Phosphorylation of PP2Ac by PKC is a key regulatory step in the PP2A-switch-dependent AKT dephosphorylation that leads to apoptosis.

BACKGROUND: Although GqPCR activation often leads to cell survival by activating the PI3K/AKT pathway, it was previously shown that in several cell types AKT activity is reduced and leads to JNK activation and apoptosis. The mechanism of AKT inactivation in these cells involves an IGBP1-coupled PP2Ac switch that induces the dephosphorylation and inactivation of both PI3K and AKT. However, the machinery involved in the initiation of PP2A switch is not known. METHODS: We used phospho-mass spectrometry to identify the phosphorylation site of PP2Ac, and raised specific antibodies to follow the regulation of this phosphorylation. Other phosphorylations were monitored by commercial antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein-protein interactions. Apoptosis was detected by a TUNEL assay as well as PARP1 cleavage using SDS-PAGE and Western blotting. RESULTS: We identified Ser24 as a phosphorylation site in PP2Ac. The phosphorylation is mediated mainly by classical PKCs (PKCα and PKCß) but not by novel PKCs (PKCδ and PKCε). By replacing the phosphorylated residue with either unphosphorylatable or phosphomimetic residues (S24A and S24E), we found that this phosphorylation event is necessary and sufficient to mediate the PP2A switch, which ultimately induces AKT inactivation, and a robust JNK-dependent apoptosis. CONCLUSION: Our results show that the PP2A switch is induced by PKC-mediated phosphorylation of Ser24-PP2Ac and that this phosphorylation leads to apoptosis upon GqPCR induction of various cells. We propose that this mechanism may provide an unexpected way to treat some cancer types or problems in the endocrine machinery.

GqPCR-stimulated dephosphorylation of AKT is induced by an IGBP1-mediated PP2A switch.

BACKGROUND: G protein-coupled receptors (GPCRs) usually regulate cellular processes via activation of intracellular signaling pathways. However, we have previously shown that in several cell lines, GqPCRs induce immediate inactivation of the AKT pathway, which leads to JNK-dependent apoptosis. This apoptosis-inducing AKT inactivation is essential for physiological functions of several GqPCRs, including those for PGF2α and GnRH. METHODS: Here we used kinase activity assays of PI3K and followed phosphorylation state of proteins using specific antibodies. In addition, we used coimmunoprecipitation and proximity ligation assays to follow protein-protein interactions. Apoptosis was detected by TUNEL assay and PARP1 cleavage. RESULTS: We identified the mechanism that allows the unique stimulated inactivation of AKT and show that the main regulator of this process is the phosphatase PP2A, operating with the non-canonical regulatory subunit IGBP1. In resting cells, an IGBP1-PP2Ac dimer binds to PI3K, dephosphorylates the inhibitory pSer608-p85 of PI3K and thus maintains its high basal activity. Upon GqPCR activation, the PP2Ac-IGBP1 dimer detaches from PI3K and thus allows the inhibitory dephosphorylation. At this stage, the free PP2Ac together with IGBP1 and PP2Aa binds to AKT, causing its dephosphorylation and inactivation. CONCLUSION: Our results show a stimulated shift of PP2Ac from PI3K to AKT termed "PP2A switch" that represses the PI3K/AKT pathway, providing a unique mechanism of GPCR-stimulated dephosphorylation. Video Abstract.

C-Src is Activated by the EGF Receptor in a Pathway that Mediates JNK and ERK Activation by Gonadotropin-Releasing Hormone in COS7 Cells.

The key participants in G-protein-coupled receptor (GPCR) signaling are the mitogen-activated protein kinase (MAPK) signaling cascades. The mechanisms involved in the activation of the above cascades by GPCRs are not fully elucidated. The prototypical GPCR is the receptor for gonadotropin-releasing hormone (GnRHR), which serves as a key regulator of the reproductive system. Here, we expressed GnRHR in COS7 cells and found that GnRHR transmits its signals to MAPKs mainly via Gαi and the EGF receptor, without the involvement of Hb-EGF or PKCs. The main pathway that leads to JNK activation downstream of the EGF receptor involves a sequential activation of c-Src and PI3K. ERK activation by GnRHR is mediated by the EGF receptor, which activates Ras either directly or via c-Src. Beside the main pathway, the dissociated Gßγ and ß-arrestin may initiate additional (albeit minor) pathways that lead to MAPK activation in the transfected COS7 cells. The pathways detected are significantly different from those in other GnRHR-bearing cells, indicating that GnRH can utilize various signaling mechanisms for MAPK activation. The unique pathway elucidated here, in which c-Src and PI3K are sequentially activated downstream of the EGF receptor, may serve as a prototype of signaling mechanisms by GnRHR and additional GPCRs in various cell types.

Gq-Induced Apoptosis is Mediated by AKT Inhibition That Leads to PKC-Induced JNK Activation.

BACKGROUND/AIMS: Gq protein-coupled receptors (GqPCRs) regulate various cellular processes including mainly proliferation and differentiation. In a previous study, we found that in prostate cancer cells, the GqPCR of GnRH induces apoptosis by reducing the PKC-dependent AKT activity and elevating JNK phosphorylation. Since it was thought that GqPCR induces mainly activation of AKT, we undertook to examine how general is this phenomenon and understand its signaling. METHODS: We used various cells to follow the phosphorylation of signaling components using western blotting. RESULTS: In a screen of 21 cell lines, we found that PKC activation results in the reduction of AKT activity, which correlates nicely to JNK activation and in some cases to apoptosis. To further understand the signaling pathways involved in this stimulation, we studied in detail the SVOG-4O and αT3-1 cells. We found that PGF2α and GnRH agonist (GnRH-a) indeed induce significant Gq- and PKC- dependent apoptosis in these cells. This is mediated by two signaling branches downstream of PKC, which converge at the level of MLK3 upstream of JNK. One branch consists on c-Src activation of the JNK cascade and the second involves reduction of AKT activity that alleviates its inhibitory effect on MLK3, to allow the flow of the c-Src signal to JNK. At the MAPKK level, we found that the signal is transmitted by MKK7 and not MKK4. CONCLUSION: Our results present a general mechanism that mediates a GqPCR-induced, death receptors-independent, apoptosis in physiological, as well as cancer-related systems.

Differential roles of PKC isoforms (PKCs) in GnRH stimulation of MAPK phosphorylation in gonadotrope derived cells.

The role of protein kinase C (PKC) isoforms (PKCs) in GnRH-stimulated MAPK [ERK1/2, JNK1/2 and p38) phosphorylation was examined in gonadotrope derived cells. GnRH induced a protracted activation of ERK1/2 and a slower and more transient activation of JNK1/2 and p38MAPK. Gonadotropes express conventional PKCα and PKCßII, novel PKCδ, PKCε and PKCθ, and atypical PKC-ι/λ. The use of green fluorescent protein (GFP)-PKCs constructs revealed that GnRH induced rapid translocation of PKCα and PKCßII to the plasma membrane, followed by their redistribution to the cytosol. PKCδ and PKCε localized to the cytoplasm and Golgi, followed by the rapid redistribution by GnRH of PKCδ to the perinuclear zone and of PKCε to the plasma membrane. The use of dominant negatives for PKCs and peptide inhibitors for the receptors for activated C kinase (RACKs) has revealed differential role for PKCα, PKCßII, PKCδ and PKCε in ERK1/2, JNK1/2 and p38MAPK phosphorylation in a ligand-and cell context-dependent manner. The paradoxical findings that PKCs activated by GnRH and PMA play a differential role in MAPKs phosphorylation may be explained by persistent vs. transient redistribution of selected PKCs or redistribution of a given PKC to the perinuclear zone vs. the plasma membrane. Thus, we have identified the PKCs involved in GnRH stimulated MAPKs phosphorylation in gonadotrope derived cells. Once activated, the MAPKs will mediate the transcription of the gonadotropin subunits and GnRH receptor genes.

GnRH Induces ERK-Dependent Bleb Formation in Gonadotrope Cells, Involving Recruitment of Members of a GnRH Receptor-Associated Signalosome to the Blebs.

We have previously described a signaling complex (signalosome) associated with the GnRH receptor (GnRHR). We now report that GnRH induces bleb formation in the gonadotrope-derived LßT2 cells. The blebs appear within ~2 min at a turnover rate of ~2-3 blebs/min and last for at least 90 min. Formation of the blebs requires active ERK1/2 and RhoA-ROCK but not active c-Src. Although the following ligands stimulate ERK1/2 in LßT2 cells: EGF > GnRH > PMA > cyclic adenosine monophosphate (cAMP), they produced little or no effect on bleb formation as compared to the robust effect of GnRH (GnRH > PMA > cAMP > EGF), indicating that ERK1/2 is required but not sufficient for bleb formation possibly due to compartmentalization. Members of the above mentioned signalosome are recruited to the blebs, some during bleb formation (GnRHR, c-Src, ERK1/2, focal adhesion kinase, paxillin, and tubulin), and some during bleb retraction (vinculin), while F-actin decorates the blebs during retraction. Fluorescence intensity measurements for the above proteins across the cells showed higher intensity in the blebs vs. intracellular area. Moreover, GnRH induces blebs in primary cultures of rat pituitary cells and isolated mouse gonadotropes in an ERK1/2-dependent manner. The novel signalosome-bleb pathway suggests that as with the signalosome, the blebs are apparently involved in cell migration. Hence, we have extended the potential candidates which are involved in the blebs life cycle in general and for the GnRHR in particular.

Differential roles of PKC isoforms (PKCs) and Ca2+ in GnRH and phorbol 12-myristate 13-acetate (PMA) stimulation of p38MAPK phosphorylation in immortalized gonadotrope cells.

We examined the role of PKCs and Ca2+ in GnRH-stimulated p38MAPK phosphorylation in the gonadotrope derived αT3-1 and LßT2 cell lines. GnRH induced a slow and rapid increase in p38MAPK phosphorylation in αT3-1 and LßT2 cells respectively, while PMA gave a slow response. The use of dominant negatives for PKCs and peptide inhibitors for the receptors for activated C kinase (RACKs), has revealed differential role for PKCα, PKCßII, PKCδ and PKCε in p38MAPK phosphorylation in a ligand-and cell context-dependent manner. The paradoxical findings that PKCs activated by GnRH and PMA play a differential role in p38MAPK phosphorylation may be explained by differential localization of the PKCs. Basal, GnRH- and PMA- stimulation of p38MAPK phosphorylation in αT3-1 cells is mediated by Ca2+ influx via voltage-gated Ca2+ channels and Ca2+ mobilization, while in the differentiated LßT2 gonadotrope cells it is mediated only by Ca2+ mobilization. p38MAPK resides in the cell membrane and is relocated to the nucleus by GnRH (∼5 min). Thus, we have identified the PKCs and the Ca2+ pools involved in GnRH stimulated p38MAPK phosphorylation.

A-Kinase Anchoring Protein 4 (AKAP4) is an ERK1/2 substrate and a switch molecule between cAMP/PKA and PKC/ERK1/2 in human spermatozoa.

Mammalian spermatozoa undergo capacitation and acrosome reaction in order to fertilize the egg. The PKC-ERK1/2 pathway plays an important role in human spermatozoa motility, capacitation and the acrosome reaction. Here we demonstrate that ERK1/2 phosphorylates proAKAP4 on Thr265 in human spermatozoa in vitro and in vivo. Cyclic AMP (cAMP) had no effect on ERK1/2 activity in human spermatozoa, but stimulated the MAPK in mouse pituitary LßT2 gonadotrope cells. cAMP via PKA attenuates PKC-dependent ERK1/2 activation only in the presence of proAKAP4. St-HT31, which disrupts PKA-regulatory subunit II (PKA-RII) binding to AKAP abrogates the inhibitory effect of cAMP in human spermatozoa and in HEK293T cells expressing proAKAP4. In transfected HEK293T cells, PMA relocated proAKAP4, but not proAKAP4-T265A to the Golgi in an ERK1/2-dependnet manner. Similarly, AKAP4 is localized to the spermatozoa principal piece and is relocated to the mid-piece and the postacrosomal region by PMA. Furthermore, using capacitated sperm we found that cAMP reduced PMA-induced ERK1/2 activation and acrosome reaction. Thus, the physiological role of the negative crosstalk between the cAMP/PKA/AKAP4 and the PKC/ERK1/2 pathways is to regulate capacitation and acrosome reaction.

Role of PI4K and PI3K-AKT in ERK1/2 activation by GnRH in the pituitary gonadotropes.

The role of PI4K and PI3K-AKT in ERK1/2 activation by GnRH was examined. A relatively long preincubation (60 min) with wortmannin (10 nM and 10 µM), and LY294002 (10 µM and 100 µM) (doses known to inhibit PI3K and PI4K, respectively), were required to inhibit GnRH-and PMA-stimulated ERK1/2 activity in αT3-1 and LßT2 gonadotrope cells. A similar preincubation protocol was required to demonstrate inhibition of IGF-1-stimulated AKT activation lending support for the need of prolonged incubation (60 min) with wortmannin in contrast to other cellular systems. To rule out that the inhibitors acted upon PI(4,5)P2 levels, we followed the [Ca(2+)]i response to GnRH and found that wortmannin has no significant effect on GnRH-induced [Ca(2+)]i responses. Surprisingly, GnRH and PMA reduced, while IGF-1 increased AKT phosphorylation. We suggest that PI3K inhibits GnRH-stimulated αGSU activity, has no effect upon GnRH-stimulated LHß activity and enhanced the GnRH-stimulated FSHß transcription. Hence, PI4K and PI3K-AKT play a role in GnRH to ERK1/2 signaling, while PI3K may regulate also GnRH-induced gonadotropin gene expression.

Differential signaling of the GnRH receptor in pituitary gonadotrope cell lines and prostate cancer cell lines.

The GnRH receptor (GnRHR) mediates the pituitary functions of GnRH, as well as its anti-proliferative effects in sex hormone-dependent cancer cells. Here we compare the signaling of GnRHR in pituitary gonadotrope cell lines vs. prostate cancer cell lines. We first noticed that the expression level of PKCα, PKCßII and PKCε is much higher in αT3-1 and LßT2 gonadotrope cell lines vs. LNCaP and DU-145 cell lines, while the opposite is seen for PKCδ. Activation of PKCα, PKCßII and PKCε by GnRH is relatively transient in αT3-1 and LßT2 gonadotrope cell lines and more prolonged in LNCaP and DU-145 cell lines. On the otherhand, the activation and re-distribution of the above PKCs by PMA was similar for both gonadotrope cell lines and prostate cancer cell lines. Activation of ERK1/2 by GnRH and PMA was robust in the gonadotrope cell lines, with a smaller effect observed in the prostate cancer cell lines. The Ca(2+) ionophore A23187 stimulated ERK1/2 in gonadotrope cell lines but not in prostate cancer cell lines. GnRH, PMA and A23187 stimulated JNK activity in gonadotrope cell lines, with a more sustained effect in prostate cancer cell lines. Sustained activation of p38 was observed for PMA and A23187 in Du-145 cells, while p38 activation by GnRH, PMA and A23187 in LßT2 cells was transient. Thus, differential expression and re-distribution of PKCs by GnRH and the transient vs. the more sustained nature of the activation of the PKC-MAPK cascade by GnRH in gonadotrope cell lines vs. prostate cancer cell lines respectively, may provide the mechanistic basis for the cell context-dependent differential biological responses observed in GnRH interaction with pituitary gonadotropes vs. prostate cancer cells.

Interactions of the GnRH receptor with heterotrimeric G proteins.

G protein-coupled receptors (GPCRs) are the largest class of integral membrane protein receptors in the human genome. We examined here the reports whether the GnRH receptor (GnRHR) interacts with a single or multiple types of G proteins. It seems that the GnRHR, as other GPCRs, alternates between various conformations and is stabilized by its ligands, other modulators and intracellular partners in selective conformations culminating in coupling with a single type or multiple G proteins in a cell- and context-specific manner.

Calcineurin mediates the gonadotropin-releasing hormone effect on expression of both subunits of the follicle-stimulating hormone through distinct mechanisms.

Gonadotropin-releasing hormone (GnRH) regulates the expression of all three gonadotropin genes, encoding the common α subunit (αGSU) and hormone-specific ß subunits, through the activation of several signal transduction pathways. We have shown that GnRH also upregulates calcineurin, and we hypothesized that calcineurin mediates the effects of GnRH on the transcription of the αGSU and follicle-stimulating hormone ß (FSHß) genes through two of its targets: nuclear factor of activated T cells (NFAT) and CREB-regulated transcription coactivator (TORC). We show that calcineurin is essential for GnRH-induced expression of both genes but that NFAT and TORC1 play quite distinct roles in activating each gene. GnRH induces calcineurin-dependent nuclear import of NFAT3, which activates the αGSU promoter, while TORC1 also mediates GnRH activation of this promoter, but not through CREB. GnRH initially stimulates the degradation of TORC1 but protects the N terminus of the newly synthesized protein to enhance its activity. Calcineurin induces Nur77 expression, likely via NFAT3, and Nur77 interacts synergistically with TORC1 and CREB to increase FSHß promoter activity. Although TORC plays a role in the basal activity of the FSHß promoter, it does not interact with phosphorylated CREB and probably does not play a major role in direct GnRH signaling to this gene. TORC may be part of an alternatively regulated pathway, possibly involving cross talk with other stimulatory hormones.

Insight: prolonged vaginal bleeding during central precocious puberty therapy with a long-acting gonadotropin-releasing hormone agonist: a proposed mechanism and management plan.

We have previously described our data collected after administration of gonadotropin releasing hormone-agonist (GnRH-a) to delay sexual maturation, in premenarchal girls suffering from idiopathic central precocious puberty.(1) We have explained the recurrent episodes of bleeding due to discontinuation of the estrogen support of the proliferative and stable endometrium. The recognition in recent years of the extra-pituitary functions of GnRH-a, the ability of GnRH to stimulate prostaglandin production and the known role of prostaglandins in irregular vaginal bleeding prompted us to seek alternative explanations to our data. We suggest considering a potential clinical use of combination therapies of GnRH agonists and prostanoid receptor antagonists to treat central precocious puberty.

Gq protein-induced apoptosis is mediated by AKT kinase inhibition that leads to protein kinase C-induced c-Jun N-terminal kinase activation.

G(q) protein-coupled receptors (G(q)PCRs) regulate various cellular processes, including mainly proliferation and differentiation. In a previous study we found that in prostate cancer cells, the G(q)PCR of gonadotropin-releasing hormone (GnRH) induces apoptosis by reducing the PKC-dependent AKT activity and elevating JNK phosphorylation. Because it was thought that G(q)PCRs mainly induce activation of AKT, we first undertook to examine how general this phenomenon is. In a screen of 21 cell lines we found that PKC activation results in the reduction of AKT activity, which correlates nicely with JNK activation and in some cases with apoptosis. To understand further the signaling pathways involved in this stimulation, we studied in detail SVOG-4O and αT3-1 cells. We found that prostaglandin F2α and GnRH agonist (GnRH-a) indeed induce significant Gα(q)- and PKC-dependent apoptosis in these cells. This is mediated by two signaling branches downstream of PKC, which converge at the level of MLK3 upstream of JNK. One branch consists of c-Src activation of the JNK cascade, and the second involves reduction of AKT activity that alleviates its inhibitory effect on MLK3 to allow the flow of the c-Src signal to JNK. At the MAPKK level, we found that the signal is transmitted by MKK7 and not MKK4. Our results present a general mechanism that mediates a G(q)PCR-induced, death receptor-independent, apoptosis in physiological, as well as cancer-related systems.

Differential role of PKC isoforms in GnRH and phorbol 12-myristate 13-acetate activation of extracellular signal-regulated kinase and Jun N-terminal kinase.

GnRH is the first key hormone of reproduction. The role of protein kinase C (PKC) isoforms in GnRH-stimulated MAPK [ERK and Jun N-terminal kinase (JNK)] was examined in the αT3-1 and LßT2 gonadotrope cells. Incubation of the cells with GnRH resulted in a protracted activation of ERK1/2 and a slower and more transient activation of JNK1/2. Gonadotropes express conventional PKCα and conventional PKCßII, novel PKCδ, novel PKCε, and novel PKCθ, and atypical PKC-ι/λ. The use of green fluorescent protein-PKC constructs revealed that GnRH induced rapid translocation of PKCα and PKCßII to the plasma membrane, followed by their redistribution to the cytosol. PKCδ and PKCε localized to the cytoplasm and Golgi, followed by the rapid redistribution by GnRH of PKCδ to the perinuclear zone and of PKCε to the plasma membrane. Interestingly, PKCα, PKCßII, and PKCε translocation to the plasma membrane was more pronounced and more prolonged in phorbol-12-myristate-13-acetate (PMA) than in GnRH-treated cells. The use of selective inhibitors and dominant-negative plasmids for the various PKCs has revealed that PKCßII, PKCδ, and PKCε mediate ERK2 activation by GnRH, whereas PKCα, PKCßII, PKCδ, and PKCε mediate ERK2 activation by PMA. Also, PKCα, PKCßII, PKCδ, and PKCε are involved in GnRH and PMA stimulation of JNK1 in a cell-context-dependent manner. We present preliminary evidence that persistent vs. transient redistribution of selected PKCs or redistribution of a given PKC to the perinuclear zone vs. the plasma membrane may dictate its selective role in ERK or JNK activation. Thus, we have described the contribution of selective PKCs to ERK and JNK activation by GnRH.

Elucidation of mechanisms of the reciprocal cross talk between gonadotropin-releasing hormone and prostaglandin receptors.

We recently described a novel GnRH receptor signaling pathway mediated by the prostaglandins (PGs) F(2alpha) and PGI(2), which acts through an autocrine/paracrine modality to limit autoregulation of the GnRH receptor and inhibit LH but not FSH release. Here we further explore the cross talk between GnRH and the PG receptors. GnRH stimulates arachidonic acid (AA) release from LbetaT2 gonadotrope cells via the Ca(2+)-independent phospholipase A(2) (iPLA(2)) and not via the more common Ca(2+)-dependent cytosolic phospholipase A(2)alpha (cPLA(2)alpha). AA release was followed by a marked induction of cyclooxygenase (COX)-1 and COX-2 by GnRH via the protein kinase C/c-Src/phosphatidylinositol 3-kinase/MAPK pathway. COX-2 transcription by GnRH is mediated by the two nuclear factor-kappaB sites and the CCAAT/enhancer-binding protein site within its promoter. Indeed, GnRH stimulates p65/RelA phosphorylation (22-fold) in LbetaT2 cells and the two nuclear factor-kappaB sites apparently act as a composite response element. Although GnRH stimulates cAMP formation in LbetaT2 cells, we found no role for cAMP acting via the cAMP response element site in the COX-2 promoter. PGF(2alpha), PGI(2), or PGE(2) had no effect on GnRH-stimulated ERK, c-Jun N-terminal kinase, and p38MAPK activation or on GnRH- and high K(+)-stimulated intracellular Ca(2+) elevation in LbetaT2 and gonadotropes in primary culture. Although, PGF(2alpha), PGI(2), and PGE(2) reduced GnRH-stimulated cAMP formation, we could not correlate it to the inhibition of GnRH receptor expression, which is exerted only by PGF(2alpha) and PGI(2.) Hence, the inhibition by PGF(2alpha) and PGI(2) of the autoregulation of GnRH receptor expression is most likely mediated via inhibition of GnRH-stimulated phosphoinositide turnover and not by inhibition of Ca(2+) elevation and MAPK activation.