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Endocrinology Vol. 142, No. 12 5145-5148
Copyright © 2001 by The Endocrine Society

INTRACELLULAR SIGNAL SYSTEMS

Estradiol (E2) Elicits Src Phosphorylation in the Mouse Neocortex: The Initial Event in E2 Activation of the MAPK Cascade?

Imam S. Nethrapalli, Meharvan Singh, Xiaoping Guan, QiongFen Guo, Dennis B. Lubahn, Kenneth S. Korach and C. Dominique Toran-Allerand

Departments of Anatomy and Cell Biology, Obstetrics and Gynecology (I.S.N., M.S., X.G., Q.G., C.D.T.-A.), and Neurology (C.D.T.-A.) and Centers for Neurobiology and Behavior (C.D.T.-A.) and Reproductive Sciences (I.S.N., M.S., X.G., Q.G., C.D.T.-A.), Columbia University College of Physicians and Surgeons, New York, New York 10032; Departments of Biochemistry and Child Health, University of Missouri (D.B.L.), Columbia, Missouri 65211; and Laboratory of Reproductive and Developmental Toxicology, National Institute of Environmental Health Sciences (K.S.K.), Research Triangle Park, North Carolina 27709

Address all correspondence and requests for reprints to: Dr. Dominique Toran-Allerand, Department of Anatomy and Cell Biology, Columbia University College of Physicians and Surgeons, 630 West 168th Street, Black Building 1615, New York, New York 10032. E-mail: cdt2{at}columbia.edu


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In neocortical explants, E2 activates various signaling components of the MAPK cascade, including B-Raf and MAPK kinase-dependent ERK, suggesting a possible role in the differentiative actions of E2 in the brain. To further characterize the signaling pathways activated by E2, we determined whether c-Src, a member of the Src family of nonreceptor tyrosine kinases and an important modulator of both the MAPK cascade and neuronal differentiation, may play a role in E2 signaling. The present studies show for the first time in the brain that E2 elicits phosphorylation of c-Src on three functionally critical tyrosine residues (Y220, Y423, and Y534), and that this phosphorylation occurs despite disruption of ER{alpha} (in ER knockout mice). PP2, a Src family kinase inhibitor, suppressed not only E2-induced phosphorylation of c-Src, but ERK phosphorylation as well, suggesting that c-Src may be an upstream regulator of E2 signaling. E2-induced phosphorylation of c-Src is associated with increased tyrosine phosphorylation of Shc, increased association of Shc with Grb2, and induction of Ras, but not Rap1, activation. Together, these data provide evidence that E2 activates a novel c-Src-dependent signal transduction pathway in the developing brain.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
WE SHOWED PREVIOUSLY in rodent neocortical explant slices derived from wild-type and ER{alpha} gene-disrupted mice (ERKO) that estradiol (E2) elicits the rapid and sustained activation of B-Raf and ERK (1), two signaling components within the MAPK cascade. Sustained activation of the MAPK cascade has been closely associated with neuronal differentiation (2), suggesting that this pathway may mediate some of the differentiative actions of E2 in the brain (3). The upstream components necessary for activation of the MAPK cascade, however, remain ill defined. In the present study we determined whether c-Src, a member of the Src family of nonreceptor tyrosine kinases and an important modulator of neuronal differentiation (4, 5), might be involved in E2 activation of the MAPK pathway.

c-Src is activated through direct interaction with membrane-associated receptors and serves as an intermediate for a diverse group of signal transduction pathways (for review, see Ref. 6), including the MAPK cascade (7). As most signaling kinases, the activity of c-Src is regulated by both phosphorylation and dephosphorylation events. In the chick, c-Src is maintained in an inactive state through interaction of its SH2 domain with a C-terminal phosphotyrosine (Y527), whereas dephosphorylation of this residue leads to Src autophosphorylation on Y416, and thus Src activation (6).

Here we demonstrate for the first time that E2 induces phosphorylation of three critical tyrosine residues of mouse c-Src (Y220, Y423, and Y534; functionally homologous to Y213, Y416, and Y527 in the chick) and elicits signaling events that include phosphorylation of the adaptor protein Shc and activation of Ras, the initial signaling kinase of the MAPK cascade. These data identify upstream components of E2 signaling in the developing brain and establish a potential link between E2-induced c-Src and ERK activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Tissue culture
Organotypic explants were derived from approximately 360-µm thick, hemi-coronal slices of postnatal d 2 (P2) frontal and cingulate cerebral cortex (day of birth = P1) obtained from pups of both sexes born to timed pregnant C57BL/6 mice (The Jackson Laboratory). Previous experiments have determined that neocortical cultures derived from both sexes respond equally to E2 with respect to activation of the MAPK cascade (Toran-Allerand, C. D., unpublished observations). P3 explants from ERKO (8) were obtained from our breeding colony established from mice provided by D. B. Lubahn, University of Missouri (Columbia, MO), and identified by genotyping (9) as either wild-type or homozygous for the ER{alpha} gene disruption. Cultures were prepared and maintained in 2 nM 17ß-E2, as previously described (1) All procedures involving animals were approved by the institutional animal care and use committee at Columbia University.

Treatment of cultures
After 6 d in culture, a 24-h washout period (as described in Ref. 1) was performed, consisting of omitting exogenously added E2. The following day, explants were pulsed with 10 nM 17ß-E2 for 60 min. To determine the effect of the Src family kinase inhibitor PP2 on E2-induced Src and ERK phosphorylation, cultures were pretreated with either PP2 (10 µM) or vehicle control (0.1% dimethylsulfoxide) for 60 min before treating the cultures with 60 min of 10 nM E2 in the continued presence of the inhibitor or vehicle control.

Western blot analysis
Explant cultures were harvested into lysis buffer and subjected to SDS-PAGE, as previously described (1). After transfer onto polyvinylidene difluoride membranes (Bio-Rad Laboratories, Inc., Hercules, CA), blots were blocked with 3% BSA (fraction V), and probed with the following antibodies: for ERK phosphorylation, rabbit antiphospho-MAPK [dual phosphospecific (Thr202/Tyr204), 1:1000; New England Biolabs, Inc., Beverly, MA]; for ERK protein assessment, goat anti-ERK1 (1:1000) and goat anti-ERK2 (1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA); for mouse c-Src phosphorylation, rabbit antiphosphoY215-specific antibody, rabbit antiphosphoY418-specific antibody, and rabbit antiphosphoY529-specific antibody (corresponding to tyrosine residues Y220, Y423, and Y534 of mouse c-Src, respectively; 1:1000, Biosource International, Camarillo, CA). Application of the horseradish peroxidase-conjugated secondary antibody and subsequent signal detection, using enzyme-linked chemiluminescence, was carried out, as previously described (1). The data presented are representative of two or three independent experiments.

Immunoprecipitation of Shc and Grb2
Neocortical lysates were immunoprecipitated with rabbit anti-Shc antibodies (1 µg IgG; Santa Cruz Biotechnology, Inc.) as previously described (1) using antirabbit IgG-precoated magnetic beads (Dynal, Oslo, Norway), and processed using the Western blot procedure described above. Blots were probed with a mouse antiphosphotyrosine (4G10) antibody (1 µg/ml; Upstate Biotechnology, Inc., Lake Placid, NY) or the Grb2 antibody (sc-255, 1:500; Santa Cruz Biotechnology, Inc.).

Assays for Ras and Rap1 activities
Determination of Ras activity was performed according to the method and reagents provided in the Ras activation assay kit (Upstate Biotechnology, Inc.). The Ras assay takes advantage of the Ras-binding domain (RBD) of Raf-1 as a means to precipitate selectively activated (GTP-bound) Ras. Briefly, neocortical lysates were precipitated with the Raf-1 RBD-agarose conjugate overnight at 4 C. Precipitates were then washed with an Mg2+ lysis buffer [25 mM HEPES (pH 7.5), 150 mM NaCl, 1% Igepal CA-630, 10 mM MgCl2, 1 mM EDTA, 10% glycerol, 25 mM NaF, 1 mM Na3VO4, 10 µg/ml aprotinin, and 10 µg/ml leupeptin], resuspended in Laemmli buffer, boiled, and separated using SDS-PAGE. Resulting blots were probed for Ras, using mouse anti-Ras (clone RAS10) antibody (1 µg/ml).

Similarly, for the determination of Rap1 activity, a glutathione-S-transferase fusion protein conjugated to the Rap-binding domain of Ral (RalGDS-RBD, gift from Dr. J. Bos, University Medical Center Utrecht, Utrecht, The Netherlands) was precoupled to glutathione-agarose beads and used to precipitate activated Rap1. The precipitate was then subjected to Western analysis and probed with a Rap1 antibody (sc-65, Santa Cruz Biotechnology, Inc.).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
E2 elicits the phosphorylation of c-Src in both wild-type and ERKO mice
To determine whether E2 induces c-Src phosphorylation, we used phosphotyrosine-specific Src antibodies generated against three functionally important tyrosine residues of c-Src. Figure 1Go shows that E2 elicited phosphorylation of c-Src on Y220, Y423, and Y534 in neocortical explants obtained from both wild-type and ERKO mice. Time-course analysis (data not shown) revealed that phosphorylation of Y220, Y423, and Y534 occurred within 15 min of E2 treatment, becoming maximal after 60 min. Y220 and Y423 phosphorylation declined thereafter, whereas phosphorylation on Y534 persisted for up to 2 h. We also evaluated whether PP2, a Src family kinase inhibitor, could inhibit E2-induced c-Src phosphorylation. As expected, PP2 blocked both basal and E2-elicited c-Src phosphorylation (Fig. 1Go).



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  		Figure 1. E2 elicits the phosphorylation of c-Src. The effect of E2 on the tyrosine phosphorylation of c-Src was evaluated using Western analysis. The first three blots show that E2 elicited phosphorylation of c-Src on Y220, Y423, and Y534 in neocortical explants from both wild-type and ERKO mice, an effect that was blocked by the Src inhibitor, PP2. The lowest blot represents reprobing of the upper blot for total ERK protein to verify equal loading across lanes. The bar graphs show the densitometric representation of the blots.

 
PP2 inhibits E2-induced ERK phosphorylation
To determine whether c-Src inhibition would also impair the phosphorylation of downstream signaling elements, we evaluated whether PP2 altered the effectiveness of E2 in eliciting ERK phosphorylation. Pretreatment of neocortical cultures with PP2 blocked E2 induction of ERK phosphorylation completely and reduced basal ERK phosphorylation as well (Fig. 2AGo).



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  		Figure 2. Western blot showing the effect of the Src family tyrosine kinase inhibitor, PP2, on E2-induced ERK phosphorylation (A) and the effect of E2 on Shc phosphorylation and Grb2 association (B). A, PP2 pretreatment resulted in the inhibition of E2-induced ERK phosphorylation. B, Neocortical lysates were immunoprecipitated with an anti-Shc antibody and probed for phosphotyrosine (B, upper blot), Shc (B, middle blot), or Grb2 (B, bottom blot). The bar graphs show densitometric analysis of the immunoblots.

 
E2 elicits the phosphorylation of Shc and results in increased association of Shc with Grb2
To investigate whether downstream targets of Src that may link Src activity to the MAPK cascade are also activated by E2 treatment, we evaluated the effect of E2 on Shc and its association with Grb2. Shc is a downstream target of c-Src and upon phosphorylation links receptor tyrosine kinases to the SH2-containing Grb2 adaptor protein and recruits the Grb2/Sos complex. Figure 2BGo demonstrates that E2 treatment for 60 min resulted in an increase in tyrosine phosphorylation of immunoprecipitated Shc (top panel). Time-course analysis (data not shown) demonstrated that E2-induced Shc phosphorylation occurred within 5 min and persisted up to 60 min after E2 treatment. Shc phosphorylation correlated with an increase in Grb2 association (Fig. 2BGo, bottom panel).

E2 elicits Ras, but not Rap1, activation
As Shc phosphorylation and the recruitment of Grb2 are associated with activation of Ras and subsequent ERK activation (10), we also determined the effect of E2 on Ras. E2 elicited a modest, but reproducible, increase in activated (GTP-bound) Ras (Fig. 3AGo). In contrast, the activity of the structurally homologous guanine nucleotide exchange factor, Rap1, was unaffected (Fig. 3BGo).



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  		Figure 3. Effect of E2 on Ras and Rap1 activities. Neocortical lysates precipitated with Raf-1 RBD-agarose were subjected to SDS-PAGE, and the resulting blot was probed for Ras protein, revealing an increase in Ras activity in response to E2 (A). For the evaluation of Rap1 activity, a similar method was employed, using a glutathione-S-transferase fusion protein conjugated to the Rap-binding domain of Ral (RalGDS-RBD). After SDS-PAGE, the blot was probed for Rap1 protein, revealing that E2 treatment was without effect on Rap1 activity (B).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study we demonstrate for the first time in the brain that E2 elicits the phosphorylation of c-Src on three functionally critical tyrosine residues (Y220, Y423, and Y534). Phosphorylation of Y423 within the catalytic domain of c-Src usually requires dephosphorylation of Y534 (6). Interestingly, in the neocortical explants, E2 elicited the phosphorylation of Y423 without an accompanying decrease in Y534 phosphorylation. This mechanism is consistent with that proposed for PDGF activation of chick c-Src, where dephosphorylation on Y527 (analogous to mouse Y534) also does not occur. Instead, platelet-derived growth factor elicited phosphorylation of Y213 (analogous to mouse Y220), which resides in the SH2 domain of Src, and activates c-Src by preventing inhibitory interaction with the C-terminal phosphotyrosine (Y527) (11). Other members of the Src family, including Lck and Fyn, have been shown to exhibit increased activities despite hyperphosphorylation of their C-terminal negative regulatory sites (12, 13). Thus, the ability of E2 to elicit phosphorylation of both Y220 and Y423 without a decrease in Y534 phosphorylation, may reflect a mechanism of c-Src activation similar to that observed after PDGF treatment or that seen with Lck and Fyn.

We also found that PP2, a Src family kinase inhibitor, blocked basal and E2 induction of both c-Src and ERK phosphorylation. This observation is consistent with a report that the related Src inhibitor, PP1, prevents E2 induction of ERK phosphorylation in cultures of rat primary cortical neurons (14). As PP1 and PP2 both block other members of the Src family of kinases, these data do not establish per se that ERK phosphorylation is mediated via c-Src. In fact, we also found that Fyn, another member of the Src family of tyrosine kinases, is also phosphorylated upon E2 treatment (data not shown). This observation has prompted the study of additional Src family members expressed in the brain, such as Lck, Lyn, Yes, and Bsk as potential mediators of E2’s differentiative actions. Nevertheless, the fact that phosphorylation of c-Src is elicited by E2 strongly suggests that E2-induced ERK activation is mediated at least in part via c-Src-dependent mechanisms. The inhibition of basal phosphorylation by PP2 may simply be a result of inhibiting concurrent signaling elicited by endogenously released growth factors.

Of particular note was the finding that the ability of E2 to elicit c-Src phosphorylation was unaltered in ERKO mice, arguing against the involvement of ER{alpha} in this response. Previous work from this laboratory suggested that a novel receptor mechanism, pharmacologically distinct from either ER{alpha} or ERß, mediates E2 activation of the MAPK cascade in the developing brain (9, 15). This receptor system may also be responsible for E2 activation of c-Src. Interestingly, the AR has also been implicated in E2 signaling in models where E2 exerts a proliferative effect (16, 17). However, this mechanism is much less likely in the differentiating, nonproliferative neocortex, where most androgen actions are mediated by prior aromatization to E2.

Finally, we reported that the effect of E2 on c-Src correlated with the phosphorylation of Shc, resulting in the association of Shc with Grb2, and consequently, the activation of Ras. These effects of E2 on Shc, Grb2, and Ras are consistent with the hypothesis that E2 activation of Shc may be involved in neuronal differentiation, as overexpression of Shc has been shown to elicit neuronal differentiation in a Ras-dependent manner (18). In contrast, Rap1, which has been implicated in prolonged activation of the MAPK pathway (19), was not activated. Although cAMP-dependent protein kinase (PKA) is involved in activation of Rap1 (20), our preliminary data suggest that PKA is not required for E2-induced ERK activation (Sétáló, Jr., G., and C. D. Toran-Allerand, unpublished observations), consistent with the lack of effect of E2 on Rap1 activity.

These data extend previous findings from this laboratory (1, 9) and suggest that E2 may activate the MAPK cascade in the developing brain via a mechanism involving non-ER{alpha}-dependent c-Src phosphorylation and subsequent activation of Shc and Ras, leading to increased ERK phosphorylation. E2-induced activation of this pathway may underlie some of the rapid, nongenomic effects of E2 in the brain. In addition, as activated ERK translocates into the nucleus to modulate transcription, this mechanism may explain E2-induced regulation of non-E2 response element (ERE)-containing genes as well. Thus, E2-induced activation of the MAPK cascade may serve as a mechanism underlying its broad range of effects, from differentiative actions during development to neuroprotective effects in the adult.


   Acknowledgments
 
We thank Drs. J. S. Bos (University Medical Center Utrecht, Utrecht, The Netherlands) for his generous gift of the RalGDS RBD construct, and Alexei Morazov (Columbia University, New York, NY) for his expert technical advice regarding the Rap1 assay. We are also indebted to Dr. Neil J. MacLusky (Columbia University) for providing valuable discussions and constructive criticisms.


   Footnotes
 
This work was supported in part by grants from NIH (NIA), NIMH, NSF, the Alzheimer’s Association/T. L. L. Temple Foundation Discovery Award, and an ADAMHA Research Scientist Award (to C.D.T.-A.).

Abbreviations: E2, Estradiol; ERKO, ER{alpha} gene-disrupted mice; P1, P2, postnatal day 1 and 2; RBD, Ras-binding domain.

Received April 30, 2001.

Accepted for publication August 16, 2001.


   References
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 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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