Potentiation of Estrogen Receptor Activation Function 1 (AF-1) by Src/JNK through a Serine 118-Independent Pathway

Weijun Feng, Paul Webb, Phuong Nguyen, Xiaohong Liu, Jiandong Li, Michael Karin and Peter J. Kushner

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


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Estrogen receptor (ER) is activated either by ligand or by signals from tyrosine kinase-linked cell surface receptors. We investigated whether the nonreceptor Src tyrosine kinase could affect ER activity. Expression of constitutively active Src or stimulation of the endogenous Src/JNK pathway enhances transcriptional activation by the estrogen-ER complex and strongly stimulates the otherwise weak activation by the unliganded ER and the tamoxifen-ER complex. Src affects ER activation function 1 (AF-1), and not ER AF-2, and does so through its tyrosine kinase activity. This effect of Src is mediated partly through a Raf/mitogen-activated ERK kinase/extracellular signal-regulated kinase (Raf/MEK/ERK) signaling cascade and partly through a MEKK/JNKK/JNK cascade. Although, as previously shown, Src action through activated ERK stimulates AF-1 by phosphorylation at S118, Src action through activated JNK neither leads to phosphorylation of S118 nor requires S118 for its action. We therefore suggest that the Src/JNK pathway enhances AF-1 activity by modification of ER AF-1-associated proteins. Src potentiates activation functions in CREB-binding protein (CBP) and glucocorticoid receptor interacting protein 1 (GRIP1), and we discuss the possibility that the Src/JNK pathway enhances the activity of these coactivators, which are known to mediate AF-1 action.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
The action of estrogen receptor-{alpha} (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 {alpha}-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.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Src Potentiates ER AF-1
Src activity in breast cancer cells and cell lines is often elevated up to 30-fold (49). To examine the effects of elevated Src activity on ER action, we transfected expression vectors for v-Src and control vectors into HeLa cells along with expression vectors for ER and an ERE responsive reporter gene (ERE:HSV-TK-CAT). v-Src is a viral derivative of cellular Src (c-Src) which has at least 10-fold higher kinase activity than c-Src. Figure 1AGo 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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Figure 1. v-Src Potentiates ER AF-1 Transcriptional Activity

A, Activity of an ERE-CAT reporter gene in HeLa cells transfected with expression vectors for wild-type ER, the ER G400V derivative, and v-Src and treated with estradiol (E2), tamoxifen (Tam), and ICI 182,780 (ICI). B, Western blots were performed upon extracts of cells that were transfected with different amounts of ER expression vector, or control vector, ±transfected v-Src. The lower panel shows transcriptional activation of the ERE responsive reporter gene as a function of ER levels ± transfected v-Src. C, Activity of the ERE reporter gene in HeLa cells transfected with an expression vector for ER, the indicated amount of expression vector for v-Src after treatment with tamoxifen (closed circles) or vehicle (open boxes). Fold inductions were determined by comparing the relative CAT activity in the presence of v-Src to that in the absence of v-Src (set as 1-fold). D, Activity of the ERE-CAT reporter gene in MCF-7 breast tumor cells that express endogenous ERs. E, Activity of a reporter gene with five GAL4 response elements in cells transfected with expression vectors for GAL4 fusion proteins to the ER A/B region (GAL4-AB), to the ER ligand binding domain (GAL-LBD), or to the herpes virus VP16 activation domain (GAL4-VP16) in the presence or absence of cotransfected v-Src. Fold inductions were determined as in Fig. 1CGo.

 
To confirm that v-Src effects upon ER activity could not be simply explained by increases in ER levels, we performed Western blots on extracts of cells that had been transfected with different amounts of ER in the presence and absence of transfected v-Src. Figure 1BGo 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. 1BGo, 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. 1CGo). We then examined v-Src action upon the ERE responsive reporter in MCF-7 breast tumor cells, which express endogenous ER (Fig. 1DGo). 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. 1EGo, 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 2AGo 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. 2BGo). 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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Figure 2. Src Action upon ER Is Independent of Tyrosine 537

A, Activity of the ERE-CAT reporter was determined in the presence of empty pSG5 expression vector, or expression vectors for ER, ERG400V, ERY537R, and ERY537S, both in the presence of an empty expression vector or an expression vector for v-Src. B, A similar experiment performed with empty CMV expression vector or expression vectors for wild-type ER and the Y537F derivative.

 
Src Kinase Activity Is Required for Potentiation of ER AF-1
To investigate whether elevated Src kinase activity itself, and not some other feature of v-Src, leads to potentiation of ER action, we examined the effect of various Src expression vectors and tyrosine kinase inhibitors upon ER AF-1 activity. Wild type c-Src and constitutively activated c-Src (Y527F) potentiated the activity of the GAL-ER(A/B) fusion protein by 2~3- and 5-fold, respectively (Fig. 3Go). 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. 3Go). 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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Figure 3. Src Action on ER AF-1 Requires Src Tyrosine Kinase Function

Shown is fold induction of the GALRE reporter gene activated by GAL-ER(A/B) in the absence of Src (normalized to 1) and by c-Src, by constitutively active c-Src [c-Src (Y527F)], by an inactive derivative of c-Src [c-Src(K295R, Y527F)], or by v-Src in the absence or presence of tyrosine kinase inhibitors (genistein or herbimycin). The effect of the inhibitors on v-Src-enhanced and basal stimulation by GAL-Elk1is shown in the inset.

 
Src Potentiates AF-1 through Both the Ras- Raf1-MEK-ERK and the Rac-MEKK-JNKK- JNK Pathways
Src tyrosine kinase might affect AF-1 either directly or indirectly through the numerous signal transduction cascades that Src is known to initiate. While Src is generally associated with the plasma membrane, and ER is generally nuclear, these locations are not rigid. In particular, there are reports of ER associated with the cell surface (67, 68). Thus, there is nothing a priori to exclude the possibility that Src might directly phosphorylate the ER AF-1 domain. We therefore tested whether a glutathione-S-transferase (GST)-ER(A/B) fusion protein was a substrate for activated c-Src kinase. GST-ER(A/B) was efficiently phosphorylated by activated ERK2 MAP kinase, consistent with other studies (37), but not by activated c-Src kinase (Fig. 4AGo). 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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Figure 4. v-Src Activates AF-1 through ERK and JNK MAP Kinases

A, The ER A/B domain is a target for ERK but not Src kinase. Assay of kinase activity using purified Src and ERK kinases, a peptide Src substrate (amino acids 6–20 of cdc-2), PHAS-I (a peptide ERK substrate), and purified GST-ER(A/B) fusion protein, as indicated. B, v-Src action on AF-1 is partly blocked by dominant negative Ras [Ras(S17N)], Raf1 [Raf(1–257)], MEK [MEK(K97R)], or the PD 98059 compound that blocks activation of ERK MAP kinases. Induction of a GALRE reporter gene activated with GAL-ER(A/B) and v-Src in the presence of the indicated dominant negative or drug inhibitor. Inset on right shows the expected complete blockade of EGF action on Elk-1 by the dominant negatives and PD compound. C, v-Src action on AF-1 is also blocked by dominant negative Rac [Rac(S17N)] or MEKK [MEKK(K432M)]. An assay similar to B of reporter gene response to Src in the presence of the indicated dominant negative inhibitor. D, TNF{alpha} and Src action on AF-1 is blocked by dominant negative JNKK(K116R). Induction of the GALRE reporter gene activated by GAL-ER(A/B) in HeLa cells cotransfected with dominant negative JNKK [JNKK(K116R)] as indicated. After 20 h incubation, the cells were treated with TNF{alpha} for 1 h or transfected with v-Src expression vector, as indicated, and then incubated for 6 h before assay. E, ERK2 and JNK1, but not p38, cooperate with Src to activate AF-1. Induction of the GALRE reporter gene activated by GAL-ER(A/B) in the presence of activated Src and elevated ERK2, JNK1, or p38 as indicated.

 
Because Src is known to cause MAP kinase activation, and the well known pathway of Ras, Raf-1, MEK, and ERK activation enhances AF-1 activity through serine 118 phosphorylation, we asked whether v-Src might enhance ER activity by initiating the Raf-1/MEK/ERK cascade. We first examined the effect of various inhibitors of this pathway upon EGF stimulation of the transcription factor ELK-1, which responds even more dramatically to EGF than does the ER. As shown in Fig. 4BGo, 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. 4CGo). 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-{alpha} (TNF{alpha}) (Fig. 4DGo). 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. 4EGo). 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. 5AGo). 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. 5BGo 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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Figure 5. Src-Activated ERKs, but not JNKs, Require S118 in the A/B Domain to Activate AF- 1

A, Src potentiation of AF-1 is in part independent of S118. Induction of the ERE responsive reporter gene activated by ER, or ERS118E and ERS118R, with and without transfected v-Src. B, Induction of the GALRE reporter gene activated by GAL-ER(A/B) or the indicated mutant thereof, with v-Src. v-Src induction of wild-type GAL-ER(A/B) was set as 100%. C, Src activation of AF-1 that is independent of S118 is resistant to PD 98059, the ERK pathway inhibitor. Src effects are shown as fold induction of reporter gene activity. D, Src potentiation on ER(A/B) with mutations in S118 serine cluster cooperates with JNK but not ERK kinases. Induction of the GALRE reporter gene activated with GAL-ER(A/B) S118, v-Src, and vectors for JNK1 or ERK2, as indicated.

 
It is noteworthy that Src has a major effect on AF-1, even in the presence of the triple serine mutation. To test whether this effect is mediated by the non-ERK (that is, JNK) part of the Src-AF-1 pathway, we examined Src activation of wild-type and triple mutant GAL-ER(A/B) in the presence of PD 98059. This inhibitor reduced Src potentiation of AF-1 activity by about 50%, as did the mutation of the three serines (Fig. 5CGo). 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. 5DGo, 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. 4EGo). 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. 6AGo). 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 ({Delta}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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Figure 6. Src Potentiates Transcription from Two Separable Subdomains of AF-1

A, Fold induction that v-Src elicits from the ERE reporter gene activated by the indicated ER derivative in the presence of tamoxifen. B, The sensitivity of v-Src enhancement of tamoxifen response to PD 98059 was determined in the presence of the indicated ER derivative in the presence of tamoxifen. All values (after subtraction of backgrounds) were normalized to that obtained in the presence of fully wild-type ER, which was set at 100%.

 
Control transfections revealed that PD 98059 completely failed to inhibit the activity of the ER truncation that only contained the N-terminal AF-1 subdomain (amino acids 1–94, Fig. 6BGo). 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{alpha}, 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. 7Go, 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. 4AGo). We conclude that activated JNKs do not directly phosphorylate the ER AF-1 domain.



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Figure 7. ER(A/B) Is Not Phosphorylated by JNK1 in Vitro

GST-cJun or GST ER(A/B) was in vitro phosphorylated by immunopurified JNK1 kinase extracted from HeLa cells treated with TNF{alpha} (a Src/JNK activator) and analyzed on 10% SDS-PAGE with autoradiography. Control experiments (see Fig. 3Go) indicate that GST-ER(A/B) is a good substrate for ERKs.

 
Src Can Potentiate AF-1 in Conditions Where AF-1 Is Mediated by the GRIP1/CBP Complex
Our results show that Src stimulates ER AF-1 activity through JNK kinases and that the JNKs do not phosphorylate the AF-1 domain. This suggests that the action of the JNK pathway on AF-1 may be mediated by phosphorylation of another protein that mediates ER activity. Basal AF-1 activity is mediated by a complex of a p160 such as GRIP1, along with p300/CBP (24). The essential contact for this action is between the N-terminal subdomain of AF-1 and the C-terminal domain of GRIP1. We therefore asked whether v-Src enhanced AF-1 under conditions in which AF-1 is itself enhanced by overexpression of GRIP1 and CBP.

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. 8AGo, 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. 8AGo, 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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Figure 8. Src/JNK Potentiates ER AF-1 Mediated by GRIP1 and CBP

A, Elevated GRIP1 or CBP cooperate with Src in activation of AF-1. Left panel shows induction of the GALRE reporter gene activated with ER-GAL(A/B) and the effects of transfected v-Src GRIP1 or CBP as indicated. Right panel shows the substantial effects of GRIP1 on v-Src potentiation of the triple serine ER-AF-1 mutant even in the presence of PD 98059 as indicated. B, A mutant of GRIP1 deficient for mediating basal AF-1 acts as a partial dominant negative for Src action on AF-1. Response of the reporter gene to v-Src in the presence of elevated amounts of the indicated derivative of GRIP1. GRIP1(1–1,121) is missing amino acids 1,122–1,462 and does not bind to ER(A/B) or mediate AF-1 action in the absence of Src. C, CBP and GRIP1 activation functions are potentiated by Src. Activity of the GALRE:luciferase reporter gene activated by GAL4 fusions to CBP or GRIP1 as indicated and cotransfected with expression vector for v-Src.

 
As noted above, both AF-1 and AF-2 recruit the p160-CBP complex, but the two ER domains contact different surfaces on the coactivators. AF-1 contacts the C-terminal domain of GRIP1 (24), whereas AF-2 contacts one of the multiple p160 NR Boxes, which are located in the middle portion of GRIP1 (13, 26, 27, 28, 29). Thus, deletion of the C terminus of GRIP1 prevents AF-1 activity without affecting AF-2. To test whether AF-1/GRIP1 contacts were needed for v-Src enhancement of AF-1, we overexpressed a C-terminal deletion of GRIP1 (1–1,121 aa), which lacks the site for AF-1 binding (24). The GRIP1 mutant missing the C terminus failed to cooperate with v-Src to enhance AF-1 and reduced activation by v-Src (Fig. 8BGo). 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. 8CGo). 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).


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
We were encouraged to investigate whether elevated Src potentiates ER action by two observations. First, in addition to estrogen, ER activity is also stimulated by signaling cascades initiated at tyrosine kinases. Second, Src tyrosine kinase activity is often elevated in breast cancers, whose proliferation is stimulated by ER activation. Our studies indicate that expression of constitutively active forms of Src or cell stimulation with the cytokine TNF{alpha}, 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. 9Go), 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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Figure 9. Pathways of v-Src Stimulation of ER(A/B) Transcriptional Activity

One pathway leads from Src through Ras, Raf, MEK, and ERKs and results in phosphorylation of the ER(A/B) on S118. The second pathway leads to Rac, MEKK, JNKK, and JNKs. JNKs do not phosphorylate S118. Among many possibilities JNKs may target coactivators that mediate AF-1 activity. This is indicated by an arrow from JNK to coactivators. That it is merely a possibility is indicated by the question mark.

 
Finally, we speculate that Src enhancement of AF-1 may have consequences for cellular responses to estrogen. Breast cancer samples and cell lines almost invariably have elevated Src tyrosine kinase activity (50). Some of those tumors and cell lines also express ER and are stimulated to grow by estrogens. In such cases, ER is believed to enhance proliferation by enhancing the expression of target genes encoding regulators of proliferation. We showed here that overexpression of v-Src leads to increases in ER transcriptional activity in breast cells, and that this effect occurs in the presence of estradiol, the absence of hormone and, most strikingly, in the presence of tamoxifen. Thus, increases in Src kinase activity could, via stimulation of ER transcriptional potency, enhance the ability of ER to induce cellular proliferation in breast cancer and breast cancer cell lines. Moreover, increased Src kinase activity could also lead to increased tamoxifen agonist activity, which might play a role in the development of tamoxifen resistance. We note that the tyrosine kinase inhibitors genistein and Herbimycin A inhibit cellular proliferation in response to estrogen (80, 81, 82, 83). Both of these inhibitors block Src activity in breast cancer cells and also inhibit Src effects upon ER AF-1. Thus, it is conceivable that both drugs could inhibit estrogen-induced proliferation via inhibition of Src effects upon the ER. Taken together, our results suggest that it is important to investigate the relationship of Src activity and ER action in breast cancer and during tumor progression.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Mammalian Reporter Genes and Expression Vectors
EREII-HSV-TK-CAT, a reporter gene containing two vitellogenin EREs upstream of the herpes simplex virus TR proximal promoter (-109/+45), has been previously described (84). The GAL4 responsive reporter gene GK1 contains five GAL4 response elements upstream of a minimal adenovirus E1b promoter that has been previously described (85).

ER expression vectors pSG5-ER, pSG5-ERG400V have been described previously (86). pSG5-ER{Delta}(A/B), and {Delta}1–100, {Delta}1–116, {Delta}130–184, and {Delta}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{alpha}-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 ({gamma}-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
 
We thank David Stokoe for helpful comments on the manuscript.


    FOOTNOTES
 
Address requests for reprints to: Peter Kushner, Metabolic Research Unit 1119 HSW, University of California, San Francisco, CA 94143-0540. E-mail: kushner{at}itsa.ucsf.edu

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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