genes among species is unknown yet. To find out physiological functional clue
of E9730, the yeast two-hybrid system was used to screen the E9730-interacting
protein(s), and one clone containing a cDNA insert with almost the entire
coding sequence (amino acids 39–335) of human Jab1 (Jun-activating domain
binding protein 1) that interacted specifically with E9730 was identified.
A specific association between Jab1 and E9730 was shown by co-immunoprecipitation
and co-localization experiments. Furthermore, the data indicated that E9730
appeared to enhance Jab1-induced AP-1 activity in a concentration-dependent
manner and Jab1 may be involved in the intracellular signaling transduction
from E9730 to AP-1.
Key words human E9730 gene; CSN5/Jab1;
protein-protein interaction; signaling transduction
The completion of the genome sequences indicates the
advent of post genomic era [1], the challenge ahead is to use the data to
interpret the function of the proteins, the cells, and the organisms [2]. In
China, cloning and functional analysis of the expressed sequence tags (ESTs)
from cDNA libraries from various tissues, such as CD34+ hematopoietic
stem/progenitor cell [3], and fetal liver [4] is the foundation of novel gene
functional studies. Human fetal liver aged 22 week of gestation (HFL22w) is a
major site of fetal hemopoesis in man, and is at the critical turning point
between immigration and emigration of the hemopoetic system. Recently, more
than 14,000 expressed sequence tags (ESTs) of human fetal liver cDNA library
have been sequenced and analyzed by searching the nonredundant GenBank database
with the Blast program, and a number of new genes with important biological
functions have been characterized in our laboratory. Among these ESTs, an
insert clone-FLE9730 was selected and the novel gene was named as E9730
according to its clone number. Following routine bioinformatic strategy, the
full-length cDNA was obtained. After the sequence analysis, a leucine zipper
sequence pattern was found in the C-terminus of E9730 polypeptides. Here, we
report the use of the yeast two-hybrid system to search the E9730- interacting
protein(s), and a positive clone containing a cDNA insert with almost the
entire coding sequence (amino acids 39–335) of human Jab1 (Jun-activating
domain binding protein 1) was identified. We further verified that E9730
interacted specifically with Jab1 in vivo to enhance AP-1 activity.
Materials and Methods
Construction of expression plasmids
To express E9730 proteins in cells, the cDNA at the EcoRI-BamHI
sites was subcloned into the pGBKT7 vector (Clontech), at the EcoRI-BamHI
sites was subcloned into the pCMV-Myc vector (Clontech) and the cDNA at the EcoRI-
XhoI sites was subcloned into the pEGFP-N1 vector (Clontech). All
subcloned inserts were confirmed by sequencing. c-Jun, RFP-Jab1, Jab1
full-length antisense cDNA (pEF-BOS-antisense-Jab1) and control antisense cDNA
(pEF-BOS-antisense-IL12Rβ2 ) are gifts from Dr. Lu [5].
Yeast two-hybrid screen
The MatchmakerTM two-hybrid system 3 (Clontech) was used according
to the protocols provided by the manufacturer. The resulting bait plasmids
pGBKT7-E9730 was used to screen the premade human testis cDNA library
(Clontech) by the yeast two-hybrid method following the manufacturer’s
protocols. The part of coding sequence of the human Jab 1 (Jun-activating
domain binding protein 1) cDNA was isolated from the human testis cDNA library
by sequencing method.
Cell culture, transfection and co-immuno-precipitation
assay
COS7 cells were cultured at 37 ℃ under 5% CO2 in high glucose Dulbecco’s modified Eagle’s medium (DMEM,
Gibcol BRI Inc.) supplemented with penicillin (50 mg/L), streptomycin (50
mg/L), and 10% heat-inactivated fetal calf serum (Hyclone). Cell pass aging was
performed using trypsin-EDTA. Transfections were performed using LipofectAMINE
(Life Technologies, Inc.). Co-immunoprecipitation assay was carried out
following the protocol of protein G-agarose (Roche Molecular Biochemicals):
COS7 cells were co-transfected with pEGFPN1- E9730, pEGFP-N1 mock vector and
pCMV-Myc-Jab 1 plasmid. After 48 h, the cells were harvested and lyzed in 100
µl of lysis buffer for 1 h on ice. The anti-GFP antibody (Clontech) was used in
immunoprecipitation reactions, and the anti-Myc antibody (Clontech) was used in
the Western-blot analysis.
Transient cell transfection and cell co-localization
COS7 cells were maintained in Dulbecco’s modified Eagle’s
high glucose medium (Gibco-BRL) supplemented with 10% fetal bovine serum
(Hyclone). 105 cells were seeded per well of a six-well dish, and the cells were
transiently co-transfected with expression vector together with plasmid
encoding green fluorescent fusion protein (pGFPE9730, 1 mg) and red fluorescent
fusion protein (pREDJab1, 1 mg) using LipofectAMINE (Life Technologies, Inc.).
24 h after transfection, cells were observed by fluorescent microscopy.
Transient cell transfection and luciferase assays
COS7 cells were plated in 24 wells at a density of 5×104 cells/well
and co-transfected with AP-1 driven luciferase reporter gene (Stratagene),
together with the indicated cDNAs, using the Lipofectamine 2000 reagent
(Invitrogen). The AP-1 driven luciferase reporter gene is a reporter plasmid
with firefly luciferase gene driven by several copies of AP-1 enhancer. The
indicated cDNAs were respectively pCMV-Myc-c-Jun coding for c-Jun, pcDNA3-Jab1
coding for Jab1 and pCMV-Myc-E9730 coding for E9730. Total amount of plasmid
DNA was kept constant by the addition of appropriate amount of mock plasmids in
all transfections. 24 h after transfection, the cells were lysed and assayed
for luciferase activity using reporter gene assay kits (Promega). The
luciferase activity was normalized for transfection efficiency using a
co-transfected pRLTK expression vector. All reporter experiments were performed
in triplicate and repeated in at least three independent
occasions.
Results
E9730 Interacts Specifically with Jab1 in vivo
To find out physiological function of E9730, the yeast
two-hybrid system was used to search for the E9730-interacting protein(s), and
several types of clones that interacted specifically with E9730 were
identified. One positive clone contained a cDNA insert with almost the entire
coding sequence (amino acids 39–335) of human Jab1 (Jun-activating domain
binding protein 1). We further verified specific association between Jab1 and
E9730 protein by co-immunoprecipitation, and found a Jab1- E9730 complex in
co-transfected COS7 cells (Fig. 1).
Co-localization of E9730 with Jab1 within COS7 cells.
The association between E9730 and Jab1 was further
confirmed in co-localization of the two proteins in vivo by two-color
fluorescence microscopy analysis. When GFP-E9730 and RFP-Jab1 were
co-transfected into COS7 cells, the E9730 protein distributed in cytoplasm and
nucleus (Fig. 2A), in addition, RFP-Jab1 proteins was diffusely presented in
cytoplasma and in nucleus (Fig. 2B) co-localized with GFP-E9730 (Fig. 2D), the
nuclei were stained with DAPI (Fig. 2C). The fact that E9730 colocalized with
Jab1 implicated their physiological interaction.
Fig.
1 E9730 interacts specifically with Jab1 in vivo
COS7 cells were co-transfected with Myc-tagged Jab1 and GFP-tagged E9730
or GFP control vector. The cell lysates were incubated with anti-GFP antibody
in the protein A/G-Sepharose beads. The immuno complex was analyzed by
immunoblotting with anti-Myc antibody. 1, One-tenth of the total cell lysate is
used in immunoprecipitation; 2, an indication of the relative expression level
for Myctagged Jab1; 4, and 5, are samples from cells expressing Myc-Jab1/GFP
and Myc- Jab1/GFP-E9730, respectively; 3 is a control for lane 5 using rabbit
mock antibody (normal IgG). A duplicate blot was probed with anti-GFP antibody
to monitor the amount of E9730 protein precipitated in each reaction (bottom).
Fig. 2 Cellular colocalization of Jab1 with E9730
COS7 cells were cotransfected with RFP-Jab1 and GFP-E9730 constructs. The
nuclei were stained by DAPI (blue). Arrowheads indicate the field of
colocalization. All cell samples were visualized by confocal microscopy
(Bio-Rad).
Enhanced AP-1 activity caused by the specific interaction
of E9730 proteins with Jab1
As Jab1 could activate the AP-1 dependent promoter,we
examined whether E9730 could modulate Jab1-induced AP-1 activity. COS7 cells
were transfected with an AP-1-driven luciferase reporter gene in the presence
of c-Jun, Jab1, and E9730, and the reporter gene activity was measured.
Overexpression of Jab1 caused an increase in the relative activation levels of
the AP-1 reporter. However, the expression of E9730 protein further enhanced
AP-1 reporter gene activity induced by co-transfected Jab1, and the increase of
Jab1-induced AP-1 activity by E9730 appeared to be concentration-dependent
(Fig. 3A). In addition, E9730 enhanced endogenous Jab1-mediated activation of
AP-1 reporter gene activity in COS7 cells. COS7 cells were co-transfected with
AP-1-driven luciferase reporter, c-Jun, HPO and one of the antisenses. The
lower panel indicates the reduction of endogenous Jab1 by Jab1 antisense or
IL-12R β2 subunit
antisense (control antisense). All luciferase activity was normalized using a
co-transfected pRL-TK expression vector. Results of at least three independent
experiments performed in triplicate are shown in Fig. 3B. Taken together, these
data suggest that Jab1 was involved in the intracellular signal transduction
from E9730 to AP-1.
E9730 enhances endogenous phospho-c-Jun levels
We further studied the endogenous expression of phospho–c-Jun
proteins in COS7 cells co-transfected under the same conditions. The results
indicated that Jab1 increased the level of phosphorylated c-Jun slightly, E9730
increased the function of Jab1 to the control level. However, neither Jab1 nor
E9730 had any effect on the expression of c-Jun. Taken together, these data
indicated that E9730 interacted with Jab1 to increase AP-1 transcription
activity through phosphorylation of c-Jun (Fig. 4).
Plasmids encoding, c-Jun, Jab1, E9730 were cotransfected
into COS7 cells alone or in combination. Expression of c-Jun, p-c-Jun and β-Actin in COS7 cells
lysates containing equivalent amount of protein (10 μg per lane) were
examined by Western blot analysis with antibodies specific for different
proteins and detected with ECL kit (Amersham Pharmacia). Equal loading was
confirmed by β-Actin reacting with anti-Actin antibody.
Fig. 3 Enhanced AP-1 activity caused by the interaction
of E9730 with Jab1
(A) E9730 enhances Jab1-mediated activation of AP-1 reporter gene activity
in COS 7 cells. COS7 cells were co-transfected with AP-1-driven luciferase
reporter, c-Jun, Jab1, and E9730. (B) E9730 enhances endogenous Jab1-mediated
activation of AP-1 reporter gene activity in COS7 cells. AP-1-driven luciferase
reporter, c-Jun, E9730 and one of the antisenses were co-transfected into COS 7
cells. The lower panel indicates the reduction of endogenous Jab1 by Jab1
antisense cDNAs. Data shown are representative examples of at least three
independent experiments performed in duplicates.
Fig. 4 Expression of p-cJun, cJun and β-Actin in COS7 cells transfected with E9730, Jab1 and
c-Jun
We found that E9730 not only located in the cytoplasm but
also in the nuclei. It will thus be interesting to examine whether E9730 plays
an important role in cross-talk between cytoplasmic signal transduction and
nuclear signal transduction.
Discussion
Jab1 belongs to subunit 5 of COP9 signalsome complex
[6,7], Jab1/CSN5 has been implicated in numerous signaling pathways including
those that regulate light signaling in plants, larval development in
Drosophila, and integrin signaling, cell cycle control, and steroid hormone
signaling in a number of systems. However, the general role of the CSN complex,
and the specific role of Jab1/CSN5, remains obscure. Jab1/CSN5 has been
characterized not only in plants involved in light-mediated signal transduction
[8], but also in animals to have kinase activity that phosphorylates IkBa and
c-Jun [6, 7]. It seems that Jab1 can be regulated by different proteins and
exhibit different functions. Jab1 can interact with p27kip1 for protein
degradation [9,10,11], with integrin LFA-1 to induce AP-1 activation [12]. HPO
also triggers AP-1 pathway through intracellular interaction with Jab1 [5], but
another cytokine MIF can bind to Jab1 and regulate Jab1-controlled pathways
[13]. Our data described that E9730 interacted specifically with Jab1 in vivo
and enhanced AP-1 activity in accordance with the increasing level of c-Jun
phosphorylation. The most interesting observations in the present studies is
the enhanced regulation of AP-1 activity by the interaction of E9730 with Jab1,
and investigations are currently in progress to characterize the interaction of
E9730 with other signaling molecules and the role of E9730 in the downstream
signaling events.
Jab1 has been shown to potentiate AP1-mediated transactivation
of its target genes that may affect cell growth and neoplasia [14,15]. In
addition to AP1, Jab1 may also act on other transcription activation factors,
at least in part, through SRC-1 interacting with nuclear receptors and NFκB, and activating transcription at the corresponding target genes [16]. As
we know, Jab1 is localized in signalosomes, but the mechanism through which
Jab1 activate AP-1, SRC-1 and NFκB activities remains unclear. It was proposed that Jab1
stabilized AP1-TRE (TPA-response element) complexes. It is thus possible that
both types of multiprotein complexes (signalosome and nuclear receptors complex)
cross-talk in the cells. Larger structures, associating weakly or transiently
with the COP9 signalosome, have been really observed [17].
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Received: August 12, 2003 Accepted: October 23, 2003
* Corresponding author: Tel, 86-10-68171208; Fax, 86-10-68214653; E-mail, [email protected]