Follicle-Stimulating Hormone (FSH)-dependent Regulation of Extracellular Regulated Kinase (ERK) Phosphorylation by the Mitogen-activated Protein (MAP) Kinase Phosphatase MKP3 *

Within the ovarian follicle, granulosa cells (GCs) surround and support immature oocytes. FSH promotes the differentiation and proliferation of GCs and is essential for fertility. We recently reported that ERK activation is necessary for FSH to induce key genes that define the preovulatory GC. This research focused on the phosphoregulation by FSH of ERK within GCs. FSH-stimulated ERK phosphorylation on Thr202/Tyr204 was PKA-dependent, but MEK(Ser217/Ser221) phosphorylation was not regulated; rather, MEK was already active. However, treatment of GCs with the EGF receptor inhibitor AG1478, a dominant-negative RAS, an Src homology 2 domain-containing Tyr phosphatase inhibitor (NSC 87877), or the MEK inhibitor PD98059 blocked FSH-dependent ERK(Thr202/Tyr204) phosphorylation, demonstrating the requirement for upstream pathway components. We hypothesized that FSH via PKA enhances ERK phosphorylation by inhibiting the activity of a protein phosphatase that constitutively dephosphorylates ERK in the absence of FSH, allowing MEK-phosphorylated ERK to accumulate in the presence of FSH because of inactivation of the phosphatase. GCs treated with different phosphatase inhibitors permitted elimination of both Ser/Thr and Tyr phosphatases and implicated dual specificity phosphatases (DUSPs) in the dephosphorylation of ERK. Treatment with MAP kinase phosphatase (MKP3, DUSP6) inhibitors increased ERK(Thr202/Tyr204) phosphorylation in the absence of FSH to levels comparable with ERK phosphorylated in the presence of FSH. ERK co-immunoprecipitated with Myc-FLAG-tagged MKP3(DUSP6). GCs treated with MKP3(DUSP6) inhibitors blocked and PKA inhibitors enhanced dephosphorylation of recombinant ERK2-GST in an in vitro phosphatase assay. Together, these results suggest that FSH-stimulated ERK activation in GCs requires the PKA-dependent inactivation of MKP3(DUSP6).

FSH acts selectively on granulosa cells (GCs) 2 contained within ovarian follicles to promote the proliferation and differ-entiation of GCs to a preovulatory phenotype (reviewed in Ref. 1) as well as meiotic competency of the enclosed oocyte (2,3). Luteinizing hormone then initiates meiosis and promotes ovulation and differentiation of remaining follicular cells into luteal cells. Although it is well recognized that the ERK signaling pathway plays a predominant role in the ovulatory response to luteinizing hormone in preovulatory GCs (4), the functional significance of the ERK signaling pathway in immature GCs is less understood. We recently showed that the ERK signaling pathway in immature GCs is required for the induction of at least a subset of FSH gene targets that define the mature preovulatory GC, including Inha (which encodes the ␣ subunit of the hormone inhibin), Lhcgr (which encodes the luteinizing hormone receptor), Egfr (which encodes the EGF receptor (EGFR) required for ovulation), and Cyp19a1 (which encodes the rate-limiting enzyme in estrogen biosynthesis) (5). ERK-dependent gene expression in immature GCs is mediated in part by phosphorylation of the transcriptional activator Y-box binding protein 1 (YB-1) on Ser 102 (5). Based on the relevance of the ERK signaling pathway to immature GC maturation, we sought to better understand the mechanism by which FSH activates ERK.
ERK is canonically activated by receptor tyrosine kinases (RTKs). Ligand-dependent activation of RTKs recruits the RAS guanine exchange factor SOS to the plasma membrane, resulting in RAS and, subsequently, RAF activation (reviewed in Ref. 6). The Ser/Thr kinase RAF then phosphorylates/activates the dual specificity kinase MEK that phosphorylates ERK. ERK then phosphorylates downstream kinases like ribosomal S6 kinase (RSK-2) (7). Phosphorylation of ERK by MEK on both Thr 202 and Tyr 204 is required for ERK activation; hence, dephosphorylation of either site by protein-tyrosine phosphatases (PTPs), Ser/Thr phosphatases, or dual specificity phosphatases (DUSPs) inactivates ERK (reviewed in Ref. 8).
In GCs, FSH activates ERK in a PKA-dependent manner (9 -12). We reported previously that the canonical pathway upstream of ERK is constitutively active in immature GCs but that ERK phosphorylation/activity is restrained by a PTP (10). We hypothesized that FSH inactivated this phosphatase, thereby permitting the accumulation of MEK-phosphorylated/ active ERK. We identified the PTP as a 100-kDa putative member of the PTP-straital-enriched protein tyrosine phosphatase (STEP)-like (SL) family (13,14) based on the following criteria: Western blotting of GC extracts using rabbit polyclonal anti-PTP-SL antibody (15) revealed a signal at 100 kDa; an in-gel PTP assay detected a signal at 100 kDa in concentrated ovarian extracts; an anti-PTP-SL-reactive signal at 100 kDa was selectively immunoprecipitated with ERK-conjugated agarose from GC extracts; in GCs loaded with 32 Pi, FSH stimulated the phosphorylation of a protein at 100 kDa immunoprecipitated with anti-PTP-SL antibody that was inhibited by the PKA, PKC, PKG (ACG) kinase family inhibitor H89 (16); and in an ERKagarose immunoprecipitation, the anti-PTL-SL-reactive band at 100 kDa was reduced by ϳ50% in GCs treated with FSH. Previous results by Pulido and co-workers (17,18) showed that PTP-SL was phosphorylated on Ser 231 in a PKA-dependent manner, relieving inhibition of ERK and its restriction to the cytoplasm and allowing ERK to translocate to the nucleus.
However, our recent results question our interpretation of our previous results. As we show below, the pan-PTP inhibitor Na 2 VO 3 (19,20), which should inhibit PTP-SL (15), does not block FSH-stimulated ERK phosphorylation. PTP-SL has a molecular weight of ϳ55 kDa, not 100 kDa (21). A second antibody directed against PTPBR7, another phosphatase sharing protein sequence homology with PTP-SL except for a 127amino acid insertion in the N terminus (14), does not detect a signal on Western blotting analyses of GC extracts at 100 kDa (10). Although the Ptprr gene that encodes PTP-SL is expressed in GCs (based on RNA sequencing results (22)), we thus question whether the PTP activity at 100 kDa that is detected by an anti-PTL-SL antibody is the phosphatase that constitutively dephosphorylates MEK-phosphorylated ERK in the absence of FSH.
The results below confirm that the ERK signaling pathway upstream of MEK is constitutively active in GCs, that ERK is constitutively phosphorylated by MEK but actively dephosphorylated in the absence of FSH, that MEK-phosphorylated ERK accumulates in the presence of FSH because of the inactivation of a phosphatase, and that FSH-stimulated ERK but not MEK phosphorylation is PKA-dependent. FSH-stimulated ERK activation is not inhibited either by inhibitors of the Ser/Thr protein phosphatase PP1 or PP2 or by the pan-PTP inhibitor Na 2 VO 3 . Rather, using a panel of DUSP inhibitors, our results show that inhibitors of MKP3(DUSP6) selectively enhance the phosphorylation of ERK in the absence of FSH to levels equivalent to those of ERK phosphorylated in the presence of FSH. ERK co-immunoprecipitates with Myc-FLAG-tagged MKP3(DUSP6), MKP3(DUSP6) inhibitors block ERK phosphatase activity in GC extracts in the absence of FSH in an in vitro phosphatase assay, and the selective PKA inhibitor PKI blocks the inactivation of the ERK phosphatase in the presence of FSH in an in vitro phosphatase assay. Together, these results suggest that ERK in GCs is maintained in a dephosphorylated state in the absence of FSH by MKP3(DUSP6) and that PKA inhibits the activity of this DUSP.

Results
Both Canonical Signaling to MEK and PKA Activity Are Required for FSH-stimulated Phosphorylation of ERK-(Thr 202 /Tyr 204 )-We initially sought to confirm that the canonical ERK signaling pathway in GCs upstream of MEK is tonically active and independent of FSH. Our previous results demonstrated an apparent requirement for the tyrosine kinase activity of the EGFR based on results showing that the EGFR inhibitor AG1478 (23) blocked FSH-stimulated ERK(Thr 202 / Tyr 204 ) phosphorylation. However, we did not evaluate the effect of AG1478 on MEK(Ser 217 /Ser 221 ) phosphorylation (10). To this end, GCs were pretreated with AG1478, followed by treatment without or with FSH. The results (Fig. 1A) show that MEK(Ser 217 /Ser 221 ) phosphorylation is readily detected in vehicle-treated GCs and independent of FSH, whereas ERK-(Thr 202 /Tyr 204 ) phosphorylation depends on FSH, and that AG1478 not only abolishes ERK(Thr 202 /Tyr 204 ) phosphorylation (95.1% Ϯ 0.8% inhibition, n ϭ 5) but also abolishes MEK-(Ser 217 /Ser 221 ) phosphorylation (89.0% Ϯ 3.8% inhibition, n ϭ 2). AKT(Ser 308 ) phosphorylation served as a negative control, as FSH-dependent activation of the PI3K/AKT pathway is independent of the EGFR in GCs. 3 These results indicate that the signaling pathway that promotes MEK phosphorylation is active in the absence of FSH and that this pathway requires the tyrosine kinase activity of the EGFR.
Although AG1478 primarily inhibits the kinase activity of the EGFR, it is also reported to inhibit non-kinase targets (24). As the EGFR canonically activates RAS, we utilized two additional approaches to confirm that the signaling pathway downstream of the EGFR was required for FSH to activate ERK and was tonically active in the absence of FSH. We transduced GCs with a dominant negative adenoviral (Ad)-(S17N)-RAS or control Ad-GFP and then treated GCs with vehicle or FSH. The results (Fig. 1B) show that the dominant negative RAS blocked MEK-(Ser 217 /Ser 221 ) phosphorylation (75.2% Ϯ 4.7% inhibition, n ϭ 3) both in the absence and presence of FSH as well as FSHstimulated ERK(Thr 202 /Tyr 204 ) phosphorylation (78.3% Ϯ 10.0% inhibition, n ϭ 4). cAMP-response element-binding protein(Ser 133 ) phosphorylation served as a negative control that is independent of EGFR/RAS signaling (25). The tyrosine phosphatase SRC homology 2 domain-containing tyrosine phosphatase (SHP2) is also required for RTK signaling into MEK/ ERK and is generally believed to contribute to the activation of RAS (26 -28). We determined whether SHP2 activity was required for the phosphorylation of MEK(Ser 217 /Ser 221 ) by pretreating GCs without or with NSC 87877, a compound that selectively inhibits SHP1 (which is not expressed in rat GCs based on RNA sequencing results (22)) and SHP2 (29). NSC 87877 markedly attenuated MEK(Ser 217 /Ser 221 ) phosphorylation (82.8% Ϯ 2.5% inhibition, n ϭ 2) in vehicle-and FSHtreated GCs and, hence, similarly reduced FSH-stimulated ERK(Thr 202 /Tyr 204 ) phosphorylation ( Fig. 1C) (92.9% Ϯ 5.5% inhibition, n ϭ 5). GAB2(Ser 159 ) phosphorylation served as a negative control that is independent of SHP2 signaling (30); FSH-stimulated GAB2(Tyr 452 ) dephosphory-lation shows the selectivity of the SHP2 PTP activity. Taken together, these results show that the signaling pathway that promotes MEK(Ser 217 /Ser 221 ) phosphorylation in GCs is tonically active in the absence of FSH and requires the EGFR, RAS, and SHP2.
FSH-stimulated ERK(Thr 202 /Tyr 204 ) phosphorylation is also recognized to be dependent on PKA (9,10,12). In the following experiment, we sought to confirm that, although the PKA inhibitor PKI, which functions as a PKA catalytic subunit pseudosubstrate (31), blocks FSH-stimulated ERK(Thr 202 /Tyr 204 ) phosphorylation, it does not affect MEK(Ser 217 /Ser 221 ) phosphorylation. As shown in Fig. 2  A, GCs were treated for 15 min without (DMSO) or with 250 nM AG1478, an EGFR kinase inhibitor, followed by treatment without (veh) or with 50 ng/ml FSH for 15 min. Samples were heat-denatured after collection in SDS sample buffer, and proteins were separated by SDS/PAGE. A blot of whole cell extracts was probed with the indicated antibodies. The results are representative of five independent experiments. B, GCs were transduced with Ad-GFP or Ad-(S17N)RAS overnight, followed by treatment without (veh) or with FSH 15 min. Samples were collected as described in A. The results are representative of four independent experiments. The dotted lines between lanes represent cropped images. C, GCs were treated without (DMSO) or with 20 mM NSC 87877, a SHP2 inhibitor, for 3 h, followed by treatment without (veh) or with FSH for 15 min. Samples were collected as described in A. The results are representative of five independent experiments.  (Fig. 3C). Concomitantly, MEK(Ser 217 /Ser 221 ) phosphorylation also increased (2.5-fold increase over veh, n ϭ 2) with Na 2 VO 3 treatment. Phosphorylation of insulin receptor substrate 1 (IRS1(Tyr 989 )), an RTK target (39), served as a positive control. The target(s) of the PTP is thus upstream of MEK, likely at the level of the EGFR, and not at the level of ERK itself. Taken together, these results suggest that the phosphatase that dephosphorylates MEK-phosphorylated ERK in the absence of FSH is neither a Ser/Thr phosphatase nor a PTP. ERK Phosphorylation Is Regulated by a DUSP-The inability of selective inhibitors of PP1, PP2, or PTPs to preferentially enhance ERK phosphorylation in the absence of FSH suggested that the relevant ERK phosphatase must be a member of the family of DUSPs. Indeed, in a recent report, DUSP27 blocked ERK but not MEK phosphorylation in a rat luteal cell line in response to prolactin treatment (40). However, DUSP27 is not expressed in rat GCs based on both microarray (41) and RNA sequencing (22) (22). Only a subgroup of the DUSPs are classified as MKPs based on the presence of a CDC25-like domain that confers specificity toward

. FSH-stimulated phosphorylation of ERK1/2(Thr 202 /Tyr 204 ) is not regulated by Ser/Thr and Tyr phosphatases.
A, GCs were pretreated without (EtOH) or with 0.2 M okadaic acid, a PP2 inhibitor, for 1 h, followed by treatment without (veh) and with FSH for 15 min. Samples were collected as described in Fig. 1A. The results are representative of three independent experiments. The results for pGSK3␤ and its S6 loading control were published previously (58). B, GCs were pretreated without (EtOH) or with 1 M tautomycin, a PP1 inhibitor, for 5.5 h, followed by treatment without (veh) and with FSH for 15 min. Samples were collected as described in Fig. 1A. The results are representative of six independent experiments. C, GCs were pretreated without (H 2 O) or with 50 M Na 2 VO 3 , a pan tyrosine phosphatase inhibitor, for 12 h, followed by treatment without (veh) and with FSH for 30 min. Samples were collected as described in GCs were pretreated without or with NSC 663284, a selective CDC25A, CDC25B, and CDC25C inhibitor that does inhibit MKP1(DUSP1) or MKP3(DUSP6) (43,44). The results (Fig. 4A) show that ERK(Thr 202 /Tyr 204 ) phosphorylation in the absence or presence of FSH is not affected (lanes 3 and 4 versus lanes 1 FIGURE 4. DUSP inhibitor studies suggest that MKP3(DUSP6) actively dephosphorylates MEK-phosphorylated ERK1/2(Thr 202 /Tyr 204 ) in the absence of FSH. A, GCs were pretreated without (DMSO) or with 5 M NSC 663284, a selective CDC25A, CDC25B, and CDC25C inhibitor (which does not inhibit MKP1(DUSP1) or MKP3(DUSP6)) for 30 min, followed by treatment without (veh) and with FSH for 30 min. Samples were collected as described in Fig. 1A. The results are representative of three independent experiments. B, GCs were pretreated without (DMSO) or with 5 M BCI, a selective MKP1(DUSP1) and MKP3(DUSP6) inhibitor, for 30 min, followed by treatment without (veh) and with FSH for 30 min. Samples were collected as described in Fig. 1A. The results are representative of three independent experiments. C, GCs were treated without (veh) and with FSH for the indicated times. Samples were collected as described in Fig. 1A. D, GCs were pretreated without (DMSO) or with 10 M triptolide, a selective MKP1(DUSP1) inhibitor, for 1 h, followed by treatment without (veh) and with FSH for 30 min. Samples were collected as described in Fig. 1A. The results are representative of three independent experiments, except for the phospho-p38 blot, which is representative of two independent experiments. E, GCs were pretreated without (DMSO) or with 5 M NSC 295642, a selective MKP3(DUSP6) and CDC25A and CDC25B inhibitor, for 30 min, followed by treatment without (veh) and with FSH for 30 min. Samples were collected as described in Fig. 1A. The results are representative of three independent experiments. The dotted lines between lanes represent cropped images. and 2) by this competitive CDC25 inhibitor. Although we were unable to identify a positive control for this inhibitor, as rat GCs do not divide in primary culture (45)(46)(47) and, hence, do not expressthecyclinsnecessaryforcyclin-dependentkinase2phosphorylation (48), this result suggests that CDC25s are not functioning as ERK phosphatases in GCs. Additional indirect evidence that the CDC25 DUSPs are not functioning as ERK phosphatases in GCs is a report that Na 2 VO 3 inhibits CDC25 DUSPs (49), whereas this phosphatase inhibitor does not inhibit the ERK phosphatase in GCs (Fig. 3C).
We next sought to verify the expression of both MKP1(DUSP1) and MKP3(DUSP6) proteins within GCs, especially because MKP1(DUSP1) is most commonly induced as an immediate early gene upon RTK activation of ERK (as reviewed in Ref. 42). GCs were treated with vehicle or FSH for the indicated times. The results show that both MKP1(DUSP1) and MKP3(DUSP6) are readily detected in GCs, and total protein levels do not change within 60 min of FSH treatment (Fig. 4C).
ERK and MKP3(DUSP6) Interact within GCs-If MKP3-(DUSP6) is dephosphorylating MEK-phosphorylated ERK in the absence of FSH, then MKP3(DUSP6) should bind to ERK in GCs. As the MKP3(DUSP6) antibody does not readily immunoprecipitate MKP3(DUSP6) (data not shown), GCs were transiently transfected with an MKP3-Myc-FLAG plasmid to determine whether MKP3(DUPS6) and ERK interact. Immunoprecipitation of ERK2 from vehicle-and FSH-treated GCs showed the presence of the FLAG-tagged MKP3(DUPS6) in ERK2-immunoprecipitated samples but not in IgG controls (Fig. 5A). Conversely, immunoprecipitation of Myc-tagged MKP3 with anti-Myc antibody from vehicle-and FSH-treated cells showed that the anti-Myc antibody selectively pulls down ERK (Fig. 5B). These results suggest that MKP3(DUSP6) and ERK1/2 interact within GCs independent of FSH treatment.
ERK(Thr 202 /Tyr 204 ) Dephosphorylation Is Dependent on MKP3(DUSP6) Activity-In the following experiments, we sought to demonstrate that MKP3(DUSP6), but not MKP1(DUSP1), is able to dephosphorylate ERK in vitro. Using the MKP1 and MKP3 inhibitors discussed above, whole GC lysate phosphatase assays were performed using GST-tagged phospho-ERK2. GCs were pretreated without or with the indicated inhibitors, followed by treatment with vehicle or FSH. Lysates were then assayed for phosphatase activity, evidenced by their ability to dephosphorylate GST-tagged phospho-ERK2, as detected by Western blotting with phospho-ERK-(Thr 202 /Tyr 204 ) antibody. Pretreatment of GCs with BCI, the dual MKP1(DUSP1) and MKP3(DUSP6) inhibitor, blocked the DUSP that dephosphorylated ERK(Thr 202 /Tyr 204 ) in the absence of FSH (Fig. 6A, compare lanes 3 and 1). Equivalent results were obtained upon pretreatment of GCs with NSC 295642, the MKP3(DUSP6) inhibitor (Fig. 6B). Taken together, these results support the conclusion that MKP3(DUSP6) is the DUSP that dephosphorylates ERK in the absence of FSH.
Inactivation of the ERK Phosphatase by FSH Is Dependent upon PKA Activity-We sought to determine whether the ability of MKP3(DUSP6) to dephosphorylate ERK(Thr 202 /Tyr 204 ) in vitro was dependent on PKA using the selective PKA inhibitor PKI (31). GCs were transduced with Ad-E or Ad-PKI overnight, treated with vehicle or FSH, and whole GC lysate phosphatase assays were performed using GST-tagged phospho-ERK2 as described above. Transduction of GCs with Ad-PKI promoted the dephosphorylation of ERK2(Thr 202 /Tyr 204 )-GST in both vehicle-and FSH-treated cells (Fig. 7, compare  lanes 3 and 4 with lanes 1 and 2). These data indicate that FSH-dependent PKA activation is necessary for inactivation of the ERK DUSP.

Discussion
Our results confirm that the canonical signaling pathway upstream of ERK, which includes the EGFR, RAS, SHP2, and MEK, is constitutively active in GCs and that the activity of these upstream components is necessary but not sufficient for FSH-dependent ERK(Thr 202 /Tyr 204 ) phosphorylation, as modeled in Fig. 8. FSH-dependent ERK(Thr 202 /Tyr 204 ) phosphorylation also requires the inhibition of a DUSP, most likely MKP3(DUPS6), based on the ability of MKP3(DUPS6) inhibitors to raise ERK(Thr 202 /Tyr 204 ) phosphorylation in the absence of FSH to levels comparable with those of FSH-treated cells and on the loss of an FSH response in the presence of the inhibitors. Additionally, ERK selectively co-immunoprecipitates with tagged-MKP3(DUPS6), and tagged-MKP3(DUSP6) selectively co-immunoprecipitates with ERK. The in vitro phosphatase assay results show that MKP3(DUPS6) inhibitors block dephosphorylation of ERK2-GST in the absence of FSH and that the selective PKA inhibitor PKI abrogates the ability of FSH to inhibit the ERK DUSP. Together, these results strongly suggest that ERK is dephosphorylated in the absence of FSH by MKP3(DUPS6) and that FSH via PKA endorses the MEK-dependent phosphorylation of ERK by inhibiting this DUSP.
Recent studies suggest that the majority of the responses in GCs elicited by FSH are mediated by PKA, based not only on the ability of PKA inhibitors to block FSH-activated signaling pathways and gene expression (reviewed in Ref. 1) but also on the ability of a constitutively active PKA catalytic subunit mutant to mimic both acute FSH signaling and delayed gene expression (12,41). Acute signaling responses include not only direct phosphorylation of target proteins by PKA, including cAMP-response element-binding protein (55), histone H3 (56), and ␤-catenin (57), but also activation by PKA of classical RTKactivated pathways such as the PI3K/AKT and MEK/ERK pathways (modeled in Fig. 8) (5, 10, 30). However, until recently, the mechanisms by which PKA accomplished the regulation of these canonical signaling pathways remained elusive. Our results indicate that, through regulation of MKP3(DUPS6), FSH is able promote the accumulation of MEK-phosphorylated ERK(Thr 202 /Tyr 204 ), whereas the upstream components of the MEK/ERK signaling cascade remain active and unregulated by FSH treatment. Interestingly, our laboratory recently published  . Active ERK2-GST dephosphorylation is dependent on PKA activity. GCs were transduced with Ad-Empty (E) or Ad-PKI overnight. Following the medium replacement outlined under "Experimental Procedures," cells were treated without (veh) and with FSH for 15 min. Samples were collected as described in Fig. 6A. The results are representative of two independent experiments. The dotted lines between lanes represent cropped images.
results demonstrating that FSH activates the PI3K/AKT signaling pathway by promoting the PKA-dependent activation of PP1, which sensitizes IRS1 to the RTK activity of the insulinlike growth factor 1 receptor to activate PI3K in GCs (58). We also recently reported that FSH, in a PKA-, ERK-, and RSK-2dependent manner, promotes phosphorylation of the transcriptional factor YB-1 on Ser 102 , which appears to be mediated primarily by the ability of RSK-2 to inhibit the phosphatase activity of PP1 (5). Collectively, these results indicate that FSH, via PKA, regulates at least two established RTK signaling cascades through regulation of phosphatases, seemingly hijacking these cascades. FSH, via PKA, also seems to enhance the transcriptional events by promoting the phosphorylation of at least one transcriptional factor via the inhibition of a phosphatase (5). Together, these results indicate that, at least in GCs, PKA either directly phosphorylates target proteins or enhances the phosphorylation of key proteins in RTK pathways by regulating the activity of protein phosphatases. MKP3(DUPS6) is a dual specificity phosphatase and member of the MKP subfamily whose substrates are the MAPKs (ERK, p38 MAPK, and JNK) (reviewed in Ref. 59). MKP3(DUPS6) has been shown to have a substrate preference for ERK over p38 MAPK and JNK (60 -62). Regulation of MKP3(DUPS6) expression is most often placed downstream of ERK signaling (63,64) and is thought to constitute a negative feedback loop to downregulate mitogenic signaling. Consistent with this conclusion, Mkp3/Dusp6 is often induced following ERK activation (65) and requires regulation by the transcription factor Ets1 (64,65). Upon expression, MKP3 is catalytically activated via its physical association with ERK (62,66,67). We showed that MKP3-(DUSP6) protein is readily expressed in GCs and that levels remain relatively constant following FSH treatment, giving no indication of a large change in total protein expression (Fig. 4C).
These results indicate that, in GCs at the time points checked, initial regulation of MKP3(DUSP6) is not at the transcriptional level, as protein levels remain stable up to 1 h following treatment with FSH.
It is our hypothesis that MKP3(DUSP6) becomes phosphorylated in GCs in a FSH-and PKA-dependent manner, resulting in its inactivation. Although it is possible that MKP3(DUSP6) becomes phosphorylated on Ser 159 and Ser 197 in response to the FSH/PKA-dependent activation of ERK in GCs, possibly sustaining the inactivation of MKP3(DUSP6), the initial inactivation of MKP3(DUPS6) must be mediated by another PKAdependent event. As casein kinase 2␣ is a constitutively active kinase (71), it does not appear to be a PKA-regulated candidate to inhibit MKP3(DUPS6) in GCs. Although FSH activates PI3K in a PKA-dependent manner (30,58), phosphorylation of YB-1(Ser 102 ) downstream of ERK and RSK-2 is not inhibited by the PI3K inhibitor wortmannin (5). These results thus indicate that PKA activation of the PI3K pathway in GCs is not the mechanism by which PKA inactivates MKP3(DUPS6). Moreover, if MKP3(DUSP6) is degraded in response to FSH, reduced protein levels of MKP3(DUSP6) are not detected within the first hour following FSH treatment of GCs (Fig. 4C).
There are no reports, to our knowledge, that MKP3(DUPS6) is directly phosphorylated by PKA or that PKA can regulate its activity. Rat MKP3(DUSP6) contains one potential, albeit weak, PKA phosphorylation motif (RR/XX/S/T): RSVThr 302 V. Consistent with our results, we thus hypothesize that MKP3(DUSP6) is phosphorylated by PKA on Thr 302 , resulting in an inhibition of its phosphatase activity. However, our inability to directly immunoprecipitate MKP3(DUSP6) and a lack of phosphospecific antibodies to MKP3(Thr 302 ) have prevented us from directly testing this hypothesis.
Although our results support MKP3(DUSP6) as the relevant DUSP that regulates the activity of ERK in response to FSH in GCs, the MKP3(DUSP6) global knockout mouse is fertile (72). We predict that another ERK-selective DUSP, such as MKPX/ PYST2(DUSP7) (73), is able to compensate for the absence of MKP3(DUSP6) in GCs. The MKPX/PYST2(DUSP7) (The Jackson Laboratory, Bar Harbor, ME) and MKP1(DUSP1) (74) global knockout mice are also fertile. Formal proof that MKP3(DUSP6) is the DUSP that dephosphorylates constitutively phosphorylated ERK in GCs requires a genetic approach in which at least MKP3(DUSP6) and MKPX/PYST2(DUSP7) are deleted.
In summary, we have shown that FSH-dependent ERK-(Thr 202 /Tyr 204 ) phosphorylation is PKA-dependent as well as dependent on the constitutively active canonical upstream MEK/ERK signaling components that include the EGFR, RAS, The results presented here support the contributions of constitutively active pathway components upstream of ERK, the ability of MKP3(DUSP6) to dephosphorylate MEK-phosphorylated ERK in the absence of FSH, and the ability of FSH, in a PKA-dependent manner, to inhibit the phosphatase activity of the likely ERK DUSP, MKP3. The ability of ERK to promote phosphorylation/activation of the transcriptional factor YB1 is based on a prior publication (5). The ability of PKA to sensitize IRS1 to the tyrosine kinase activity of the insulin-like growth factor 1 receptor (IGF 1 R) is also based on prior publications (30,58).
SHP2, and MEK. We conclude that, in the absence of FSH, constitutively phosphorylated ERK is maintained in a dephosphorylated state by a DUSP, most likely by MKP3(DUPS6). FSH in a PKA-dependent manner inactivates the DUSP, allowing for the accumulation of MEK-phosphorylated ERK.

Experimental Procedures
Materials-The following were purchased: ovine FSH Animals-Sprague-Dawley, CD-outbred rats (breeders from Charles River Laboratories) were from a breeder colony maintained by our laboratory in a pathogen-free facility at Washington State University. The facility is maintained in accordance with the Guidelines for the Care and Use of Laboratory Animals using protocols approved by the Washington State University Institutional Animal Care and Use Committee.
GC Cultures-Immature female rats were primed with subcutaneous injections of 1.5 mg of estradiol-17␤ (E2) in propylene glycol on postnatal days 21-23. Ovaries were collected following 3 days of injections. GCs were collected by puncturing individual follicles using 27-gauge needles (75). Cells were plated on fibronectin-coated plates at a density of ϳ1 ϫ 10 6 cells/ml of serum-free medium supplemented with 1 nM E2, 100 units/ml penicillin (P), and 100 g/ml streptomycin (S). The indicated treatments were added to cells ϳ20 h following plating and terminated by aspirating the medium and washing once with PBS, followed by sample collection.
Western Blotting-Total cell extracts were collected by scraping cells in a SDS sample buffer (76) at 50 l/1 ϫ 10 6 cells, followed by heat denaturation. Equal protein loading was accomplished by plating equal numbers of cells and collecting in a standardized SDS collection volume. Equal volumes of protein extract were loaded per gel lane, and equal loading was verified by probing for total SHP2 or AKT, as indicated. Pro-teins were separated by SDS/PAGE and transferred onto either a Hybond C-extra or Protran (Amersham Biosciences) nitrocellulose membrane (56). Western blotting analyses were scanned using an Epson Perfection V500 scanner and Adobe Photoshop CS2 9.0 software with minimal processing and quantified using Quantity One software (Bio-Rad). Experimental densitometric values were divided by load control protein values and expressed relative to vehicle values.
Adenoviral Transductions of GCs-Transduction with adenoviruses was done as described previously (47). Briefly, GCs were plated in 35-mm plates at 1.5 ϫ 10 6 cells/2 ml in DMEM/F12 ϩ E/PS. Four hours after plating, the indicated adenoviruses were added to the cells. The next morning, the adenovirus was removed, the cells were washed with PBS, and fresh DMEM/F12 ϩ E/PS was added. The cells were treated as indicated. Adenoviral optical particle unit concentration per milliliter of viral stock was calculated from the A 260 as described previously (30). Results are expressed as optical particle unit per cell and based on the number of GCs plated and the volume of virus added. Ad-PKI was kindly provided by Marco Conti (University of California, San Francisco, CA) (77). Ad-(S17N)-RAS was kindly provided by Valina Dawson (Johns Hopkins University School of Medicine, Baltimore, MD) (78).
Transfection of GCs-Full-length MKP3-Myc-FLAG was purchased from OriGene (Rockville, MD). Cells were plated on fibronectin-coated plates at a density of 1 ϫ 10 6 cells/ml in OptiMEM ϩ P/S ϩ E2 with expression constructs (500 ng/1 ϫ 10 6 cells) and transfected using Lipofectamine 2000 (Invitrogen) according to the instructions of the manufacturer. After 6 h of incubation, the medium was removed, and fresh DMEM ϩ P/S ϩ E2 was added. Cells were treated as indicated following an ϳ16-h recovery period.
Whole Cell Lysate Phosphatase Assay-The protocol was adapted from a previous report (79) as detailed below. Cells were treated as indicated and then collected in 0.2 ml of phosphatase assay buffer (10 mM EDTA, 10 mM EGTA, 50 mM HEPES (pH 7.6), 1 mM phenylmethylsulfonyl fluoride, 0.1 mg/ml leupeptin, and 1 mg/ml aprotinin). Lysates were subjected to five rounds of freezing/thawing, followed aspiration through a 25-guage needle or sonication. Lysates were clarified by centrifugation at 13,000 ϫ g for 30 min at 4°C. To assess phosphatase activity, 50 ng of active ERK2-GST (EMD Millipore, 14-173) was added to each sample, and samples were incubated by rotating for 30 min at 30°C. The reaction was halted by adding 100 l of 3ϫ SDS sample buffer (76), and samples were subjected to SDS/PAGE and Western blotting.
Statistics-Results were analyzed using GraphPad Prism, and significance was determined using either a one-way analysis of variance with Tukey's multiple comparison test or one-tailed Student's t test.