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Metabolic Research Unit (W.F., P.W., P.N., P.J.K.) and
Department of Anatomy (J.L.) University of California San
Francisco, California 94143
Department of Pharmacology
(X.H.L., M.K.) University of California San Diego La Jolla,
California 92093
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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(hereafter, ER) is regulated
both by binding of ligand and by inputs from signal transduction
cascades. Binding of estrogen to ER frees it from a complex with heat
shock proteins and allows ER to bind estrogen response elements (EREs)
in the promoter region of target genes (for review see Ref. 1). ER then
stimulates transcription via the concerted action of the AF-1
activation function in its amino terminus and the hormone-dependent
activation function, AF-2, that lies within the ligand-binding domain
(LBD). The antiestrogen tamoxifen allows release from heat shock
proteins and ERE binding, but blocks AF-2 (reviewed in Refs. 1, 2).
Tamoxifen allows weak AF-1 activity, but in many cases this is
insufficient to increase gene expression (3). Other antiestrogens, such
as raloxifene and ICI 182,780 (ICI), allow neither AF-1 nor AF-2
activity (4, 5). Both activation functions work by recruiting a
coactivator complex to the promoter (reviewed in Refs. 1, 6, 7).
The complex contains a p160 protein, such as SRC-1(N-CoA1) (8, 9),
glucocorticoid receptor interacting protein 1 (GRIP1) (TIF2, N-CoA2)
(10, 11, 12), or p/CIP (AIB1, ACTR, TRAM-1, RAC3) (13, 14, 15, 16, 17), p300/CBP (CREB
binding protein) (9, 18, 19, 20), and p/CAF (21, 22, 23) (for review see Ref.
7). AF-1 binds the C terminus of the p160 component (24), whereas
estrogen-bound AF-2 binds tightly to a short -helices with the
consensus LXXLL (the NR boxes) that are repeated several times within
each p160 (13, 25, 26, 27, 28, 29). The coactivator complex, once recruited,
stimulates transcription via its histone acetyl-transferase activity,
which is thought to remodel chromatin and allow access to the
transcriptional template, and by interactions with the basal
transcription machinery (15, 30, 31, 32). ER activity is also stimulated by signaling pathways that are activated when growth factors, such as epidermal growth factor (EGF) and insulin-like growth factor I (IGF-I), bind their tyrosine kinase-linked receptors. Growth factors are sometimes sufficient to activate ER in the apparent absence of ligand (33, 34, 35, 36). More commonly, growth factors synergize with ligand by enhancing AF-1 activity. EGF binding to the EGF receptor results in sequential activation of Ras, Raf, MEK, and the mitogen-activated protein (MAP) kinases ERK1 and ERK2, which phosphorylate ER at serine 118 in the N-terminal domain and potentiate AF-1 activity (36, 37, 38). Mutation of S118 to alanine blocks ER phosphorylation by MAP kinases and potentiation of AF-1 action by growth factors (36, 37, 38). Furthermore, the S118A mutation also reduces basal phosphorylation of S118 by unspecified kinases (38) and decreases basal AF-1 activity (39, 40). Phosphorylated ER AF-1 shows enhanced binding to the p68 RNA helicase, which is thought to account for its enhanced transcriptional activity (41). AF-1/GRIP1 interactions map to the N-terminal region of the ER AB domain, which has not been implicated in growth factor enhancement of AF-1 activity, and are unaffected by mutations in serine 118 (24).
ER is likely to be subject to signal transduction inputs during normal development and repair processes and may also be subject to abnormal stimulation by such pathways in pathological states, such as cancer. Many breast tumors exhibit elevated expression of growth factors such as EGF, Her2/neu, IGFs, and their receptors (42, 43, 44, 45). Furthermore, more than 80% of primary breast cancers show increased activity of the nonreceptor Src tyrosine kinase activity compared with normal breast tissue (Refs. 46, 47, 48, 49 ; reviewed in Ref. 50). Elevated Src activity leads to activation of multiple signal transduction cascades (reviewed in Refs. 51, 52, 53) that, in turn, activate both the ERK and JNK subgroups of MAP kinases (for examples see Refs. 54, 55). Src activates ERKs presumably via Ras and the Raf/MEK/ERK kinase cascade (see, for example, Ref. 54). It also activates JNKs, presumably via Rac-1 and related GTPases, and the sequential activation of the MEKK/JNKK/JNK kinase cascade (56). In light of these reports we investigated whether activated Src enhances ER action. We find that Src does so and that it specifically enhances ER AF-1 activity via two independent mechanisms. One involves phosphorylation at S118 via Src activation of the Raf1-MEK-ERK pathway. The other is mediated by Src activation of the MEKK-JNKK-JNK pathway, the target of which does not appear to be S118, or even ER. We discuss the possibility that JNKs target one of the several coactivators that associate with the ER.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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shows that v-Src potentiated ER transcriptional activity by 2-fold in
the presence of 17ß-estradiol (E2), a primary
estrogen agonist activating both AF-1 and AF-2. V-Src also potentiated
ER activity in the absence of ligand by about 2-fold. More strikingly,
v-Src enhanced ER transcriptional activity in the presence of tamoxifen
(Tam), an ER ligand that inhibits AF-2 but allows AF-1 activity (3), by
about 15-fold. V-Src had no effect upon ER transcriptional activity in
the presence of ICI 182,780, an ER ligand that blocks the activities of
both AF-1 and AF-2 (3, 57, 58, 59). Thus, v-Src increases ER
transcriptional activity in the presence of estradiol and the absence
of ligand, but shows larger effects in the presence of tamoxifen. We
then examined v-Src effects upon ERG400V, an ER mutant that lacks
constitutive activity but is otherwise normal (60). Here, v-Src showed
modest potentiation of estrogen response and a much larger potentiation
of tamoxifen response. Thus, v-Src truly enhances the transcriptional
activity of the tamoxifen-ER complex above basal. Over the course of
this study, v-Src enhanced the overall levels of estrogen response by
between 50% and 4-fold, but consistently yielded larger enhancements
of tamoxifen response. Thus, v-Src increased the overall level of
tamoxifen response from a tiny percentage of estrogen response to
15–40% of estrogen response.
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shows
that the amount of ER increased as a function of transfected ER
expression vector in the absence of v-Src, and that v-Src increased ER
levels by about 3-fold. In parallel, v-Src gave much stronger
potentiation of ER action at the ERE responsive reporter gene, even at
ER levels that were optimal for estrogen and tamoxifen response (Fig. 1B, lower panel). In particular, the ER activity obtained
with 3 µg of transfected ER and v-Src exceeded the ER activity
obtained with 10 µg of transfected ER and no v-Src, even though the
former contained lower amounts of ER protein. Thus, v-Src increases ER
transcriptional activity, especially in the presence of tamoxifen.
We then asked whether v-Src enhancement of ER activity might occur
under more physiological conditions. We first examined the amount of
v-Src required for enhancement of ER activity. Tamoxifen response
increased with as little as 300–600 ng of transfected v-Src expression
vector (Fig. 1C). We then examined v-Src action upon the ERE responsive
reporter in MCF-7 breast tumor cells, which express endogenous ER (Fig. 1D). Here, ER showed significant constitutive activity, which was
further elevated by addition of estradiol but completely suppressed by
tamoxifen and ICI. In the presence of transfected v-Src, both the
constitutive and estrogen-dependent transcriptional activity were
modestly elevated and, once again, tamoxifen-dependent transcriptional
enhancement was strongly increased. Thus, v-Src enhances the activity
of the estrogen-ER and tamoxifen-ER complexes in breast cells, just as
it does in HeLa cells.
That the ER response to v-Src overexpression is more dramatic in the
presence of tamoxifen than in the presence of estrogen, and that there
is no response in the presence of ICI, suggests that ER AF-1 might be a
primary action target of v-Src. To investigate whether the target of
v-Src action was AF-1 or AF-2, we examined the effect of v-Src
overexpression upon the activity of a reporter gene with a promoter
containing a TATA box and multiple binding sites for the yeast GAL4
protein (5xGALRE-E1b-tata-LUC). This reporter was then activated with
the DNA binding domain of GAL4 fused to the ER A/B region containing
AF-1 [GAL4-ER(A/B)], to the ER LBD containing AF-2 [GAL4-ER(LBD)],
or to VP16 (GAL4-VP16). As shown in Fig. 1E, v-Src potentiated the
GAL4-ER(A/B) transcriptional function approximately 30- to 40-fold, but
had only a minimal effect on GAL4-LBD or GAL4-VP16. Like v-Src action
upon the tamoxifen-liganded ER, v-Src potentiation of AF-1 activity
could be detected at optimal levels of transfected GAL4-ER(A/B)
expression vector (data not shown). Thus, v-Src strongly enhances ER
AF-1 activity. These observations are consistent with the strong
activation of the tamoxifen-bound ER by v-Src and confirm that the A/B
domain of ER, which contains AF-1, is the v-Src target.
Src Action upon ER Is Independent of Tyrosine 537
Some reports have suggested that Src directly phosphorylates ER at
tyrosine 537 (Y537) within the ER-LBD (61, 62, 63), although the
physiological significance of this effect is unclear. In fact, some
Y537 mutations were later shown to enhance ER activity in the absence
of hormone, by allowing ligand-independent interactions of ER AF-2 with
its target coactivators (64, 65, 66). To determine whether Y537
phosphorylation was required for v-Src action upon the ER, we examined
the effect of v-Src overexpression upon several ERs bearing mutations
at Y537. We chose Y537R, which shows similar constitutive activity to
wild-type ER, and Y537S and Y537F, which show either enhanced or
reduced constitutive activity, respectively.
Figure 2A shows that ER and ER-G400V
elicited strong estrogen responses and weak tamoxifen responses from
the ERE responsive reporter. In parallel, ERY537R showed comparable
constitutive activity to wild-type ER, and ERY537S showed the expected
increase in constitutive activity, but both mutants showed similar
levels of activity to wild-type ER in the presence of estrogen and
tamoxifen. In the presence of v-Src, wild-type ER and each Y537 mutant
(Y537R and Y537S) showed a modest enhancement of transcriptional
activity in the presence and absence of estradiol and a larger
enhancement of transcriptional activity in the presence of tamoxifen.
Thus, v-Src effects upon the estrogen-ER complex, the unliganded ER,
and the tamoxifen-ER complex are all independent of a requirement for
ER phosphorylation at tyrosine 537. v-Src also enhanced estradiol and
tamoxifen activation in the presence of the ERY537F mutant, which lacks
constitutive activity (Fig. 2B). This confirms that v-Src enhances the
activity of the tamoxifen-ER complex over basal, even when tyrosine 537
is mutated. We conclude that v-Src enhancement of ER activity occurs
through effects upon AF-1, and not through effects upon tyrosine 537 in
the ER-LBD.
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3- and 5-fold, respectively (Fig. 3). The kinase-inactive mutant Src
(Y295K, Y527F), in contrast, was unable to enhance AF-1. Moreover,
enhancement of ER AF-1 by Src was blocked, and even reduced below basal
levels, by genistein and herbymicin A, two inhibitors of Src tyrosine
kinase activity (Fig. 3). Control experiments indicated that both of
these inhibitors were without effect on GAL-VP16 (data not shown).
These results indicate that the intrinsic tyrosine kinase, and not
another feature of the v-Src molecule, mediates the enhancement of ER
activities. That the kinase inhibitors reduce AF-1 level below basal
may further suggest that endogenous Src tyrosine kinase activity might
underlie basal AF-1 activity.
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). Thus, ER is not a
direct substrate of Src. We infer that Src must activate ER AF-1
indirectly through phosphorylation cascades.
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, left, each of these reagents nearly abolished the EGF
activation of a GAL-Elk1 fusion protein. PD 98059, a specific inhibitor
of MEK activation (69), also blocked EGF activation of GAL-Elk1. Thus,
these inhibitors work effectively to block transcription mediated by
the ERK pathway in these transfected cells. In contrast, only modest
(20–40%, shown) decreases in v-Src effects on GAL-ER (A/B) were
detected. Even high levels of transiently transfected expression
vectors for each dominant negative protein produced no more than
30–40% inhibition of Src action (data not shown). Similar modest
inhibition was obtained with PD 98059. These results indicate that Src
potentiation of ER AF-1 is only partly mediated by the Ras/Raf/MEK/ERK
pathway and suggest that another pathway must also be involved.
Because the Rac-MEKK-JNKK-JNK pathway is also activated by v-Src, we
then asked whether this pathway might mediate v-Src effects upon the
ER. Dominant negative Rac(S17N) or MEKK1(K432M), strongly inhibited
v-Src activation of ER AF-1 (Fig. 4C). In a separate experiment a
vector for dominant negative JNKK4(K116R) eliminated the induction
mediated by short-term exposure to v-Src expression or stimulation of
the endogenous Src/JNK pathway treatment with the cytokine tumor
necrosis factor- (TNF) (Fig. 4D). These observations indicate
that the route from Src to AF-1 proceeds through both the Ras-Raf-MEK
and the Rac-MEKK-JNKK pathways and that the ERK and JNK kinases are the
true effectors of Src action.
The role of the ERK and JNK kinases was then confirmed by examining the
effect of elevated expression of these kinases on Src action.
Overexpressed JNK1 and ERK2 synergized with v-Src to activate ER AF-1
(Fig. 4E). Neither JNK nor ERK was sufficient to enhance AF-1 activity
in the absence of v-Src, suggesting that both required inputs from
upstream kinase cascades to enhance AF-1 activity. In contrast,
overexpression of another MAP kinase, p38, which is also activated by
JNKK1 (70), inhibited, rather than synergized with, the v-Src
activation of ER AF-1. While the mechanism of this dominant negative
effect of p38 is unknown, the fact that p38 acts as a dominant negative
indicates that p38 does not mediate Src activation of AF-1. In summary,
these studies indicate that the route from Src to AF-1 proceeds through
both a Ras-Raf-MEK-ERK and a Rac-MEKK-JNKK-JNK pathway.
Src Potentiates AF-1 Both by ERK-Mediated Phosphorylation of S118
and by a JNK- Mediated, S118 Independent, Pathway
As noted previously, activation of the Ras-Raf-MEK-ERK pathway
leads to phosphorylation of ER S118 by ERKs. We therefore investigated
whether S118, or the nearby S104 and S106, played similar roles in Src
activation of AF-1. We first examined the effect of S118 mutations to
glutamic acid (E), which mimics the negative charge of phosphorylated
serine and allows stronger ER AF-1 binding to p68 RNA helicase (36, 41), or arginine (R), which blocks S118 phosphorylation and shows
robust inhibition of ER AF-1 activity (71). As expected, ERS118E showed
a slight increase in tamoxifen response relative to ER, and ERS118R
showed no tamoxifen response (Fig. 5A).
In parallel, transfected v-Src gave a much stronger enhancement of
tamoxifen response, suggesting that the increased negative charge of
the ER-S118E mutant was insufficient to mimic the enhancement of AF-1
activity that is obtained with v-Src. Furthermore, ERS118E-dependent
tamoxifen responses were still strongly enhanced by v-Src, and ERS118R
only reduced the overall level of tamoxifen response in the presence of
v-Src by about 50%. v-Src also potentiated the tamoxifen responses
that were obtained in the presence of an ERS118A mutant (data not
shown). Together, these results suggest that v-Src potentiation of AF-1
activity is partially insensitive to mutation of S118. Mutation of S118
to alanine also decreased the ability of v-Src to potentiate
GAL-ER(A/B) by 20 to 40% (Fig. 5B and data not shown). There was no
further decrease in AF-1 activity when all three serines were mutated
to alanine (S104, 106, 118A). Thus, v-Src enhancement of isolated AF-1
is also partially insensitive to mutation of the AF-1 phosphorylation
sites.
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). However, PD 98059 had no effect on the residual Src
activation of the triple serine mutant. This result indicates that the
portion of the Src effect that is independent of S118 phosphorylation
is also ERK independent. Because JNK activation is not blocked by PD
98059 (Ref. 69 and data not shown), we infer that the
serine-independent portion of AF-1 activation is likely to be the
portion that is mediated by JNKs.
To examine the role of JNKs more directly, we examined the ability of
Src and overexpressed JNK to cooperate in AF-1 activation. As shown in
Fig. 5D, v-Src and JNK cooperated to enhance the activity of GAL-A/B
S118A mutant even more strongly than v-Src alone. Thus, the Src-JNK
pathway enhances AF-1 activity in a manner that is independent of
phosphorylation at S118. Surprisingly, ERK1 now specifically inhibited
v-Src action at the GAL-A/B S118A mutant, much as p38 inhibited v-Src
action upon wild-type AF-1 (Fig. 4E). Again, we do not have a ready
explanation for this effect but speculate that transfected ERKs could
inhibit other second messenger inputs to AF-1 and that this effect is
only detectable when their stimulatory effect upon ER AF-1 activity is
abolished. Nonetheless, taken together, our results suggest that v-Src
enhances AF-1 activity via two independent pathways, one that involves
ERKs and targets the AF-1 serine cluster and one that involves JNKs and
is independent of the serine cluster.
Src Activates Two Separate Subdomains of AF-1 That Show
Differential Sensitivity to Inhibitors of MAP Kinases
AF-1 is complex, and it has been suggested that AF-1 is comprised
of independent subdomains (39, 57, 72). We therefore tested some of
these candidate subdomains for response to Src. We transfected
expression vectors for ERs with specific deletions of the N-terminal
(AB) domain into HeLa cells and examined whether they would elicit a
tamoxifen response from the ERE-chloramphenicol acetyltransferase (CAT)
reporter gene in the presence or absence of cotransfected v-Src (Fig. 6A). As expected, v-Src enhanced the
tamoxifen response obtained with wild-type ER by about 8-fold and
showed no effect upon an ER truncation that lacked the A/B domain
(
AB, 185). Transfected v-Src also enhanced the tamoxifen response
obtained with an ER truncation that contained amino acids 1–129 by
7-fold. This truncation contains all of the ER sequences that are
required for AF-1 activity in HeLa cells (57), so this result
underscores the notion that v-Src acts upon AF-1. Transfected v-Src
enhanced the tamoxifen responses obtained with ERs bearing either the
N-terminal (amino acids 1–94) or C-terminal (amino acids 101–185)
AF-1 subdomains by about 5- fold. Furthermore, Src responsiveness of
the C-terminal AF-1 subdomain was lost when the region from 101 to 117,
which contains the serine cluster, was deleted (117). Together, these
observations indicate that v-Src targets two separate subdomains in
AF-1. One lies between amino acids 1–94, and a second lies between
amino acids 101–129, which overlaps the serine cluster. Although the
role of the serine cluster as a target of second messengers is well
established, this is the first direct indication that the N-terminal
region of AF-1 (amino acids 1–94) contributes to second messenger
stimulation of AF-1 activity.
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). Thus, v-Src
action upon the N-terminal AF-1 subdomain does not proceed through MAP
kinase activation, which implies that v-Src action upon this region
proceeds through JNK kinases.
Activated JNKs Do Not Phosphorylate the AF-1 Domain, Suggesting an
Indirect Mode of Action
The above studies indicate that Src activates AF-1 through at
least two pathways, one of which is mediated through JNK and does not
require S118. To investigate whether JNK might phosphorylate another
site within AF-1, we tested the ability of JNKs extracted from cells
that had been treated with TNF, the proinflammatory cytokine and
activator of Src/JNK (70), for their ability to phosphorylate the AF-1
domain in vitro. Activated JNKs were able to efficiently
phosphorylate a c-Jun substrate in vitro (Fig. 7, top) but were unable to
phosphorylate an AF-1 substrate, even though this substrate had been
efficiently phosphorylated by activated ERK as shown earlier (Fig. 4A).
We conclude that activated JNKs do not directly phosphorylate the ER
AF-1 domain.
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We first cotransfected expression vectors for GRIP1 or CBP, along with
vectors for GAL-AF-1 and v-Src, and asked whether the coactivators
would potentiate v-Src action on the GALRE:luc reporter gene.
Overexpression of GRIP1 further enhanced v-Src potentiation of ER AF-1
(7- to 9-fold, Fig. 8A, left).
CBP also slightly increased Src effects on AF-1, similar to its action
on basal AF-1 (24). Thus, v-Src synergizes with overexpressed GRIP1 and
CBP, suggesting that Src activation of AF-1 can occur when AF-1
activity is mediated by contacts with GRIP1 and CBP. We further
examined whether the S118-independent and PD 98059-resistant component
of Src action also synergizes with GRIP1. GRIP1 synergized with v-Src
to activate GAL-AF-1 with the triple serine mutation S104, 106, 118 A
(Fig. 8A, right) both with and without PD 98059. We conclude
that the S118-independent and PD-resistant component of the Src pathway
of AF-1 potentiation is active when AF-1 is mediated by the p160
coactivator GRIP1.
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). Similar observations were also obtained in the
presence of tamoxifen-activated ER (data not shown). Thus, the
C-terminus-deleted GRIP1 has a dominant negative effect on Src
activation of AF-1. Taken together, these observations confirm
that Src can potentiate AF-1 under conditions where AF-1 activity is
mediated by contacts with the GRIP1 C terminus. It may also indicate
that Src activation of AF-1, like basal AF-1 activity, requires the
GRIP1/CBP complex or its functional equivalent.
Src Potentiates CBP and GRIP1 Activation Functions
The above studies suggest that Src potentiation of ER AF-1 is
compatible with circumstances in which AF-1 activity is mediated by
contacts with GRIP1 and CBP, but the studies are neutral as to whether
Src affects the activities of GRIP1 and CBP or some other target. To
gain insight into this latter question, we investigated how Src
affected the transcriptional activation functions of CBP and GRIP1.
GRIP1 or CBP fused to the DNA binding domain of GAL4 activate
transcription when they are tethered to the GALRE reporter gene (Fig. 8C). Cotransfection of v-Src greatly increased both the transcriptional
activity of CBP and GRIP1. We conclude that v-Src has the potential to
change the activities of the GRIP1/CBP coactivator complex. Below we
discuss the possibility that this complex may be one of the candidates
for a Src/JNK target (see Discussion).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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, both of which lead
to JNK activation (56, 73), enhances activation of reporter gene
expression by the estrogen-ER complex and powerfully enhances the
otherwise weak regulation by the tamoxifen-ER complex. These effects
are observed both in transiently transfected HeLa cells and in MCF-7
breast cells that express endogenous ERs. Src action upon the ER is
mediated through a robust activation of the ER AF-1 function. This is
seen most easily when the AF-1 region is removed from the remainder of
the ER and fused to a heterologous DNA binding domain from the yeast
GAL4 protein. Src action did not require the integrity of tyrosine 537
in the ER-LBD, which has been reported to be directly phosphorylated by
Src (61, 62, 63). Src tyrosine kinase activity is required for its action
upon AF-1, as mutations and drugs that inactivate the kinase block the
ability of Src to stimulate ER activity. Thus, taken together, our
results suggest that elevated Src kinase activity results in elevated
ER AF-1 activity. The mechanism of Src enhancement of AF-1 activity is unusual. Previous studies have demonstrated that growth factors enhance AF-1 activity via a signal transduction pathway that is mediated by Ras, Raf, MEK, and the ERK kinases. We therefore expected that Src stimulation of ER activity might proceed through a similar pathway. However, transfection of dominant negative Ras, Raf1, and MEK mutants, or treatment of cells with PD 98059, which blocks Raf1 inputs to MEK, only partially inhibited Src potentiation of AF-1. This suggested that Src also enhanced AF-1 activity via a second pathway and, indeed, dominant negative versions of Rac, MEKK, and JNKK also inhibited Src enhancement of AF-1 activity. Src cooperates both with overexpressed ERK and overexpressed JNK to generate even higher AF-1 activity. Thus, the Src pathway leading to potentiation of AF-1 proceeds through JNKs and through ERKs.
Although the pathway from Src to enhancement of AF-1 activity clearly runs through Rac/MEKK/JNKK and JNKs, it is unclear how the JNKs bring about the enhancement of AF-1 activity. One possibility is that the JNKs directly phosphorylate the AF-1 domain and thereby modify its function, as previously shown for transcription factors c-jun (74, 75) and Elk-1 (76). Our in vitro studies weigh against this possibility, as they indicate that JNKs fail to bind AF-1 (data not shown) and that they also fail to phosphorylate the ER A/B domain in vitro. Although it is possible that these failures reflect a requirement for an accessory protein not supplied in vitro, it is most likely an indication that the JNKs cannot efficiently phosphorylate the AF-1 domain, as activated JNKs require no accessory proteins to phosphorylate other substrates (75, 77, 78). Our in vivo studies also suggest that JNK action is independent of direct ER phosphorylation. The major site of phosphorylation in the AF-1 region is S118, and mutation of this residue to alanine blocks AF-1 phosphorylation and enhancement of AF-1 activity by EGF, activated Ras, and other activators of ERK kinases. JNKs do not activate AF-1 via phosphorylation at S118. Replacement of S118 with alanine only partly reduces Src stimulation of AF-1 activity, and the residual activation is mediated through an ERK-independent pathway. We infer that this residual action is due to Src-activated JNK and this has been confirmed with overexpressed JNKs. In addition, the Src-to-JNK cascade targets at least two subdomains within AF-1, from amino acids 1–94, and 101–129. The first of these domains contains no potential sites for JNK phosphorylation. Furthermore, replacement of the serine cluster, including the MAP kinase phosphorylation site (S118), has no effect on the response of AF-1 to JNK activation.
How does Src-activated JNK enhance AF-1? One attractive possibility is that activated JNKs target a protein that, itself, affects AF-1 action. Recent studies indicate that AF-1 works by recruiting p160/CBP coactivators by means of a direct contact between AF-1 and the C terminus of the p160 (24). We have confirmed that Src enhancement of AF-1 activity can also occur in the presence of overexpressed GRIP1 and CBP, and that this enhancement requires the C terminus of GRIP1. Thus, one possibility is that the AF-1 mediating activities of the p160/CBP complex are the target for JNK. Accordingly, v-Src was able to enhance the transcriptional activity of both GRIP1 and CBP when they were directly tethered to DNA. One obvious question is that if, indeed, v-Src does enhance the activity of the GRIP1/CBP complex, and given that both AF-1 and AF-2 work by binding a GRIP1/CBP complex, then why would v-Src preferentially enhance AF-1 activity? We have previously shown that the AF-1 and AF-2 functions of different nuclear receptors both bind to p160s, but bind to different surfaces and require different transcriptional outputs (24, 79). Thus, v-Src could preferentially affect AF-1 activity by preferentially affecting a subset of p160 transcriptional inputs or outputs that are required for AF-1 action.
We stress that the sole positive evidence for Src/JNK targeting of the
coactivator complex is that Src potentiates the transcriptional
activation functions of both GRIP1 and CBP when they are fused to GAL4
and tethered to a promoter. This experiment is only suggestive.
While JNKs might phosphorylate GRIP1 itself, or another component of
the complex (Fig. 9), there are other,
equally likely, possibilities. For example, JNKs might phosphorylate
and modify the activities of corepressors that are suspected of
modulating AF-1 action. Exploratory studies are underway to examine
these and other possibilities.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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ER expression vectors pSG5-ER, pSG5-ERG400V have been described
previously (86). pSG5-ER(A/B), and 1–100, 1–116,
130–184, and 95–184 have been described previously (57).
pSG5-GAL4, -GAL4-ER(A/B), -GAL4-ER(LBD), -GAL4-VP16 expression vectors
have been described (57, 87). The pSG5-ERY537R, Y537S mutants were
obtained from Dr. G. Greene, University of Chicago. The pCMV-ERY537F
mutant was obtained form Dr. B. Katzenellen-bogen, University of
Illinois. The pSG5-GAL4-ER(A/B) mutants in which each the
phosphorylation sites at serine (S) 104, S106, S118, were mutated to
alanine (A) were created by synthesizing double-stranded
oligonucleotides that encode the mutant sequence and using Quick Change
Site-directed Mutagenesis Kits (Stratagene, La Jolla, CA).
The mutated sequences were verified by DNA sequencing using Sequenase
Kits (Stratagene). The GAL-4-ER(A/B) mutant containing the
triple phosphorylation site mutation (S104, 106, 118A) was made by
multiple rounds of mutagenesis.
Many signal transduction molecule expression vectors were kindly
provided as follows: pCMV-v-Src (Dr. M. Bishop, University of
California, San Francisco, CA); pCMV-c-SrcRF(K295R,Y527F) and
pCMV-c-Src (Dr. Joan Brugge, Ariad Pharmaceuticals, Inc., Cambridge,
MA); Constitutively activated pCMV-c-Src (Y527F) (Dr. Tony Hunter, Salk
Institute for Biological Studies, La Jolla, CA); dominant negative
pcDNA3-Rac1(S17N) and -Raf(1–257) (Dr. H. Goldberg, University of
Toronto, Toronto, Ontario, Canada); pCMV-Flag-p38 (Dr. Roger Davis,
University of Massachusetts Medical Center, Boston, MA). Dominant
negative Ras(S17N), MEK(K97R), pSR-JNKK(K116R), MEKK(K432M),
HA-JNK1, and HA-ERK2 have been described previously (56). Coactivator
expression vector pCMV-CBP was a gift from Dr. M. Rosenfeld (University
of California San Diego, La Jolla, CA). pSG5-GRIP1 has been
previously described (29).
Cell Culture and Transfection
HeLa cells were maintained and transfected as previously
described (86). Briefly, around 70% confluent HeLa cells were
transfected with 5 µg of (ERE)2-TK-CAT or 5x
GAL4-Luc reporter gene, 1 µg ß-galactosidase plasmid, 1 µg ER
expression vector, and other coactivator and signal molecule expression
vectors (2 µg) as indicated in the figure legends. After 20 h
incubation, cells were lysed and CAT, LUC, and ß-galactosidase assays
were performed using standard methods. The ß-galactosidase activity
was used to correct the variations of transfection efficiency in CAT
and LUC activities. The hormones (10 nM estradiol, ICI, and
5 µM tamoxifen) and kinase inhibitors [300
µM genistein (Sigma, St. Louis, MO), or 1
µM herbimycin A (Sigma), and 100 µm PD
98059 (Calbiochem)] were added immediately after
transfection. CAT and LUC activities represented the averages from
triplicate wells with less than 20% deviation. Experiments were
repeated at least three times.
Western Blots
HeLa cells were transfected with ER and v-Src expression
vectors, or empty control vectors, and allowed to stand overnight. Half
were prepared for Western blot analysis, and the remaining half was
used for standard CAT and ß-galactosidase assays. The following day,
the transfected cells were washed in cold PBS and treated with 1 ml of
luciferase lysis buffer on ice for 5 min. The lysate was scraped off
the dish, transferred to Eppendorf (Madison, WI) tubes,
and pelleted in an Eppendorf microfuge for 15 min at 4 C.
Protein contents were determined and 15 µg of cell proteins were
separated on a 10% SDS-polyacrylamide gel and transferred to a
premoistened Immuno-Blot polyvinylidene difluoride
membrane (Bio-Rad Laboratories, Inc. Hercules CA),
overnight at 90 mA, 30V, using a standard transfer apparatus. After
transfer, the membrane was incubated at room temperature in 5% nonfat
milk in PBS-T (1xPBS, 0.1% Tween-20) for 1 h, and washed twice
in PBS-T for 10 min. The primary anti-ER antibody (HC-20, Santa Cruz Biotechnology, Inc.,, Santa Cruz, CA) was diluted 1:2,000
in PBS-T and incubated with the membrane for 1 h, followed by
PBS-T washes, once for 15 min and then twice for 5 min. The membrane
was then incubated for 45 min with horseradish peroxidase-conjugated
antirabbit IgG (Santa Cruz Biotechnology, Inc.) diluted
1:2,000 in PBS-T, followed by PBS-T washes, once for 15 min and four
times for 5 min in PBS-T. After the last wash, the membrane was
developed according to manufacturer’s instructions with a standard ECL
kit (Amersham Pharmacia Biotech, Arlington Heights, IL),
covered with Saran wrap and exposed to x-ray film.
GST-Fusion Proteins
GST-ER(A/B) and GST-cJun (amino acids 1–79, from Dr. A.
DeFranco, UCSF) fusion protein were prepared as previously described
(86). Briefly, bacteria (500 ml LB media) expressing the fusion
proteins were resuspended in 15 ml of TST buffer (0.5 M
Tris, 1.5 M NaCl, 0.5% Tween 20, pH 7.5) and sonicated
mildly for 2–3 min in ice. The debris was pelleted at 12,000 rpm for
1 h in an ss34 rotor. The supernatant was rotated gently for
2 h in a cold room with 0.5 ml of glutathione sepharose 4B beads
that had been prewashed with 5–10 vol of TST buffer. GST-fusion
protein beads were washed with 10–20 vol PBS 0.01% Nonidet P-40 and
resuspended in 1:1 vol of 20 mM HEPES, 150 mM
KCl, 5 mM MgCl2, 10% glycerol, 1
mM dithiothreitol, 0.2 mM phenylmethylsulfonyl
fluoride, and protease inhibitors, pH 7.9, for storage at 4 C until
use.
In Vitro Kinase Assay
Src, MAP, and JNK kinase assays were carried out following the
manufacturer’s protocol (Upstate Biotechnology, Inc.,
Lake Placid, NY) with purified Src, MAP, and JNK kinases, as provided.
Briefly, purified Src, MAP, and JNK kinases were mixed with the
corresponding substrates or GST-ER(A/B), the reaction was started by
adding the corresponding reaction buffer containing
(
-32P)ATP, and then mixed gently and incubated
at 30 C for 15–30 min. After addition of 40% trichloroacetic acid or
2x SDS-PAGE loading buffer to stop the reaction, the phosphorylated
kinase substrates and GST-ER(A/B) were detected by liquid scintillation
counter or autoradiography of 10–12% SDS-PAGE.
|
ACKNOWLEDGMENTS |
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FOOTNOTES |
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This work was supported by Public Health Service Grants DK-51083 and CA-80210 (to P.J.K.) and by grants from the California Breast Cancer Research Program (to P.J.K. and M.K.).
Peter Kushner wishes to inform readers that he has significant financial holdings in and is a consultant to KaroBio AB, a pharmaceutical company with interests in nuclear receptors.
Received for publication April 7, 1999. Revision received October 5, 2000. Accepted for publication October 9, 2000.
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