(Received for publication, December 11, 1995; and in revised form, January 30, 1996)
From the
Human T-cell leukemia virus type I Tax is a pleiotropic gene
regulator that functions through CREB/ATF- and NF-B-mediated
pathways. In most contexts, Tax is a potent gene activator. Here, we
describe an unexpected finding of Myc repression by Tax. In cells that
overexpress human T-cell leukemia virus type I Tax, the detection of
c-Myc protein in the nucleus by a monoclonal antibody was masked. Tax
prevented immunological visualization of a Myc epitope contained within
amino acids 45-104, resulting in interference with Myc function
in transcription and in anchorage-independent cell growth. Tax did not
affect steady-state protein levels since detection of c-Myc with other
antibodies was unperturbed. Four observations suggest that this Tax-Myc
interaction is mediated through CREB/ATF signal transduction. 1) Tax
point mutants, selectively defective for activation of CREB/ATF but not
NF-
B, failed to mask c-Myc; 2) masking of Myc was abolished when
Tax-expressing cells were treated with protein kinase inhibitor H-9; 3)
Tax-specific shielding of Myc is absent in cells (B1R) that are
genetically defective for cAMP signaling; and 4) forskolin treatment of
cells mimicked Tax in masking the Myc epitope. Considered collectively,
these findings suggest a regulation of Myc function at the level of
localized protein conformation.
Human T-cell leukemia virus type I (HTLV-I) ()is the
etiologic agent for adult T-cell leukemia
(ATL)(1, 2, 3) . HTLV-I encodes a 40-kDa
phosphoprotein, Tax, which is essential for viral
transcription(4, 5, 6, 7) . Tax has
been proposed to be involved in molecular events leading to ATL
(reviewed in (8, 9, 10, 11) ).
Numerous cellular findings, including the demonstration that Tax
expression leads to immortalization of T-lymphocytes(12, 13) and transformation of rat fibroblasts (14, 15) ex vivo, are consistent with this
proposition. In animals, transgenic mice that express Tax have
constitutively activated T-cells(16) , and targeted expression
of Tax to T-lymphocytes results in development of large granular
lymphocytic leukemias(17) . How Tax effects these cellular
changes is not well understood. It is, however, well demonstrated that
this viral activator modulates a variety of cellular processes through
signals transduced separately by CREB/ATF and NF-
B (reviewed in (18) ).
Transcription directed from the HTLV-I long terminal
repeat and many cellular promoters (e.g. interleukin-2,
interleukin-2R, transforming growth factor-
1, c-Fos, c-Jun,
granulocyte-macrophage colony-stimulating factor, and epidermal growth
factor receptor-1 among others(19, 20, 21) )
is potently up-regulated by Tax. Three imperfectly repeated 21-base
pair motifs, each containing a core 8-base pair cAMP-responsive
element(22, 23) , have been characterized as the cis-responsive long terminal repeat target for Tax
regulation(24, 25, 26, 27, 28) .
Tax activates the viral long terminal repeat (and cellular promoters)
by interacting with CREB/ATF (29, 30) bound at
promoter-proximal cAMP-responsive
elements(31, 32, 33, 34, 35, 36, 37, 38, 39, 40) .
Tax also activates other promoters through alternative means that are
less understood (41, 42, 43, 44) .
Additionally, another function of Tax is reflected in its ability to
induce the translocation of NF-
B from the cytoplasm into the
nucleus(45, 46, 47, 48, 49) ,
thereby modulating expression of a further class of genes including
that encoding for
interleukin-2R
(50, 51, 52, 53, 54) .
Recent evidence indicates that signaling pathways, previously thought to be discrete, are often intertwined(55, 56, 57) . Homeostatically, it makes sense that each activating event in cells should be countered by a moderating reaction(s), although in the literature, descriptions of the former far exceed those of the latter. Thus, depending on context, activators should also frequently serve as repressors (reviewed in (58) ). In HTLV-I, many aspects of disease pathogenesis suggest the existence of tight checks on viral activation function. Most notable is the fact that <5% of all infected individuals ultimately develop ATL and usually not before a latency period greater than 20-30 years(59, 60) . Hence, in vivo, disease development is slow and protracted. This contrasts sharply with the observed rapid and dramatic effects exerted by ectopic expression of HTLV-I proteins on cultured cells (discussed in (61) ). Such juxtaposition of findings suggests the existence of yet characterized biological controls on viral activation.
c-Myc is a 64-kDa nuclear phosphoprotein that regulates cell proliferation and differentiation (reviewed in (62) ). When dimerized with its heterologous partner Max, Myc is a sequence-specific DNA-binding protein (63, 64, 65) that has pleiotropic transcriptional activity(65, 66) . Deregulated expression of c-Myc is linked to the the development of many human cancers (reviewed in (62) ) and, in some context, leads to apoptosis(67, 68) . Understandably, tight control of Myc expression, which has been described at transcriptional and post-transcriptional levels (reviewed in (62) ), is essential to normal cellular metabolism.
In searching for cellular targets of HTLV-I Tax, we unexpectedly observed a novel interaction with Myc. When cells were transfected to express Tax, the nuclear detection by a monoclonal antibody directed to an N-terminal epitope of Myc was masked. This effect did not affect steady-state stability/amount of Myc since detection of protein using other antibodies was unchanged. While Tax has been shown previously to cooperate with oncogenes (e.g. ras) (69) in transformation, its ability to antagonize also a second oncoprotein through presumptive regulation at the level of local protein conformation is intriguing. We discuss the possible biological implications of this finding.
Transfection of suspension cells was achieved using Lipofectin (Life Technologies, Inc.) according to manufacturer's suggestions. Following transfection, the cells were attached to polylysine-coated glass coverslips and processed for microscopy as described for adherent cells.
When Tax-expressing HeLa cells (Fig. 1A, arrow 1) were visualized using anti-Myc monoclonal antibody C-8, no Myc fluorescence was detected in the nucleus (Fig. 1B, arrow 1). In the same field, HeLa cells that did not express Tax stained brightly for c-Myc (Fig. 1B, arrow 2). This finding was reproduced in all other fields, and quantitation of fluorescent signals showed that Tax-expressing cells (Fig. 1C, trace 1) measured less than one-tenth the staining intensity of that seen in Tax-nonexpressing counterparts (Fig. 1C, trace 2).
Figure 1:
Masking
of c-Myc by HTLV-I Tax. Tax-expressing cells were identified using
anti-Tax rabbit polyclonal antibody and Texas Red-conjugated goat
anti-rabbit (IgG) secondary antibody (A, D, and F). Myc was stained using three monoclonal antibodies, two
(C-8 and C-33) for separate epitopes in Myc (B and E)
and another (9E10) for Myc peptide, amino acids 408-439 (G). Identical fields are shown for Tax (left) and
for Myc (right). Arrows point to cells expressing
Tax, which was detected in 20% of transfected cells. Relative
expression of Myc in Tax-expressing cells (labeled as arrow 1) versus Tax-nonexpressing cells (labeled as arrow 2)
was quantitated based on fluorescent intensities and is plotted in C. In Tax-expressing cells, Myc-specific fluorescence was
absent with the C-8 monoclonal antibody (A-C). Myc was
seen when the cells were stained with the C-33 (D and E) and 9E10 (F and G) monoclonal
antibodies.
The epitope in c-Myc recognized by the C-8 monoclonal antibody was unknown. To check whether the disappearance of Myc fluorescence was due to a reduction in steady-state protein or was a result of epitope masking, two other Myc-specific antibodies were tested. Anti-Myc C-33 is a monoclonal antibody directed to an epitope different from C-8, although this reactivity has also not been mapped (Fig. 1E). Anti-Myc 9E10 recognizes amino acids 408-439 (Fig. 1G). When a population of HeLa cells transfected with a Tax vector was costained with anti-Tax and either C-33 or 9E10, clear images of c-Myc appeared in the nucleus, regardless of whether the cell expressed Tax protein or not (Fig. 1, compare arrowed cells in D and E and those in F and G). These findings contrasted with those obtained using the C-8 monoclonal antibody and suggested that Tax expression did not perturb steady-state Myc levels, but instead masked detection of one antigenic epitope.
To better
understand this masking, we mapped the epitope recognized by the C-8
monoclonal antibody. Wild-type and 10 mutant proteins that contained
selected amino acid deletions were expressed in Escherichia coli (Fig. 2A). This panel of 11 Myc polypeptides was
probed with C-8 in immunoblot assays. The results revealed that
Myc1-48 and Myc
106-143 were reactive to C-8,
while Myc
48-175 was not (Fig. 2A). Further
analysis revealed that an expressed peptide (silver staining of
proteins (Fig. 2B, left) and immunoblotting of
same proteins (Fig. 2B, right)) containing
only amino acids 45-130 was recognized by C-8 (Fig. 2B, lane 8), deductively narrowing the
epitope to amino acids 45-105. Of interest, we note that amino
acids 45-105 are wholly contained within the previously
characterized transcriptional activation domain of Myc (reviewed in (76) ).
Figure 2:
Mapping of the Myc epitope recognized by
the C-8 monoclonal antibody. A, a diagrammatic representation
of wild-type Myc and Myc deletions. The names for the constructs
incorporate the amino acid numbers demarcating the deletion boundaries.
All proteins were expressed in E. coli as fusion proteins and
resolved by SDS-polyacrylamide gel electrophoresis and then analyzed by
immunoblotting. Reactivity with the C-8 monoclonal antibody is
indicated as positive (+); absence of C-8 reactivity is indicated
as negative(-). B, silver staining (lanes
1-4) and Western blotting (lanes 5-8) of
selected polypeptides used in determining the C-8 epitope. Note that
Myc amino acids 45-130 (Myc45-130) (lanes 4 and 8) and Myc1-48 (lanes 2 and 6)
are fully reactive with C-8, whereas the glutathione S-transferase (GST) control (lanes 1 and 5) and Myc
45-175 (lanes 3 and 7)
show no reactivity with C-8.
Figure 3:
Tax represses Myc-induced
anchorage-independent cell growth. Three cell lines were established
from Rat1a (R1aH; Myc/Tax
) using
hygromycin coselection. The cell lines were characterized for
expression of Myc (RM8; Myc
/Tax
),
Myc and wild-type Tax (RM8X;
Myc
/Tax
), and Myc and mutant Tax
(RM8XG320; Myc
/TaxG320
) using
immunofluorescence and immunoblotting. A, images on the left
are stainings using anti-Tax serum, while the corresponding light-field
images are shown on the right. Expression of Tax was observed for RM8X
and RM8XG320. B, Western analyses of Tax expression. Tax (asterisks) was found in RM8X (lane 3) and RM8XG320 (lane 4). As a positive control, HeLa cells transfected with a
Tax-expressing plasmid are shown (lane 5). C, foci
formation in soft agar. Pictures show representative foci from equal
numbers of seeded R1aH, RM8, RM8X, and RM8XG320 cells. Note the large
colonies seen for RM8 and RM8XG320. These were absent for R1aH and
RM8X. D, Western analyses of Myc expression. RM8, RM8X, and
RM8XG320 expressed similar amounts of 64-kDa c-Myc after normalizing
for some intensity differences in background bands. RM8X and RM8XG320
samples were loaded in duplicates.
The
four established cell lines were assayed also for anchorage-independent
growth (Fig. 3C). When plated into soft agar, control
R1aH cells (Myc/Tax
) failed to form
significant colonies (Fig. 3C). RM8
(Myc
/Tax
), consistent with previous
findings (77) , produced many large foci (Fig. 3C). RM8XG320 (Myc
/inactive
Tax(19) ) resembled RM8 in also producing many large colonies.
However, RM8X (Myc
/Tax
) (Fig. 3C), which expressed both Myc and Tax, showed
much reduced cell masses in agar. One interpretation of these results
is that Tax, perhaps as a consequence of epitope masking, repressed the
induction by Myc of anchorage independence in cells. We caution that
our findings do not eliminate the possibility that Tax has other more
general effects on the cells, thereby reducing proliferative capacity.
Figure 4:
Repression of Myc-dependent transcription.
MLPCAT (top) contains two copies of the Myc-binding motif
positioned in the adenovirus major late promoter chloramphenicol
acetyltransferase plasmid. MLPDMCAT (bottom) is MLPCAT with
the two point-mutated Myc motifs. Top, MLPCAT was
cotransfected with pUC19, with RSVmyc, with RSVmyc + pHTLVTax (RSVmyc+Tax; see (53) ), with RSVmyc +
pHTLVTax (RSVmyc+TaxD), and with pHTLVTax (Tax). Bottom, transfections were performed with
MLPDMCAT in place of MLPCAT; otherwise the transfections are identical
to those shown above. Tax
is a Tax cDNA deleted in all amino acids
except for the N-terminal 58 residues. Data shown are averages from
experiments repeated twice.
Figure 5:
CREB/ATF-active (but not NF-B-active)
forms of Tax mask Myc. A, shown is a linear diagrammatic
representation of the Tax protein. Tax mutants used for analyses are
indicated. Asterisks indicate the relative locations of the
mutations. The mutated amino acids are numbered below each asterisk.
Some of the previously identified structural and functional domains of
Tax are indicated. B, HeLa cells were seeded onto coverslips
and transfected with wild-type Tax (panels A and B)
or Tax mutant (panels C-N) plasmids followed by
immunostaining. Identical paired fields are shown on the left
(anti-Tax) and right (anti-Myc C-8). Arrows point to
Tax-expressing cells. Tax mutants TaxC23, TaxN43, and TaxA113 (panels C-H), active for CREB/ATF but inactive for
NF-
B, masked Myc expression (see panels C and D, E and F, and G and H,
respectively). Mutants TaxS29, TaxN52, and Tax284 (panels
I-N), inactive for CREB/ATF but active for NF-
B, did
not affect Myc detection (see panels I and J, K and L, and M and N,
respectively).
If Tax represses Myc detection through activation of cAMP-dependent signaling, then one might expect that an independent means of cAMP stimulation would produce a similar picture. To investigate this possibility, we treated HeLa cells with the adenylate cyclase activator forskolin and investigated whether activation of protein kinase A (PKA) signaling, in the absence of Tax, perturbs the Myc C-8 epitope. We observed that mock-treated cells stained well with either C-8 (Fig. 6A) or pan-reactive polyclonal (Fig. 6B) serum in double simultaneous stainings. In contrast, cells incubated for 4 h with forskolin were not stained with C-8 (Fig. 6C), while the polyclonal antiserum readily visualized nuclear fluorescence of Myc in the identical cells (Fig. 6D).
Figure 6: Effect of PKA modulation on masking of Myc. HeLa cells were exposed to forskolin for 4 h, immediately fixed, and stained. Myc masking was confirmed by comparing changes in the intensity of C-8-specific fluorescence relative to fluorescence from polyclonal pan-Myc. In untreated cells (A and B), equal intensities were observed with the two antibodies. In the presence of forskolin, C-8-specific immunostaining was reduced compared with pan-Myc-specific immunostaining (compare C and D). In a converse assay, HeLa cells were seeded onto coverslips and transfected with a Tax expression vector followed by treatment with H-9 for 24 h. Tax expression was measured by immunofluorescence in treated (G) and untreated (E) cells. The same cells were simultaneously costained with the C-8 monoclonal antibody in treated (H) and untreated (F) groups. Untreated HeLa cells showed reduced C-8 immunostaining in Tax-expressing cells compared with Tax-negative cells (compare intensities of Myc staining in Tax-expressing versus Tax-nonexpressing cells; F). Treatment of cells with H-9 blocked the ability of Tax to mask Myc (compare intensities of Myc staining in Tax-expressing versus Tax-nonexpressing cells; H).
As a complementary assay, we reasoned that if cAMP-PKA stimulation through either Tax or forskolin effects masking, then inhibitors of PKA activation should blunt this effect. To test this, we examined the ability of Tax to mask the C-8 epitope in cells treated with kinase inhibitor H-9. HeLa cells seeded onto coverslips were transfected with Tax-expressing plasmid and then incubated with H-9 for 12 h. Cells were fixed and stained with antiserum. In the absence of H-9, Tax-expressing cells (Fig. 6E) showed reduced C-8 fluorescence (Fig. 6F). Tax-expressing cells treated with with H-9 (Fig. 6G) recovered the normal intensity of C-8 as compared with Tax-nonexpressing cells (Fig. 6H), confirming that protein kinase inhibitors can blunt masking of Myc.
We further studied cells that are genetically defective in the cAMP-PKA axis. The murine B1R cell line is a PKA-defective clone derived from the S49.1 lymphoma(81) . These cells fail to respond to cAMP as the result of an absence of cAMP-binding protein(82) . If cAMP-PKA signaling is required by Tax to mask Myc, then one should observe staining of Myc by C-8 despite expression of Tax in B1R cells. We, in fact, saw brightly stained Myc protein (Fig. 7E, arrowhead) in B1R cells that expressed Tax (Fig. 7D, arrowhead). In comparison, a suspension T-cell line (Jurkat) not defective in cAMP-PKA signaling, when similarly stained with C-8 (Fig. 7B), showed reduced Myc fluorescence in a cell that expressed Tax (Fig. 7A). Thus, the results from B1R (Fig. 7), considered collectively with those from Tax mutants (Fig. 5) and from forskolin- and protein kinase inhibitor-treated cells (Fig. 6), underscore a linkage between the cAMP-PKA signaling pathway and Tax masking of the Myc C-8 epitope.
Figure 7:
Tax does not mask Myc in a cell line
defective for cAMP signaling. The ability of Tax to mask Myc was
measured in the PKA-defective cell line B1R and was compared with that
in Jurkat cells. Cells grown in suspension to 5 10
cells/ml were transfected with Tax-expressing plasmid. Following
transfection, the cells were washed in PBS and were settled onto
polylysine-derivatized coverslips. The adherent cells were fixed and
stained. Each pair of panels contains dual stainings of the identical
field of view. Jurkat cells were probed with anti-Tax and anti-Myc
(C-8) to determine Myc masking in T-cells (compare A and B). Tax-expressing cells (arrowheads) displayed
reduced C-8 fluorescence. B1R cells expressing transfected Tax were
similarly probed with anti-Tax and anti-Myc (C-8). In this case,
Tax-expressing cells (D) stained strongly with the C-8
monoclonal antibody (E). Light-field views of each group are
shown in C and F.
Tax functions are complex. Examples of activation of the
HTLV-I long terminal repeat through CREB/ATF and activation of the
promoters for human immunodeficiency virus type 1, interleukin-2R,
and granulocyte-macrophage colony-stimulating factor (among others)
through NF-
B illustrate intricate interplay between divergent
signaling pathways (reviewed in (8) and (10) ). In
understanding the role of Tax in HTLV-I lymphoproliferation, one faces
the issue of a long latency between virus infection and development of
ATL. Indeed, only a small minority of infected individuals (<5%)
progress to ATL, invariably more than 20-30 years after initial
exposure(9, 59, 60) . Hence, in
vivo, Tax is only mildly oncogenic, and its transforming
properties are arguably incidental to its pleiotropic effects on
cellular genes.
Intuitively, it is reasonable that Tax cooperates with cytoplasmic oncogenes such as ras(69) in transformation. It was unexpected that Tax might antagonize the anchorage-independent cell growth and transcription functions of Myc. While the short half-lived Myc protein has been shown to be regulated at discrete stages in transcriptional initiation, transcriptional elongation, and post-transcriptional processing (reviewed in (62) ), regulation at the level of protein conformation has not, heretofore, been proposed. The region in Myc perturbed by Tax (amino acids 45-106) is contained within a highly conserved transformation/activation domain(66, 71, 78, 79, 83, 84, 85) . While we do not understand fully the mechanistic details responsible for the Tax-Myc interaction, our experimental evidence supports the likelihood that the N-terminal portion of Myc is conformationally altered by Tax, without additional effects on overall protein stability. A conformational disruption of this protein domain could reasonably account for an abrogation of Myc's transactivation and transformation properties.
p53 and Rb are two examples of regulatory proteins targeted for modulation by viral oncogenes (reviewed in (86) ). While there are many biological instances in which mechanisms such as phosphorylation and protein degradation are used to control function, one of the increasingly common regulatory themes is exemplified by formation of protein-protein complexes, such as those between p53/Rb and viral oncoproteins. Indeed, in one setting, Myc function is known to be regulated by direct binding to Rb(84, 87) . In comparison, a common mechanism of Tax action appears to be the facilitation of protein-protein dimerization. This has been illustrated well by effects of Tax on bZIP proteins (38, 39, 40) that result in the stabilized binding of these factors to cognate DNA sites(36, 39) . However, Tax has also been shown to contact directly CREB, bZIP, and other proteins(33, 34, 35, 88, 89) , and this latter ability could, in part, explain its capacity to mold conformationally receptive partners(36, 39) .
A direct protein-protein contact mechanism would be an attractive explanation for the masking of Myc by Tax. While we cannot exclude this possibility, we have, so far, been unable to recover evidence for a Tax-Myc complex by physical/genetic means (i.e. coimmunoprecipitation, protein column chromatography, and yeast two-hybrid assay; data not shown). On the other hand, our findings do establish a strong correlation between the inhibition of Myc function and Tax activity in CREB/ATF signaling. Since activation of CREB/ATF is a requirement for Tax-mediated transformation(15) , our current observations would support a proposition that embedded into activation is a countervailing signal (transmitted as antagonism of Myc) that moderates transformation. This duality of positive/negative effects emanating from a single (CREB/ATF) pathway might be one biosynthetically conservative strategy for maintaining cellular homeostasis. Indeed, from other systems, it is not unexpected that activation events in the cAMP-PKA axis also transduce repressive signals (reviewed in (90) ).
Viruses have complex symbioses with cells. On the one hand, for optimal replication of viral genomes, viruses activate cells to states of high synthetic capacity. On the other hand, such activation with accompanied viral replication results in cell death. Presumably, there is a fine balance in achieving a ``quasi-activated'' state. In terms of infected hosts, some semblance of this is probably reflected in HTLV-I seropostive, but asymptomatic, individuals(91) . Because uncontrolled activated cellular expansion results in undesired ends for the virus (e.g. aggressive expansion of HTLV-I-infected cells leads to fulminant ATL and rapid death of the patient(9, 92, 93) , or recognition of infected cells by host immune surveillance results in clearance of the virus), it makes sense for viruses to evolve balanced control of cellular metabolism. The long period (>20-30 years) of asymptomatic latency and the low (<5%) disease penetrance(59, 60) suggest that HTLV-I has very successfully evolved a coexistence with host cells. In this regard, the fact that some Tax effects activate host cell processes and other Tax effects provide balanced repression would be consistent with this evolution. Our observation of Tax-Myc masking serves an added example of this balanced regulation. Similar findings (i.e. repression of Myc activity by Tax) have been confirmed by others(89) , although with fewer molecular details.
From a mechanistic
perspective, much remains to be addressed on the ability of Tax to mask
Myc. Because immunofluorescence analysis does not provide an
unambiguous interpretation, we are pursuing through genetic and
biochemical approaches the possibility that although Tax does not
directly contact Myc, some CREB/ATF-like bZIP proteins, modulated by
Tax, could serve surrogate roles. In this regard, we have recently
isolated by the yeast two-hybrid approach three novel cDNAs that encode
cellular proteins that bind Tax. ()The biology of
Tax-cellular protein interactions is likely to be highly complex and
merits detailed investigation. We note, however, that recent findings
demonstrating that the adenovirus E1a gene product can at once
cooperate with a second oncogene to transform cells (94) and at
another serve as a tumor suppressor (95) suggest that some of
the non-intuitive observations with Tax are generally relevant and
similarly operative in other biological paradigms.