From the Fels Institute for Cancer Research and Molecular Biology and the Department of Biochemistry, Temple University School of Medicine, Philadelphia, Pennsylvania 19140
Received for publication, June 27, 2000, and in revised form, September 14, 2000
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ABSTRACT |
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GADD45, MyD118, and
CR6 (also termed GADD45 The maintenance of normal cellular homeostasis requires that
cells accurately decipher signals for
cell cycle progression, growth, terminal differentiation, and
apoptosis. Also, in recent years it has become increasingly evident
that both the molecular basis for the initial increase in the
susceptibility of malignant cells to anti-cancer agents and the
development of treatment resistance originate from genetic lesions that
alter the function of genes that play a role in normal cell
homeostasis, notably in determining cell cycle progression and the
apoptotic set point (2, 3). Thus, understanding the molecular genetic
pathways that mediate negative growth control is of high priority from
both a basic science and cancer therapeutic point of view.
To this end, cDNA clones of myeloid differentiation primary
response (MyD)1 genes,
activated in M1 myeloblastic leukemia cells, in the absence of de
novo protein synthesis, upon induc-tion of terminal
differentiation associated with growth arrest and apoptosis were
isolated and characterized (4-6). In the course of this work several
novel MyD genes were isolated including MyD118 (7). The MyD118 gene product was found to be remarkably similar to protein encoded by
GADD45, a growth arrest- and DNA damage-induced gene, regulated in part
by the p53 tumor suppressor gene (8, 9). A third member of the
MyD118/GADD45 family, CR6
(cytokine response gene 6), was originally
identified as an immediate early response gene in T cells stimulated by
interleukin-2 (10, 11). Recently, the full-length sequences of murine
and human CR6 cDNAs have been determined (12, 13). It
has become evident that the MyD118/CR6/GADD45 gene family encodes for
small (18 kDa) evolutionarily conserved proteins that are highly
homologous to each other (55-58% overall identity at the amino acid
level) (13), are highly acidic (pI~4.0-4.2), and are primarily
localized within the cell nucleus.
Data have accumulated to indicate that MyD118/CR6/GADD45 serve similar
but not identical functions along different apoptotic and growth
suppressive pathways. For example, it was observed that
MyD118, but not GADD45, is activated upon
transforming growth factor- Importantly, it became evident that individual members of the
MyD118/CR6/GADD45 family are differentially induced by a
variety of genetic and environmental stress agents, suggesting that
each gene is optimally induced by a certain subset of environmental stresses (13, 17). Short term transfection assays have revealed that
MyD118/CR6/GADD45 can synergize in suppression of colony formation by
several different human tumor cell lines (12, 18).
MyD118, CR6, and GADD45 were found to interact with several cellular
proteins. All three proteins interact with and activate the
stress-responsive MTK1/MEKK4, which is an upstream activator of the
p38/JNK kinase pathways (19, 20). Evidence also has been obtained that
MyD118 and GADD45 interact with PCNA (18, 21, 22), a normal component
of multiple quaternary complexes, including the cycling
CDKs and the CDK inhibitor p21 (23-25),
which plays a central role in DNA repair and DNA replication (26-28). MyD118 and GADD45 also were found to interact with p21 (18).
Given the central role assigned for PCNA in cell proliferation and its
identification as a cellular partner for MyD118 and GADD45, in this
work we sought to determine whether CR6 interacts with PCNA, and if so,
to dissect CR6/PCNA interacting domains and analyze their relevance for
negative growth control. We show that PCNA domains, which previously
were identified to mediate interaction with MyD118 and GADD45, also
mediate interaction with CR6. PCNA interacting domains within CR6 were
localized to the C terminus of the protein, similar to what was
observed with MyD118 and GADD45 (1). CR6, like MyD118 and GADD45, also
is shown to interact with p21. Importantly, it is shown that
interaction of CR6 with PCNA, like interactions of MyD118 and GADD45
(1), impedes its function in negative growth control.
Cell Culture, Transient Transfection, and Cytokine
Treatment--
H1299 and 293T cells were obtained from American Type
Culture Collection and were cultured at 37 °C in Dulbecco's
modified Eagle's medium (Cellgro) with 10% fetal bovine serum in a
humidified atmosphere of 10% CO2 (293T were cultured
at 5% CO2). H1299 and 293T cells were cultured to give
60-80% confluency at the time of transfection. One day before
transfection H1299 cells were plated at 1.2 million cells/100-mm dish,
and 293T cells were plated at 3 million cells/l00-mm dish. The murine
myeloid leukemic M1 cell line was grown in Dulbecco's modified
Eagle's medium supplemented with 10% heat-inactivated horse serum
(Life Technologies, Inc.). The cells were cultured in a humidified
atmosphere with 10% CO2 at 37 °C. One day before
purified human rIL-6 (100 ng/ml) (Amgen Inc) treatment, 0.1 million M1
cells were plated per 150-mm dish.
Yeast Two-hybrid Analysis--
Manipulation of Escherichia
coli and DNA was performed according to standard methods (29).
Yeast two-hybrid analysis was performed with the
CLONTECH Matchmaker Two-Hybrid System-2,
essentially as described in the CLONTECH protocol.
Full-length murine CR6 cDNA in pBluescript (12) and human PCNA in
p3038-T7hPCNA (30) were used to generate all the deletion constructs of
CR6/PCNA. Deletion mutants were constructed by taking advantage of
unique restriction enzyme sites within the coding sequence of these
cDNAs. Full-length cDNAs and truncated cDNAs encoding for
deletion peptides were cloned in-frame into pAct2 (GAL4 activation
domain: "trap") as well as into the modified pAS2.1 (GAL4
DNA-binding domain: "bait") vector. All of the constructs were
sequenced to verify in-frame cloning. Transformation into yeast strain
Y187 was performed according to CLONTECH protocol.
Yeast strain Y187 was used for testing interaction by activation of the
In Vitro Interaction Assays Using Coupled
Transcription/Translation--
For in vitro association
assays, full-length PCNA and Bax cDNA coding sequences were cloned
into pET14b (18). Full-length p21 was cloned into pcDNA3. HA-tagged
full-length CR6 and C-terminal peptide coding sequences were cloned
into HApcDNA3.1( In Vivo Interaction Assays Using Transient Transfection into 293T
or H1299 Cells--
For the in vivo interaction studies,
the HA tag epitope sequences in the mammalian expression vector
HApcDNA3.1( In Vivo Interaction Assays in IL-6-treated M1 Cells--
At
48 h after IL-6 treatment, M1 cells were harvested, washed twice
with PBS, and lysed in 0.4 ml of buffer containing protease inhibitors,
as described previously. Triplicate 1.0-ml aliquots of diluted cell
lysates (200 µg) were subjected to co-immunoprecipitation as
described above for in vitro association assay, using p21, CR6, PCNA antibodies, or nonspecific rabbit or mouse IgG. Immune complexes were separated on 15% SDS-PAGE gel. The protein bands were
transferred to nitrocellulose membrane, Western probed with p21, CR6,
PCNA antibodies, and visualized by ECL.
Colony Suppression Analysis--
A short term transfection assay
was used to assess the ability of full-length CR6 protein and N- or
C-terminal peptides to suppress colony formation in H1299 cells (18,
31). pTK-hyg was obtained from CLONTECH, Inc.
Briefly, H1299 cells were co-transfected by the LipofectAMINE method
with 0.5 µg of pTK-hyg together with 10 µg of empty
HApcDNA3.1( Apoptosis Analysis--
H1299 cells growing on coverslips in
35-mm tissue culture dishes were co-transfected with 2 µg of CR6
construct (in HApcDNA3.1( CR6 Interacts with PCNA and p21--
Previously we have
demonstrated that both MyD118 and GADD45 interact with PCNA and p21. It
was, therefore, of interest to determine whether CR6, the third member
in this related gene family, interacts with PCNA and/or p21. To this
end the yeast two-hybrid system (YTHS) was employed ("Experimental
Procedures"). Full-length CR6 was cloned into the Gal4 DNA-binding
domain of pAS2.1 YTHS vector. The CR6 construct was co-transfected into
yeast with pAct2 YTHS constructs encoding for either PCNA or p21 and
fused to the Gal4 activation domain. Interactions, either positive (+)
or negative (
To further establish that these interactions are biologically relevant,
M1 cells were treated with IL-6, and the cell lysates were obtained
from untreated cells at 0 and 48 h after addition of IL-6. The
Western blot analysis of cell lysate shows the abundance of PCNA
protein at both 0 and 48 h (Fig. 1C). CR6 and p21
proteins were absent in untreated cells but were significantly induced at 48 h, as shown in Fig. 1C. As shown in Fig.
1D, the p21 immune complex obtained from cell lysate after
48 h of IL-6 treatment contained CR6. Reciprocally, the CR6 immune
complex contained p21. It can also be seen that the PCNA immune
complex, obtained from M1 cell extracts contained CR6. Reciprocally,
the CR6 immune complex contained PCNA. Taken together these results
demonstrate in vivo association of endogenous CR6 with
endogenous p21 and PCNA.
Identification of CR6 and PCNA Interacting Domains Using the Yeast
Two-hybrid System--
Recently, we have observed that similar domains
within PCNA, MyD118, and GADD45 mediate interactions between these
proteins. Thus, it was of interest to determine whether amino acid
domains, which mediate interactions between PCNA and CR6, are similar
or different from those found to mediate interactions between PCNA, MyD118, and GADD45.
To this end, deletion constructs of PCNA cDNA, encoding for
N-terminal, middle, or C-terminal peptides of PCNA were cloned into the
Gal4 DNA-binding domain of the pAS2.1 YTHS vector. Each of these
constructs were co-transfected into yeast with pAct2 YTHS constructs
encoding CR6, fused to the Gal4 activation domain. As mentioned
earlier, interactions were ascertained by both
As shown in Fig. 2, full-length PCNA
interacted with CR6. PCNA C-terminal (PCNA/224-261), N-terminal
(PCNA/1-46), and middle (PCNA/87-127) peptides also interacted
with CR6. This indicated that these regions of PCNA contained CR6
interacting domains. Consistent with CR6 interacting domains within the
N-terminal region of PCNA, also PCNA/1-149 and PCNA/1-127 peptides
interacted with CR6. The PCNA/1-223 peptide, however, failed to
interact, implying that there is a steric hindrance domain within the
150-223 region of PCNA that inhibited binding to CR6. Consistent with this notion, the 150-223 aa hindrance domain also prevented
association of PCNA/128-261 and PCNA/150-261 C-terminal peptides with
CR6. PCNA/1-100 and PCNA/1-86 N-terminal peptides also failed to
interact with CR6. Because PCNA/1- 46 and PCNA/1-127 did interact with CR6, the data implied that the 47-86 aa region of PCNA, when
juxtaposed to the N-terminal 1-46 aa region, in the absence of the
87-127 aa region, hinders binding of PCNA peptides to CR6.
The relative binding affinities of different PCNA domains with CR6 were
quantified using the YTH liquid culture assay ("Experimental Procedures"). As shown in Fig. 2, the interaction between full-length PCNA and full-length CR6 was weaker than the interaction when using
PCNA/1-46 or PCNA/87-127 peptides. This observation is consistent with the notion that full-length PCNA in addition to CR6 interacting domain contains domains that hinder interaction.
Having mapped domains within PCNA that interact with CR6, next we
mapped domains within CR6 that mediate interaction with PCNA. The YTH
vector pAS2.1 was used to construct yeast expression vectors that
encode for N- and C-terminal deletion peptides of CR6. These deletion
peptides were tested for interaction with PCNA cloned in the YTH vector
pAct2 (Fig. 3, and see legend). It can be
seen that CR6/76-159 interacted with PCNA as well as full-length CR6,
whereas CR6/1-135 failed to interact. This indicated that the
C-terminal (76-159 aa) region of CR6 harbors a domain that is required
for interaction with PCNA.
The relative binding affinities of full-length and C-terminal peptides
of CR6 with PCNA were quantified using Analysis of CR6 and PCNA Interactions in Vitro--
To determine
whether the interacting domains identified in CR6 and PCNA by the YTH
approach are capable of associating directly in vitro,
binding assays were performed with 35S-labeled PCNA,
HA-tagged CR6 full-length protein and deletion peptides, generated by
coupled transcription/translation. The murine Bax protein was included
as an internal control to monitor the specificity of the binding assay.
Equal amounts of 35S-labeled proteins were mixed, and
following incubation, the protein mixtures were immunoprecipitated with
antibodies specific to PCNA, HA, or Bax. Because the PCNA monoclonal
antibody (PC10) used was specific for amino acids 111-120 of PCNA, it
could be used only to test for interaction of the middle domain of
PCNA. Thus, HA antibody was used to test for interactions with other
PCNA domains.
As indicated before (Fig. 1B), CR6 was contained within PCNA
immune complex, and PCNA was contained within CR6 immune complex. The
specificity of these interactions was inferred by the observation that
Bax was not contained within PCNA or CR6 immune complexes, consistent
with previous observations (33). As shown in Fig. 4A, immune complexes of
peptides corresponding to PCNA N-terminal (1-46 aa) and middle
(87-127 aa) but not the C-terminal (224-261 aa) domains also
contained CR6. This indicated that the middle and N-terminal regions of
PCNA contain domains that are capable of direct association with CR6
in vitro.
Coupled transcription/translation also was used to test N- and
C-terminal peptides of CR6 for their ability to associate with PCNA
in vitro. For in vitro association, the
N-terminal (1-76 aa) and the C-terminal (76-159 aa) peptides were
generated via transcription/translation. As shown in Fig.
4B, PCNA immune complexes contained the CR6/76-159 peptide.
Reciprocally, CR6 C-terminal (76-159 aa) immune complexes contained
PCNA. Again, the specificity of these associations was indicated by the
observation that PCNA immune complexes did not contain peptides
corresponding to N-terminal CR6/1-76 aa region, whereas N-terminal CR6
immune complex did not pull down PCNA (Fig. 4A). In
conclusion, the in vitro binding assays have shown that
domains contained within the N-terminal (1-46 aa) and middle regions
(87-127 aa) of PCNA are capable of directly associating in
vitro with domain contained within C-terminal regions of CR6
(76-159 aa).
Dissection of CR6 and PCNA Interaction in Mammalian Cells--
The
work described, so far, has identified domains within CR6 that mediate
interaction with PCNA in yeast and in vitro. To test the
biological significance of CR6 interaction with p21 and PCNA in
negative growth control, it was important to establish that these
domains, similarly mediate interaction in mammalian cells. Toward this
end, pcDNA3 expression vector encoding either for Bax (as negative
control), or p21, or HApcDNA3.1(
The results of the in vivo interaction assays are shown in
Fig. 5. As shown in Fig. 5A,
p21 immune complex contained CR6. Reciprocally, CR6 immune complex
contained p21, which confirms the in vivo association of CR6
with p21. The specificity of the interactions in 293T cells were
further demonstrated by the observation that neither CR6 nor p21 immune
complexes contained Bax. It can also be seen that PCNA immune complex,
obtained from 293T cell extracts transfected with
HA-CR6/pcDNA3.1( Interaction with PCNA Impedes CR6-mediated Colony Suppression of
Human Tumor Cells--
Previously, using a short term transfection
assay (18, 21, 31), it was shown that CR6 suppresses colony formation
of H1299 cells. Having identified the PCNA interacting domains within CR6, it was of interest to explore the significance of CR6 interactions with PCNA for negative growth control using the colony suppression assay.
To accomplish this, a hygromycin selection plasmid (pTK-hyg) together
with HApcDNA3.1(
As shown in Fig. 6, p53 suppressed colony formation by about 90%,
whereas p21 and CR6 inhibited colony formation by about 75 and 60%,
respectively. Interestingly, the C-terminal CR6 peptide, which harbors
the PCNA interacting domain, suppressed colony formation significantly
less efficiently than the full-length protein (30 and 60%,
respectively), whereas the non-PCNA interacting N-terminal CR6 peptide
suppressed colony formation more efficiently than the full-length
protein (80 and 60%, respectively). Taken together, these findings
indicate that interaction of CR6 with PCNA impedes the function of this
protein in negative growth control.
Interaction with PCNA Impedes CR6-mediated Apoptotic Cell
Death--
To further examine the role of CR6 interaction with PCNA in
H1299 colony suppression and determine whether it was due to apoptotic cell death, H1299 cells growing on coverslips were co-transfected with
limiting amount (0.2 µg) of
As shown in Fig. 7, ectopic expression of CR6 induced apoptosis in
H1299 cells. Furthermore, as observed for H1299 colony suppression,
C-terminal CR6 peptide, which harbors the PCNA interacting domain,
induced apoptosis appreciably less efficiently than the full-length
proteins. In contrast, induction of apoptosis by N-terminal CR6
peptide, which did not interact with PCNA, was significantly more
efficient than what was observed with the full-length CR6 (Fig. 7).
These findings suggest that apoptotic cell death is a major factor,
which contributes to the apparent ability of CR6 to suppress colony
formation of tumor cells. These findings also extend the notion that
interaction of CR6 with PCNA impacts negatively on the ability of this
protein to induce cellular death.
In this work we have provided evidence that CR6, like MyD118 and
GADD45, interacts both with PCNA and the cyclin-dependent kinase inhibitor p21. The physiological significance of
CR6/MyD118/GADD45 interaction with p21 for cell cycle arrest, DNA
repair, and/or DNA replication remains to be explored. Although the
endogenous CR6 is undetectable in untreated leukemic M1 cells, CR6
protein expression is induced after IL-6 treatment. The endogenous CR6 is shown to interact both with endogenous PCNA and p21 proteins. Upon
completion of this work, and consistent with our findings, interaction
of CR6 (termed OIG37) with PCNA and p21 has been reported by another
group of investigators (33).
PCNA is a normal component of multiple quaternary complexes, which
include the cycling CDKs and the CDK inhibitor p21WAFI/CIPI (34-36),
that play a pivotal role in cell cycle regulation (23, 24, 34), DNA
replication (28), and repair of damaged DNA (37). Given the central
role of PCNA in cell proliferation and that similar domains within PCNA
were observed to interact with similar amino acid regions within MyD118
and GADD45, clearly it was of interest to dissect domains that mediate
interaction between CR6 and PCNA, to determine whether these are same
or different from those observed to mediate interactions between PCNA,
MyD118, and GADD45, and also to analyze the significance of CR6-PCNA
interaction for negative growth control. To unequivocally identify
amino acid regions that mediate interactions between CR6 and PCNA,
several complementary in vivo and in vitro
methods were used, including the yeast two-hybrid system, in
vitro transcription/translation and transient expression in
various cells.
The YTH approach has led to the identification of three CR6 interacting
domains within PCNA that have been localized to the N-terminal (1-46
aa), middle (87-127 aa), and C-terminal (224-261 aa) regions of PCNA.
Evidence also was obtained for regions within PCNA, i.e.
47-86 and 150-223 aa, that compromised the binding of these PCNA
domains to CR6 (Fig. 2). It is possible that such regions may play a
physiological role in modulating PCNA interactions with CR6 in response
to or following exposure of cells to genotoxic stimuli. Only the
N-terminal and the middle domains of PCNA were found to associate with
CR6 in vitro. Thus, the apparent interaction of CR6 with
C-terminal PCNA in yeast may reflect indirect interaction. The C
terminus of PCNA contains the PCNA/PCNA interacting domain (38), and it
is possible that following co-transfection of YTH vectors into yeast,
the C-terminal region of PCNA encoded by one YTH vector interacts with
endogenous PCNA that is associated with CR6 encoded by the other YTH
vector. Another possibility is that, unlike in vitro
conditions, under in vivo conditions the C-terminal region
of PCNA may assume a tertiary structure and/or may be
post-translationally modified to facilitate interaction with CR6.
Initial information regarding the strength of interactions between PCNA
and CR6 was obtained using the yeast two-hybrid approach in a
semi-quantitative assay. Based on this assay, evidence was obtained
that the N-terminal (1-46 aa) domain of PCNA has higher binding
affinity to CR6 than the middle region (87-127 aa). The binding
affinity of the middle region with full-length CR6 is somewhat low when
compared with the binding affinity of the middle region with
full-length MyD118 and GADD45 (1). Our results regarding PCNA domains
that interact with CR6 thus indicate that similar domains within PCNA
mediate interactions with all three proteins in this family
(i.e. CR6/MyD118/GADD45) (1).
Recent work has established a central role for trimeric PCNA, a moving
platform on DNA, as a communication point and signal processing center
for a variety of cellular processes, including DNA replication,
nucleotide excision repair, post replication mismatch repair, base
excision repair, and at least one apoptotic pathway (39-41). Central
to these roles of PCNA is the interaction with the RNA polymerase
accessory factor (TFIIH) complex, (42-44) as well as its interactions
with DNA polymerase Mapping CR6 regions that interact with PCNA has shown that this protein
interacts with PCNA through its C terminus (76-159 aa). PCNA
interacting domains have been localized to the C-terminal (114-156 aa) peptide of MyD118 and to the C-terminal (95-165 aa) peptide of GADD45 (1). Thus, similar domains within the C-terminal region of CR6/MyD118/GADD45 appear to mediate interactions of these
proteins with PCNA.
Importantly, CR6 deletion peptides employed in H1299 cells for colony
suppression and apoptosis assays have provided evidence that
interaction with PCNA, in essence, impedes the function of CR6 in
negative growth control. Accordingly, the non-PCNA interacting N-terminal peptide (1-76 aa) suppressed colony formation and induced apoptosis much more efficiently than the full-length protein. Similar
observations have been made for MyD118 and GADD45 (1). Surprisingly,
C-terminal (76-159 aa) CR6 still retained some colony suppressing and
apoptotic activity, similar to what has been observed with MyD118
(114-156 aa) and GADD45 (95-165 aa). Similar results were obtained
using 293T cells (data not shown). Understanding the mechanism by which
this family of proteins exert their function and how PCNA may modulate
it should explain how C termini peptides of these proteins still retain
residual negative growth control function.
Recently, it became evident that GADD45, MyD118, and CR6, in addition
to interacting with PCNA and p21, also interact with several other
cellular proteins. It was reported that all three proteins (termed
GADD45 In summary, we have observed that the expression kinetics of CR6
differed from the expression of MyD118 and GADD45 in murine tissues, in
hematopoietic cell development, and in the response of cells to varying
genotoxic agents (12, 13, 33). CR6, however, like MyD118 and GADD45 was
found to inhibit colony formation and to interact with and activate
MTK1/MEKK4 kinase, an upstream regulator of the stress-induced p38/JNK
kinase pathways (13). Thus, to further dissect the role CR6, MyD118,
and GADD45 proteins play in negative growth control and to determine
what role the intricate network of homo- and hetero-interactions and
interactions with other cellular proteins play in cell cycle arrest,
apoptosis, and DNA repair, a battery of CR6/MyD118/GADD45 deletion
mutants (12 aa in length each) are being genetically engineered. This battery of mutants should provide valuable tools to further elucidate the physiological role that this family of acidic proteins and their
myriad of interactions with other cellular proteins play in negative
growth control and DNA repair.
,
, and
)
comprise a family of genes that encode for related proteins playing important roles in negative growth control, including growth
suppression. Data accumulated suggest that MyD118/GADD45/CR6 serve
similar but not identical functions along different apoptotic and
growth suppressive pathways. It is also apparent that individual
members of the MyD118/GADD45/CR6 family are differentially
induced by a variety of genetic and environmental stress agents. The
MyD118, CR6, and GADD45 proteins were shown to predominantly localize within the cell nucleus. Recently, we have shown that both MyD118 and
GADD45 interact with proliferating cell nuclear antigen (PCNA), a protein that plays a central role in DNA replication, DNA repair, and
cell cycle progression, as well as with the universal
cyclin-dependent kinase inhibitor p21. In this work we show
that also CR6 interacts with PCNA and p21. Moreover, it is shown that
CR6 interacts with PCNA via a domain that also mediates interaction of
both GADD45 and MyD118 with PCNA. Importantly, evidence has been
obtained that interaction of CR6 with PCNA impedes the function of this protein in negative growth control, similar to observations reported for MyD118 and GADD45 (1).
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1-induced apoptosis. On the other
hand, GADD45, but not MyD118 or CR6,
was identified as target for p53 function (14-16). All three genes
appear to be induced with different expression kinetics during terminal
hematopoietic differentiation, which is associated with growth arrest
and apoptosis (12). Also, distinct expression patterns for these genes
were observed in a variety of murine tissues (12).
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-Gal gene (blue selection). Yeast strains CG1945 or Y190 were used
to test interaction by activation of the histidine gene. Interaction
assays were conducted only with pAct2/pAS2.1 constructs that tested
negative for self-activation. Interactions either positive (+) or
negative (
) were ascertained by both
-Gal expression (blue
selection) and histidine expression (histidine selection). Quantitation
of relative binding affinities of protein and deletion peptides was
determined in yeast strain Y187. Three positive double transfectants
were selected on minimal medium plates for each transfection and grown
overnight in liquid selection minimal medium. The expression of
-Gal
was quantified, according to the CLONTECH protocol,
with O-nitrophenyl
-D-galactopyranoside as
the substrate, and the intensity of yellow color development was
measured colorimetrically at 420 nm.
), kindly provided by Dr. Dhanasekaran.
Full-length CR6 and the C-terminal peptide sequences were cloned into
the unique EcoRI site of HApcDNA3.1(
). N-terminal
peptide encoding cDNAs were generated by truncating and linearizing
full-length CR6 cDNA in HApcDNA3.1(
) with unique restriction
enzymes at the required sites, and were then used in the
transcription/translation reaction (TNT; Promega). PCNA 87-261 aa
(EcoRV-NdeI Fragment) was cloned in-frame into
the pAS2.1 vector. Subsequently, PCNA 87-127 aa
(NdeI-EcoRI fragment from pAS2.1) and PCNA
87-149 aa (NdeI-BglII fragment from pAS2.1) were excised and cloned in-frame into pET23b (Novagen), which was used in
the TNT reaction. Constructs were purified by CsCl gradient centrifugation and suspended in DNase and RNase free H2O.
The T7 based coupled transcription/translation system, with rabbit reticulocyte lysate (TNT; Promega) and Easy Tag
L-[35S]methionine (PerkinElmer Life Sciences), was
used to produce radiolabeled proteins and peptides. In vitro
interaction experiments were done as described (18). Briefly, equal
amounts of 35S-labeled PCNA, Bax, and CR6 protein/peptides
were mixed together (105 cpm each) in 200 µl of
interaction buffer A (20 mM Tris, pH 8.3, 150 mM NaCl, 1.0% Nonidet P-40, 0.1% Tween 20, and 1.0 mg/ml
bovine serum albumin). 35S-Labeled Bax was added to all
reactions as an internal negative control for interaction. Each
interaction reaction was carried out, in triplicate, for 30 min on ice.
Subsequently each reaction mix was nonspecifically immunoprecipitated
with rabbit IgG or mouse IgG for 1 h. Following pulling down of
nonspecific immune complexes with protein A/G-agarose beads (Oncogene
Sciences), supernatants were subjected to co-immunoprecipitation with
specific primary antibodies: PCNA (PC10; Santa Cruz), p21 (Ab-5;
Calbiochem), Bax (N20; Santa Cruz), and HA (Y11; Santa Cruz). Immune
complexes were pulled down with protein A/G-agarose beads. Beads were
washed four times with interaction buffer A (without bovine serum
albumin), suspended in Laemmeli protein loading buffer, heated at
75 °C for 10 min, and loaded on 15% SDS-PAGE gel for analysis.
Following electrophoresis the gels were fixed with 25% isopropanol in
10% acetic acid, subjected to fluorography with Amplify (Amersham Pharmacia Biotech), and vacuum dried before exposure to x-ray film.
) were used. 10 µg of the pcDNA3 constructs
encoding for Bax, p21, or HA-tagged CR6 full-length protein or deletion
peptides were transiently transfected into 293T or H1299 cells, using
LipofectAMINE (Life Technologies, Inc.). After 24 h, transfected
cells were trypsinized, washed twice with PBS, and lysed in 3.0 ml of
interaction buffer containing protease inhibitors including
phenylmethanesulfonyl fluoride, leupeptin, aprotonin, and pepstatin A
(Sigma). Triplicate 1.0-ml aliquots of cell lysates were subjected to
co-immunoprecipitation, as described above for the in vitro
association assay, using Bax, p21, HA, PCNA antibodies, or nonspecific
rabbit or mouse IgG. Immune complexes were separated on 15% SDS-PAGE
gel. The protein bands were transferred to nitrocellulose membrane
(Hybond-EC; Amersham Pharmacia Biotech), Western probed with HA
antibodies, and visualized by ECL (Amersham Pharmacia Biotech).
Membranes were reprobed with PCNA antibodies after stripping the blot
according to the manufacturer's protocol.
) construct (negative control) or HApcDNA3.1(
) encoding for HA-tagged full-length CR6 protein or N/C-terminal peptides. pTK-hyg was used for hygromycin selection and as an internal
normalization control for transfection efficiency. Co-transfections were performed in 60-mm tissue culture dishes. One day following transfection, the cells were washed with PBS and trypsinized. The cells
were replated in duplicates with hygromycin-containing medium (200 µg/ml) and incubated for 14 days to allow colonies to develop. Then
the medium was removed, and colonies were washed once with PBS and
fixed with 75% methanol in 25% acetic acid for 5 min, and the plates
were dried. Colonies were stained with Lillie's crystal violet (2 g of
crystal violet and 0.8 g of ammonium oxalate in 80% ethanol) for
5 min and subsequently washed with deionized water to remove excess
stain. Stained colonies containing more than 10 cells were scored and
counted. The percentage of colony formation was normalized to colonies
formed following transfection with empty HApcDNA3.1(
). pcDNA3
encoding p53 or p21 were co-transfected with pTK-hyg as positive
controls. pcDNA3 encoding for antisense CR6 was used as an
additional negative control.
)) and 0.2 µg of
-Gal expression
vector (pSV-
-Gal; Promega) using the LipofectAMINE method.
-Gal
expression and apoptosis were analyzed 72 h following
transfection. The cells were washed once with PBS and fixed for 10 min
with 0.05% glutaraldehyde in PBS. Following three washes with PBS to
remove the fixative, X-gal solution (20 mM potassium
ferricyanide, 20 mM potassium ferrocyanide, l
mM magnesium sulfate in PBS; X-gal is added to a final
concentration of 1 mg/ml just before use, from a 20 mg/ml stock
solution in N,N'-dimethyl formamide) was spread
over the dishes, and the dishes were incubated overnight at room
temperature. The next day, the X-gal solution was removed, and cell
nuclei were stained for 3 min with 0.1 µg/ml Hoechst No.33342 (Sigma)
and then washed three times with PBS. The coverslips were mounted in
Vectashield mounting medium H-1000 (Vector Labs inc. CA 94010) and
analyzed under a Leitz fluorescent microscope. The percentage of
apoptotic cells were determined by dividing the number of
-Gal
expressing blue cells that exhibit apoptotic nuclear morphology
(condensed/fragmented nucleus) by the total number of blue cells. At
least 200 cells from five randomly chosen fields were analyzed for each experiment.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
) were ascertained by both
-Gal expression (blue
color) and histidine expression (histidine selection) ("Experimental
Procedures"). Empty pAct2 vector was used as the negative control in
the YTHS for both qualitative and quantitative interaction with the
full-length CR6 in pAS2.1 vector. As shown in Fig.
1 (A and B),
full-length CR6 interacted with PCNA. Full-length CR6 interacted also
with p21. The relative binding affinities of CR6 interactions with PCNA
or p21 were quantified using the YTH liquid culture assay ("Experimental Procedures"). In this assay, the level of
-Gal expression in yeast under control of the interacting partner proteins encoded by pAct2 and pAS2.1 was quantified by measuring the intensity of yellow color development using O-nitrophenyl
-D-galactopyranoside as substrate. As shown in Fig.
1A, CR6 interacted equally strongly with PCNA and p21. To
determine whether the interactions of CR6 with PCNA and p21, identified
by the YTH approach, reflect direct association between these proteins,
in vitro binding assays were performed with
35S-labeled PCNA or p21 and full-length HA-tagged CR6
protein that were generated by coupled transcription/translation. The
murine Bax protein (pI~4.7), a bcl-2 partner protein with a mass and charge similar to MyD118, GADD45, and CR6, (pI~4.0) (32), was included as an internal control to monitor the specificity of the
binding assay. Equal amounts of 35S-labeled proteins were
mixed, and following incubation, the protein mixtures were
immunoprecipitated with antibodies specific to PCNA, p21, HA epitope,
or Bax. As shown in Fig. 1B, PCNA or p21 were contained
within HA-CR6 immune complexes, and CR6 was contained in PCNA or p21
immune complexes (Fig. 1B). The specificity of these
interactions was inferred by the observation that Bax was not contained
within PCNA, p21, or HA-CR6 immune complexes.
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Fig. 1.
Interaction of CR6 with PCNA and
p21. A, interaction of CR6 with PCNA and p21 using the
yeast two-hybrid system. Full-length CR6 cloned in pAS2.1 (Gal4 binding
domain) was tested for interaction with PCNA and p21 cloned in pAct2
(Gal4 activation domain), in the yeast two-hybrid system
("Experimental Procedures"). Positive (+) and negative ( )
interactions were ascertained by both
-Gal expression (blue color
selection) and histidine expression (histidine selection), as described
under "Experimental Procedures." Empty pAct2 was used as the
negative control to monitor for the specificity of the interactions.
Numbers indicate relative binding affinities and standard
deviations determined by using the yeast two-hybrid culture assays, as
mentioned under "Experimental Procedures." B, in vitro
analysis of CR6 interaction with PCNA and p21. In vitro
transcription/translation kit (TNT; Promega) was used to label
full-length CR6, PCNA, and p21 with [35S]methionine.
In vitro interaction, co-immunoprecipitation, 15% SDS-PAGE
analysis, and fluorography were done as described under "Experimental
Procedures." Bax was included as a negative control to monitor the
specificity of the interactions. C, induction of CR6 and p21
in IL-6-treated M1 cells. IL-6-treated M1 cells were collected at 0 and
48 h, and whole cell extracts were prepared and were subjected to
Western blot analysis, using antibodies as indicated. Actin was used as
negative control for equal loading. D, in
vivo analysis of CR6 interaction with PCNA and p21 after IL-6
induction. IL-6-treated M1 cells were collected at 48 h, and whole
cell extracts were prepared and were subjected to
co-immunoprecipitation and Western blot analysis, using antibodies as
indicated.
-Gal expression and
histidine expression ("Experimental Procedures").
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Fig. 2.
Identification of domains within PCNA that
interact with CR6 using the yeast two-hybrid system. Schematic
diagram of full-length PCNA and PCNA deletion peptides cloned in
pAS2.1, that were tested for interaction with CR6 cloned in pAct2 using
the yeast two-hybrid system ("Experimental Procedures"). Positive
(+) and negative ( ) interactions were ascertained by both
-Gal
expression (blue color selection) and histidine expression (histidine
selection), as described under "Experimental Procedures." PCNA
cDNA restriction enzyme sites used to generate truncated cDNAs
encoding for the deletion peptides are shown at the bottom of the
figure. Numbers in parentheses indicate relative
binding affinities and standard deviations, determined by using the
yeast two-hybrid liquid culture assays, as indicated under
"Experimental Procedures."
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Fig. 3.
Identification of domains within CR6 that
interact with PCNA using the yeast two-hybrid system. Schematic
diagram of full-length CR6 and CR6 deletion peptides in pAS2.1 (Gal4
binding domain) that were tested for interaction with PCNA cloned in
pAct2 (Gal4 activation domain) using yeast two-hybrid system
("Experimental Procedures"). Positive (+) and negative ( )
interactions were ascertained by both
-Gal expression (blue color
selection) and histidine expression (histidine selection), as described
under "Experimental Procedures." Numbers in
parentheses indicate relative binding affinities and
standard deviations, determined by using the yeast two-hybrid liquid
culture assays, as indicated under "Experimental Procedures."
-Gal expression in yeast and
are shown in Fig. 3. Notably, the binding affinity to PCNA of the
CR6/76-159 C-terminal peptide was higher than the full-length CR6.
Taken together these findings indicate that amino acid domains, which
mediate interactions between CR6 and PCNA, are similar to the domains
that were observed to mediate interaction between MyD118, GADD45, and
PCNA (see Ref. 1 and "Discussion").
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Fig. 4.
In vitro analysis of CR6
interaction with PCNA. A, analysis of middle domain,
N-terminal PCNA, and C-terminal PCNA for association with CR6.
B, analysis of N-terminal and C-terminal CR6 for association
with PCNA. In vitro transcription/translation kit (TNT;
Promega) was used to label the protein products with
[35S]methionine. In vitro interaction,
co-immunoprecipitation, 15% SDS-PAGE analysis, and fluorography were
done as described under "Experimental Procedures." Bax was included
as a negative control to monitor the specificity of the
interactions.
) expression vectors encoding for
full-length CR6 and deletion peptides, all with an HA tag, were
transiently transfected into 293T cells, and 24 h later cell
extracts were tested for interaction of the encoded CR6 protein with
p21 or endogenous PCNA by co-immunoprecipitation with either p21, PCNA,
or HA antibody.
), contained HA-CR6. Reciprocally, HA tag immune
complex obtained from the same cells contained PCNA (Fig.
5B). Transfection of 293T with pcDNA3.1(
) encoding for
either C-terminal or N-terminal HA-CR6 peptides has demonstrated that
PCNA immune complexes contained C-terminal but not N-terminal CR6
peptide. Likewise, HA immunocomplexes brought down PCNA from cells
transfected with pcDNA3.1(
) encoding for C-terminal but not
N-terminal CR6 (Fig. 5B). The specificity of the
interactions in 293T cells was further demonstrated by the observation
that PCNA immune complexes obtained from 293T cells transiently
transfected with Bax/pcDNA3 did not contain Bax, and Bax immune
complexes did not contain PCNA. Taken together these results indicate
that CR6 interacts with PCNA in mammalian cells via its C-terminal
region, similar to what was observed for MyD118 and GADD45 (1).
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Fig. 5.
In vivo analysis of CR6
interaction with p21 (A) and PCNA
(B). Transfection of pcDNA3 or
HApcDNA3.1( ) expression vectors encoding for p21 or HA-tagged CR6
or CR6 deletion peptides into 293T cells was performed as described
under "Experimental Procedures." Cells were collected 24 h
after transfection, whole cell extracts were prepared and were
subjected to co-immunoprecipitation and Western blot analysis, using
antibodies as indicated. Bax was used as the negative control for
transfection, co-immunoprecipitation and Western blot analysis.
), encoding for HA-CR6 deletion peptides, or
empty HApcDNA3.1(
) control plasmid, at 1:20 ratio, were
transfected into H1299 cells. Following 2 weeks of selection in
hygromycin-containing medium, surviving colonies were fixed, stained,
and scored. In all the experiments empty vector and antisense-CR6 in
pcDNA3 were used as negative control, and p53 and its target gene
p21 were used as positive controls. The results of the short term
colony suppression assays are shown in Fig.
6. Representative tissue culture plates
that were fixed, stained, and scored are shown in Fig. 6A.
Results of all experiments are summarized in Fig. 6C, where
colony formation using empty HApcDNA3.1(
) vector is the standard
for no colony suppression and is, therefore, set at 100%. Data used
for calculating colony suppression have been derived only from
experiments where the initial expression of the various proteins and
peptides did not vary significantly, as shown in Fig.
6B.
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Fig. 6.
Suppression of human H1299 lung carcinoma
colony formation by CR6 and CR6 deletion peptides. A,
colony suppression using CR6 and CR6 deletion peptides. One set of
representative results is shown here. B, Western analysis of
transfected CR6 and CR6 deletion peptides. Expression of full-length
CR6 and deletion peptides in H1299 cells, 24 h after transfection
was determined by Western blotting. C, percentage of colony
formation by CR6 and CR6 deletion peptides. Each result is mean of
three independent experiments and is plotted along with standard
deviations. Human H1299 cells were transfected with the indicated
expression vectors as described under "Experimental Procedures."
Empty pcDNA3 and antisense-CR6 were used as negative controls,
whereas pcDNA3 encoding for p53 and p21 were used as positive
controls. Two weeks after the transfection, the hygromycin-resistant
colonies were scored. The number of colonies scored in presence of
empty pcDNA3 alone is designated 100%.
-Gal expression plasmid together with
excess (2.0 µg) pcDNA3.1(
) encoding for full-length HA-CR6 or
deletion peptides. Following 72 h the transfected cells were analyzed for
-Gal expression (blue color), and nuclei of the transfected cells were stained with Hoechst to detect apoptotic morphology characterized by nuclear shrinkage and chromatin
condensation (Fig. 7). Percentage of
apoptotic cells was determined as the number of
-Gal expressing blue
cells that exhibited apoptotic nuclear morphology divided by the total
number of
-Gal expressing blue cells.
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Fig. 7.
Analysis of apoptosis induced by CR6 deletion
peptides. H1299 cells grown on coverslips were co-transfected with
2.0 µg of CR6 constructs (in pcDNA3) and 0.2 µg of -Gal
expression vector using LipofectAMINE.
-Gal expression and apoptosis
were analyzed 72 h after transfection, as described under
"Experimental Procedures." Percentage of apoptotic cells was
determined by dividing the number of
-Gal expressing (blue color)
cells exhibiting apoptotic nuclear morphology (condensed/fragmented
nucleus) by the total number of blue cells. At least 200 cells from
five randomly chosen fields were analyzed. Note that in H1299 cells,
nuclear apoptotic morphology was observed to precede membrane blebbing
and cellular shrinkage.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
, Fenl, DNA ligase I, and p21, via the
interdomain connector loop (100-150 aa) (45-48). It is clear,
therefore, that these later proteins compete for accessibility to bind
to the same domains of PCNA that have been identified to interact with
CR6, MyD118, GADD45, and p21 (1, 22, 49-51). This picture is further
complicated by the ability of CR6 to interact with p21 as well as other
members of the GADD45 family
members.2 The
biological significance of this intricate myriad of interactions for
the role CR6, MyD118, and GADD45 play in different negative growth
control pathways, and for the functions of PCNA in DNA replication and
repair remains to be elucidated.
, GADD45
, and GADD45
) interact with and activate the
stress-responsive MTK1/MEKK4 kinase that mediates activation of the
p38/JNK kinase pathway and apoptosis in response to environmental
stress stimuli (13). Evidence was obtained that GADD45 also associates
with CDK1 (cdc2) and inhibits the kinase activity of the CDK1·cyclin
B complex, implicated in GADD45 induction of a G2/M
cell cycle check point in response to certain genotoxic stresses (52,
53). In addition, it was documented that GADD45 can recognize damaged
chromatin and modify chromatin accessibility by direct interaction with
the core histones (54). Whether CR6 interacts with CDK1 and/or core
histones remains to be determined.
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FOOTNOTES |
---|
* This work was supported by National Institute of Health Grant 1RO1CA43618 (to D. A. L.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
To whom correspondence may be addressed: Fels Inst. for Cancer
Research and Molecular Biology and Department of Biochemistry, Temple University School of Medicine, 3307 N. Broad St.,
Philadelphia, PA 19140. Tel.: 215-707-6903/2; Fax: 215-707-2805;
E-mail: lieberma@unix.temple.edu.
Published, JBC Papers in Press, October 5, 2000, DOI 10.1074/jbc.M005626200
2 N. Azam, B. Hoffman, and D. A. Liebermann, unpublished results.
![]() |
ABBREVIATIONS |
---|
The abbreviations used are:
MyD, myeloid
differentiation primary response;
CDK, cyclin-dependent
kinase;
aa, amino acid(s);
HA, hemagglutinin;
-Gal,
-galactosidase;
PBS, phosphate-buffered saline;
X-gal, 5-bromo-4-chloro-3-indolyl
-D-galactopyranoside, YTH,
yeast two-hybrid;
YTHS, YTH system;
PCNA, proliferating cell nuclear
antigen;
PAGE, polyacrylamide gel electrophoresis;
IP, immunoprecipitation.
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