Negative Regulation of ERK and Elk by Protein Kinase B Modulates c-fos Transcription*

In this study, we have identified novel regulatory steps involved in the cross-talk between protein kinase B (PKB) and MAPK signaling pathways. We found that PKB down-regulates the Ras-Raf-MEK-ERK pathway by reducing the activity of ERK, which leads to inactivation of the transcription factor Elk1. In addition, PKB is able to reduce protein levels of Elk1. Both events lead to suppression of serum response element (SRE)-dependent transcription and a consequent decrease in the transcription of SRE-containing genes, such as c-fos. Because activation of the Ras/MAPK cascade is reported to increase c-fos transcription before apoptosis, our results are consistent with a specific role for PKB in promoting cell survival. Decrease in c-Fos protein levels in glioblastoma cells with constitutively active PKB provides further support for our observations. Therefore, our findings delineate a novel mechanism regulating immediate-early transcription, which may be involved in the initial steps in PKB-induced oncogenic transformation.

targets of PKB regulation are either genes whose products are involved in insulin metabolism or transcription factors regulating cell survival (for review, see Ref. 4). Thus, PKB mediates transcription of several hepatic genes by acting through the insulin response sequences within the respective promoters. Insulin-induced genes include fatty acid synthase and GLUT1, whereas insulin also represses transcription of the IGF-binding protein-1 gene (for review, see Ref. 5).
Several transcription factors are also regulated by PKB. Phosphorylation by PKB is reported to stimulate the activity of cAMP-response element-binding protein (CREB) (6). In addition, PKB participates in IB degradation, which results in NFB induction (7). This implicates PKB in cytokine-mediated immunity and anti-apoptotic signaling. On the other hand, Forkhead transcription factors, members of the FOXO subfamily, are negatively regulated when phosphorylated by PKB (for review, see Ref. 8). Phosphorylated FOXOs are inactive and sequestered in the cytoplasm away from their transcriptional targets, which prevents induction of apoptosis or cell cycle arrest by these transcription factors. Furthermore, it was reported recently that PKB phosphorylates Mdm2 and, thus, promotes its nuclear translocation. This leads to increased ubiquitination of p53 and resistance to apoptosis in some cancer cells (9,10).
Another key mediator of cellular response to external stimuli is the mitogen-activated protein kinase pathway, which is highly conserved in all eukaryotes (for review, see Refs. 11 and 12). It is activated by a GTP-binding protein, p21 Ras, which leads to recruitment of Raf (MAPKKK) to the plasma membrane. Activated Raf phosphorylates MEK (MAPKK), a dual specificity kinase that phosphorylates the Thr-X-Tyr motif in the activation loop of extracellular-signal-regulated kinase (ERK). Upon activation, ERK translocates to the nucleus and regulates the activity of many transcription factors, which results in distinct biological responses (for review, see Refs. 11 and 12).
The most-studied transcription factors regulated by MAPK are ternary complex factors (TCFs). TCF proteins belong to the ETS family of transcription factors, which have a characteristic DNA binding domain (the ETS-DBD), forming a highly conserved helix-turn-helix structural motif (for review, see Ref. 13). The C terminus of TCFs comprises a transactivation domain with multiple serine and threonine residues that can be phosphorylated by different groups of MAP kinases. Whereas serum, growth factors and TPA stimulate phosphorylation of TCFs via the Raf-MEK-ERK pathway, interleukin-1, tumor necrosis factor ␣, osmotic stress, H 2 O 2 , UV radiation, and anisomycin can induce phosphorylation of TCF through the MEK kinase (MEKK)-SEK1-JNK as well as through the TAK1-MKK3-p38 pathways (for review, see Ref. 13 and 14). The mammalian TCFs, Elk1, Sap1, and Sap2/ERP/Net, regulate transcription from the serum response element (SRE) (for re-  view, see Ref. 13). SRE is the major cis-element responsible for activation of immediate-early gene transcription in response to mitogens (for review, see Ref. 15). It functions as a target for the Ras-MAPK pathway; thus, SRE is also termed a Rasresponsive element. The c-fos SRE is constitutively occupied by a serum response factor (SRF) dimer, which binds with high affinity to the CC(A/T) 6 GG motif within the SRE. SRF dimer recruits monomeric TCF, which forms a ternary complex with SRF on SRE and promotes transcription (for review, see Ref. 13).
PKB and ERK signaling cascades are activated under similar conditions, which provides the possibility for cross-regulation. Thus, under specific conditions PKB can phosphorylate Raf1 (16,17) or B-Raf (18), which inactivates the MAPK pathway. On the other hand, PI3K can stimulate the activity of the MAPK cascade via the Rac1-PAK pathway by activating Raf (19). It has been reported recently that Rho-family GTPases, Rac1 and Cdc42, inhibit MAPK signaling at the level of Raf through PI3K/PKB activation (20). Inactivation of Rac1/Cdc42 signaling mimics the loss of cell anchorage, which activates the Raf-MEK-ERK pathway and induces apoptosis (20).
The aim of this study was to identify novel regulatory points in the cross-talk between PKB and MAPK pathways that contribute to the PKB-mediated changes in transcription. We found that PKB down-regulates the Ras-Raf-MEK-ERK pathway by reducing the activity of ERK, which leads to inactivation of transcription factor Elk1. Significantly, active PKB also indirectly induces a reduction of Elk1 protein levels. Both events lead to suppression of SRE-dependent transcription and consequently change the immediate-early gene response. Our results delineate a novel mechanism of regulating transcription from the SRE/c-fos promoters that may be involved in oncogenic transformation by PKB.
Cell Culture, Transient Transfection, and Stimulation Conditions-HEK 293 and glioblastoma cell lines LN229, U87MG, and U343MG were maintained as described previously (21). The cells were seeded at 60% confluency and transfected the following day by a modified calcium phosphate method (22). The cells were incubated for 24 h with the transfection mix, washed twice with Dulbecco's modified Eagle's medium, and serum-starved for 24 h, in some cases in the presence of 20 M UO126 (Tocris). After starvation, the cells were stimulated with 10% fetal calf serum for 6 h, or 100 ng/ml TPA or 50 ng/ml IGF-1 as indicated.
Luciferase and SEAP Assays-HEK 293 cells were lysed with 200 l of buffer (cell culture lysis reagent; Promega) supplemented with 1 M microcystin-LR (Alexis), 1 mM phenylmethylsulfonyl fluoride, and 1 mM benzamidine. The lysates were centrifuged at 15,000 ϫ g for 15 min at 4°C. Protein concentrations were determined by the method of Bradford (23). The luciferase activity was assayed according to the Promega protocol and corrected for transfection efficiency. SEAP assays were done according to the manufacturer's instructions using the Great EscAPe chemiluminescence protocol (Clontech).
Immunoprecipitation and in Vitro Kinase Assays-HEK 293 cells were lysed with lysis buffer containing 50 mM Tris-HCl (pH 7.5), 1% Nonidet P-40, 2 M microcystin-LR (Alexis), 1 mM phenylmethylsulfonyl fluoride, 1 mM benzamidine, 1 mM sodium pyrophosphate, 0.1 mM sodium orthopervanadate, and 10 mM NaF. The lysates were centrifuged at 15,000 ϫ g for 15 min at 4°C, and protein concentrations were determined as described above. The HA-tagged PKB␣ and Myc-tagged ERK2 proteins were immunoprecipitated from 100 g of cell extracts as described previously (3). The immune complexes were washed with lysis buffer containing 0.5 M NaCl followed by lysis buffer and finally with kinase buffer (50 mM Tris-HCl (pH 7.5), 1 mM dithiothreitol). In vitro kinase assays were performed for 60 min at 30°C in 50 l of reaction mixture containing 30 l of immunoprecipitate in kinase buffer, 3 g of glutathione S-transferase-ElkC as a substrate, 10 mM MgCl 2 , 1 M protein kinase A inhibitor peptide (Bachem), and 100 M [␥-32 P]ATP (Amersham Biosciences; 1000 -2000 cpm/pmol). All reactions were stopped by adding Laemmli sample buffer, resolved by SDS-10% PAGE, and transferred to Immobilon P membranes (Millipore). Phosphorylated proteins were visualized by autoradiography and quantified with ImageQuant software (Molecular Dynamics).
For endogenous PKB and ERK kinase assays, proteins were immunoprecipitated from 2 mg of cell extracts, and immune complexes were washed as above. In vitro kinase assays were performed for 30 min at 30°C with crosstide (PKB) or myelin base protein (ERK) as described (24,25).

RESULTS
Protein Kinase B Is Able to Suppress Transcription from the SRE-To investigate possible cross-talk between PKB and other signaling cascades, we performed a screen of signal transduction pathways by monitoring the transcriptional activity of several reporter constructs. For this purpose, we used the SEAP reporter system, where a specific response element is located upstream of a minimal thymidine kinase promoter fused to secreted alkaline phosphatase. Secreted phosphatase activity represents a direct readout for induction of specific signaling pathways. Thus, AP1 mirrors mitogen-activated JNK signaling, cAMP-response element (CRE) responds to polypeptide hormone-and neurotransmitter-activated protein kinase A and the stress-activated JNK/p38 pathway, NFB activity increases after IK-mediated cytokine and platelet-derived growth factor signaling, and the SRE is activated by mitogeninduced ERK/JNK pathways.
To monitor the influence of PKB on other signaling cascades, constitutively active membrane targeted myristoylated/palmitoylated PKB (m/pPKB) was co-transfected with different SEAP reporter vectors. As shown in Fig. 1A, the basal activity of the tested response elements was already high in HEK 293 cells, even in the absence of stimuli. Under these conditions, co-expression of m/pPKB led to a 20 and 30% increase in AP1and NFB-driven transcription, respectively, whereas the CRE pathway was not significantly affected by m/pPKB.
The most significant change in transcription was observed when m/pPKB was co-transfected with the SRE-dependent construct, the activity of which decreased 85% compared with the control. This suggests that PKB plays an important role in the regulation of ERK/JNK pathways and/or ternary complex factor Elk1/Sap1-mediated transcription.
To further confirm whether the SRE can be regulated by the PKB pathway, a luciferase reporter gene was constructed that contained binding sites for the TCFs and SRF as well as an AP1-binding site (Fig. 1B). As shown in Fig. 1B, the expression of increasing amounts of wild-type PKB reduced the basal level of transcription of the SRE in a dose-dependent manner in HEK 293 cells. In contrast, the same effect could not be observed with an inactive form of the kinase lacking the Cterminal 14 amino acids (PKB-KD), which indicates that the kinase activity of PKB is required for the effect on the SRE. Importantly, wild-type PKB promoted 50% inhibition at a concentration as low as 1 ng/ml transfected DNA, whereas inactive PKB had no effect on transcription.
To further test whether this effect was due to PKB kinase activity, we compared the activity of constitutively active PKB mutants with that of wild-type PKB. For this purpose, we expressed in HEK 293 cells constitutively active m/pPKB as well as the constitutively active cytosolic PKB mutant in which both the regulatory sites Thr-308 and Ser-473 were mutated to aspartate (PKB-DD). As shown in Fig. 1C, both constitutively active mutants suppressed the basal level of SRE trans-activation, whereas the inactive PKB-KD had no effect on transcription ( Fig. 1C, open bars). The effect of PKB mutants was in accordance with their previously described activity (3).
Because it is well known that mitogens induce transcription by acting on SRE, we examined whether this activation is blocked by PKB. Serum stimulation led to a significant increase in SRE transcription (Fig. 1C). However, all the active PKB mutants not only prevented activation of the SRE but also lowered its activity to below basal levels. As observed for basal conditions, PKB-KD was also unable to repress induction of the promoter by serum (Fig. 1C, black bars).
Taken together, these results demonstrate that PKB can suppress basal as well as activated transcription from SRE. These data also suggest that PKB interfered with one or more pathways involved in SRE regulation.
Protein Kinase B Down-regulates the Human c-fos Promoter-The SRE is the major cis-element activated in response to growth factors and is, thus, responsible for rapid and transient activation of immediate early genes, including c-fos and egr-1 (27). To examine the effect of PKB on SRE within a physiologically relevant promoter, we performed experiments similar to those shown in Fig. 1, with the luciferase reporter gene under the control of a 750-bp proximal human c-fos promoter containing Sis-inducible enhancer (SIS) and CRE elements in addition to SRE ( Fig. 2A).
Consistent with our results with the SRE reporter ( Fig. 1), wild-type but not kinase-inactive PKB reduced basal transcription from the c-fos promoter by 90% (Fig. 2B). The effect of PKB was dose-dependent and caused a 50% inhibition of c-fos transcription at concentrations as low as 4 ng/ml transfected expression vector. Significantly, PKB and the constitutively active mutants also abolished the activity of the c-fos promoter after serum stimulation (Fig. 2C). Moreover, reduction in c-fos transcription in HEK 293 cells correlated with the level of activity of endogenous PKB induced by upstream activators such as serum, TPA, or insulin (data not shown). Similar results were obtained with NIH 3T3 and COS-1 cells (data not shown), indicating that this regulation is not cell type-specific but may be a general mechanism of transcriptional regulation.
PKB Inhibits the Ras-Raf-MEK Pathway-We showed that PKB was able to suppress transcription mediated by SRE, which results in inhibition of its function to promote transcription of serum-responsive genes, such as c-fos. Mitogens activate immediate-early gene transcription through all three Ras-induced MAPK signaling pathways. The SRE is mainly activated FIG. 1. PKB specifically reduces transcription from the serum response element. A, HEK 293 cells were co-transfected with 100 ng/ml empty vector or a vector encoding constitutively active PKB (m/pPKB) together with 100 ng/ml SEAP reporter vectors under the control of AP1, CRE, NFB, and SRE, respectively. SEAP activity from the cells co-transfected with an empty vector was taken as 1. The experiment was performed three times, and a representative experiment is shown. PKB expression (inset) was confirmed by immunoblot analysis using an anti-HA-epitope antibody. RLU, relative light units. B, structure of the SRE luciferase reporter containing binding sites for TCF, SRF, and AP1 (upper panel). HEK 293 cells were co-transfected with an empty vector or a vector encoding either wild-type (WT) PKB or a kinase-defective mutant (PKB-KD) together with 100 ng/ml SREluciferase reporter. Luciferase activity from the cells co-transfected with an empty vector was taken as 1 and was calculated from four independent experiments. PKB expression was confirmed as in A. C, HEK 293 cells were co-transfected with 100 ng/ml empty vector or a vector encoding PKB, m/pPKB, or constitutively active PKB-DD or PKB-KD. Luciferase activity was assayed as in Fig. 1B, and the average values of four independent experiments are shown. via the pathway involving Ras, Raf, MEK, and ERK (for review, see Ref. 15). We first searched for a direct target of PKB regulation in this pathway using the c-fos promoter construct as a readout. For this purpose, HEK 293 cells were transfected with constitutively active components of the pathway (Fig. 3, A-C). As expected, constitutively active RasV12 stimulated c-fos transcription 20-fold (Fig. 3A). This was almost completely inhibited by a MEK-specific inhibitor UO126 (Fig. 3A) and indicates that the MEK-ERK signaling cascade was the main pathway involved in c-fos transcription. Constitutively active PKB, when coexpressed with Ras, displayed the same effect as the MEK inhibitor, decreasing c-fos transcription below the basal level, indicating that PKB affects the same pathway (Fig. 3A). To investigate whether PKB acts downstream or upstream of Raf, PKB was co-transfected with constitutively active Raf BXB. As shown in Fig. 3B, Raf BXB induced c-fos transcription 45-fold, which was completely abolished when UO126 was added to the cells. Similar to the experiments with RasV12, constitutively active PKB was able to decrease Rafinduced transcription from the c-fos promoter (Fig. 3B), suggesting that PKB does not act directly on Ras but, rather, downstream in the pathway.
By a similar strategy, constitutively active EE-MEK was co-transfected with PKB. Transfection of EE-MEK alone stimulated transcription 10-fold (Fig. 3C). PKB diminished the activity of the promoter to its basal level, similar to that observed with UO126 (Fig. 3C). Taken together, these data indicate a PKB target downstream of Ras, Raf, and MEK, which directed us to investigate transcription factors regulated by ERK.
PKB Suppresses TCF-dependent Transcription-The c-fos SRE is constitutively occupied by an SRF dimer, which recruits monomeric TCFs, Elk1, Sap1, or Sap2/ERP/Net, to form a ternary complex with SRF (for review, see Refs. 13 and 14). All three MAPK pathways converge on the TCFs, and ERK, JNK, and p38 are all able to phosphorylate Elk1 (for review, see Refs. 13 and 14). Therefore, we examined TCFs as possible targets of PKB downstream of MAP kinases. All three ETS-domain-containing transcription factors that can form ternary complexes on SREs were transfected with a minimal reporter containing the ETS palindromic site joined to the luciferase reporter. Both Elk1 (35-fold) and Sap1 (4-fold) activated, whereas Net inhibited (70%) transcription from the luciferase reporter ( Fig. 4A  and data not shown), as previously demonstrated (28). PKB repressed Elk-as well as Sap-induced transcription but did not interfere with Net-mediated inhibition ( Fig. 4A and data not shown). As a positive control, we used an Ets1 expression vector, which specifically binds to the ETS palindromic site and activates transcription. As shown in Fig. 4A, Ets1-induced transcription was also efficiently suppressed by PKB, which can be explained by the fact that Ets1 can be regulated by MAP kinases (29). In addition, TCF activity was sufficient to drive transcription from this reporter, indicating that the presence of SRF was not required and, therefore, strongly suggesting that inhibition of the c-fos transcription was not SRF-dependent.
Because Elk was a more potent activator of transcription than Sap and it can be regulated by all MAP kinases, we next examined whether Elk is a target of PKB. For these experiments we used an expression vector containing the C-terminal MAP kinase-inducible activation domain of Elk1 fused to the heterologous DNA binding domain of the yeast protein Gal4 (Gal.ElkC). As shown in Fig. 4B, Elk promoted transcription from the Gal4 reporter, but this was completely inhibited by PKB, whereas PKB-KD had no effect on transcription. Furthermore, UO126 only partially reduced Elk-stimulated transcription (Fig. 4B). This confirmed previous data suggesting that several pathways contribute to Elk activation (Fig. 4B). PKB, however, was able to abolish transcription completely and, thus, acts on an effector common to all these pathways (Fig. 4B).
PKB Regulates ERK Activity-We examined next whether PKB directly phosphorylates Elk1 in vitro. Under the conditions employed we could not find any evidence for Elk as a PKB substrate (data not shown), which prompted us to investigate the effect of PKB on Elk phosphorylation indirectly. ERK2 and PKB constructs were co-expressed in HEK 293 cells, and Rasactivated ERK2 was used as a positive control. In vitro kinase assays of immunoprecipitated ERK2 and PKB were performed using glutathione S-transferase-Elk (ERK) or crosstide (PKB) as a substrate. Unstimulated ERK showed low activity toward glutathione S-transferase-Elk, whereas activation by RasV12 increased phosphorylation of Elk by ERK more than 10-fold (Fig. 5A, middle panel). Constitutively active, but not inactive PKB suppressed Ras-induced ERK activity, resulting in 80% lower phosphorylation of Elk (Fig. 5A, middle panel, bar graph). The level of repression of ERK activity (Fig. 5A, middle panel, bar graph) was proportional to PKB activity (Fig. 5A,  upper panel). Levels of immunoprecipitated ERK on the beads were controlled by blotting the same autoradiograph with the Myc monoclonal antibody. This revealed similar amounts of ERK in each lane and, thus, excludes the possibility that lower ERK activity in the presence of PKB is due to the lower amount of ERK (Fig. 5A, lower panel). ERK is activated by phosphorylation on Tyr and Thr in the activation loop. This TEY phosphorylation is regularly regarded as a measure of ERK activity. Therefore, to examine the phosphorylation status of ERK2 in our experiments, the membranes were stripped and re-probed with TEY phospho-specific antibody. Surprisingly, in contrast to the observed decrease in ERK activity by PKB (Fig. 5A,  middle panel, bar graph), the level of Ras-induced phosphorylation of ERK2 was not significantly changed in the presence of PKB (Fig. 5A, lowest panel). This implies that the level of ERK activity does not always necessarily correlate with TEY phosphorylation in the activation loop. These results indicate that in some cases, despite TEY phosphorylation, ERK activity may be suppressed by a mechanism other than dephosphorylation. Therefore, direct kinase assays provide a more accurate measure of ERK activity.

FIG. 4. PKB reduces TCF-dependent transcription.
A, HEK 293 cells were co-transfected with 100 ng/ml empty vector or a vector encoding PKB together with 500 ng/ml expression vectors for ETS1p68, Elk1, Sap1a, or Net (not shown) and 500 ng/ml pGL2(Pal)8-TK-luc reporter. Luciferase activity was determined as described in Fig. 1B, and the average values from four independent experiments are shown. B, HEK 293 cells were transfected with 100 ng/ml empty vector or a vector encoding PKB together with 250 ng/ml Gal4.ElkC and 250 ng/ml (Gal4)5-TK-luc reporter. Luciferase activity was determined as described in Fig. 1B, and the average values from four independent experiments are shown. WT, wild type.

FIG. 5. PKB regulates ERK activity.
A, HEK 293 cells were transfected with 1 g/ml HA-ERK2 together with 100 ng/ml HA-PKB and/or 1 g/ml RasV12. In vitro kinase assays were performed as described under "Experimental Procedures." Quantification of three independent experiments is shown. The total levels of PKB and ERK on the beads were analyzed by immunoblotting by HA-antibody followed by stripping and blotting with pERK antibody. B, HEK 293 cells were stimulated with 100 ng/ml TPA or with 50 ng/ml IGF-1 for the indicated times. In vitro kinase assays were performed as described under "Experimental Procedures," and the average values from three independent experiments are shown.
To confirm these results, HEK 293 cells were stimulated by TPA, IGF-1, or both at various time points, and endogenous kinase assays were performed. As shown in the Fig. 5B, TPA was able to induce ERK activity 14-fold after 10 min and 25-fold after 30 min (lower panel), whereas it did not significantly affect activity of endogenous PKB. Conversely, IGF-1 predominantly induced PKB activity. When the cells were exposed to TPA and IGF-1 to activate both pathways, we observed strongly suppressed activity of ERK (66% after 10 min or 80% after 30 min) as compared with TPA stimulation (Fig.  5B, lower panel). At the same time points PKB activity was high (Fig. 5B, upper panel). Taken together, these data provide evidence for PKB-promoted inhibition of ERK kinase activity by a novel mechanism (see "Discussion").
PKB Regulates Protein Levels of Elk1-The stability of some transcription factors can be regulated by phosphorylation (for review, see Ref. 30). Thus, phosphorylation is needed not only to activate the transcription factor but also to protect it from degradation. For example, c-Jun is protected from ubiquitination when phosphorylated on Ser-73 by JNK (31). To investigate the effect of reduced ERK activity on Elk1 stability, we co-expressed Elk together with active or inactive PKB. The results in Fig. 6 show that PKB and m/pPKB decreased the amount of Elk1 protein in a dose-dependent manner. However, PKB-KD had no effect on Elk1 protein levels (Fig. 6). Cotransfection of green fluorescent protein and PKB showed that neither active nor inactive PKB significantly changed the expression levels of green fluorescent protein (data not shown), which excludes the possibility that the decreased level of Elk1 protein was an unspecific consequence of transfection. The proteasome inhibitor MG132 did not block PKB-promoted reduction in Elk levels (data not shown), suggesting that this was not proteasome-mediated.
These results were confirmed when the cells were treated with cycloheximide to inhibit protein synthesis. Under such conditions, the amount of Elk1 protein declined more rapidly when the cells were co-transfected with PKB or treated with insulin (data not shown). These findings indicate that PKB can reduce ERK activity and decrease the level of Elk1 phosphorylation, leading to reduced levels of Elk protein by a proteasome-independent mechanism.
Active PKB Can Reduce the Levels of Fos Protein in Vivo-It is well established that PKB activity can be regulated by the tumor suppressor PTEN, a phosphatase that dephosphorylates phosphatidylinositol 3,4,5-trisphosphate. By dephosphorylating phosphatidylinositol 3,4,5-trisphosphate, PTEN acts in opposition to PI3K (for review, see Ref. 32). Recently, we discovered a PKB-interacting protein, termed CTMP, that inhibits phosphorylation of PKB␣ by its upstream kinases on Ser-473 and somewhat less on Thr-308 (21). These findings identify CTMP as a specific negative regulator of PKB, which prompted us to test whether CTMP can also reverse the effect of PKB on the c-fos promoter. As shown in Fig. 7A, co-transfection of CTMP rescued the inhibitory effect of PKB and restored c-fos transcription. The control vector for CTMP (pSG5) did not change the inhibition promoted by PKB. The restoration of transcription by CTMP was dose-dependent and was observed at the level of both basal and serum-induced transcription. Moreover, CTMP even increased c-fos transcription above its initial level, suggesting that CTMP inhibits endogenous PKB, consistent with our previous observations (21).
Finally, we examined cells in which both the PKB and MAPK pathways were constitutively active in vivo. In U87MG and U343MG glioblastoma multiforme cell lines with compromised PTEN function, CTMP is expressed at low levels, which results in constitutive PKB activity. However, in LN229 glioblastoma cells with normal PTEN status, CTMP is also expressed at higher levels, thus inhibiting PKB activity (21). Moreover, because PTEN is able to down-regulate the MAPK pathway, PTEN Ϫ/Ϫ cells also have high ERK activity (for review, see Ref. 33). As shown in Fig. 7B, low PKB activity in LN229 co-relates with increased levels of c-Fos protein, as demonstrated by Western blotting. In contrast, PKB was phosphorylated and, thus, activated in both U87MG and U343MG cells, whereas the levels of c-Fos were low (Fig. 7B). These data provide evidence that in the glioblastoma cells studied c-Fos levels are inversely correlated with PKB activity. DISCUSSION Cross-talk between the MAPK and PI3K/PKB pathways apparently operates at different levels and depends on specific conditions and the cell types studied. The "classical" activation of ERK involves signaling from Ras through Raf and MEK (for review, see Ref. 11), although an alternative mechanism also exists involving activation of the Raf-MEK-ERK cascade through signaling via PI3K (19,34,35). On the other hand, under specific conditions, PKB can inhibit activity of the Ras-MAPK pathway. It is reported that PKB negatively regulates Raf1 through phosphorylation of Ser-259 (16,17) and B-Raf by phosphorylating several residues in the N-terminal regulatory domain (18). Loss of cell anchorage induces anoikis in fibroblasts. It can be mimicked by inhibition of Rac1/cdc42 signaling through PI3K and PKB, which activates Raf-MEK-ERK (20). This suggests under normal conditions Rac1/cdc42 activates PKB, which then inhibits Raf signaling (20).
In this study we identify ERK/Elk-mediated regulation of c-fos transcription as a novel target for MAPK/PKB cross-talk. The negative effect of PKB on c-fos promoter transcription that we observed does not, however, appear to involve previously described mechanisms. This is based on several lines of evidence as follows. (i) The induction of the c-fos promoter by constitutively active Raf lacking Ser-259 in the CR2 domain was efficiently down-regulated by PKB (Fig. 3B); (ii) c-fos promoter induction by constitutively active MEK was also efficiently repressed by PKB (Fig. 3C); (iii) expression of PKB was sufficient to inhibit c-fos induction by co-expressing either ERK or Jun kinase (data not shown). In our study, we identified two regulatory steps involved in the cross-talk between the Raf/ MAPK and PI3K/PKB pathways. We demonstrated that PKB can reduce activity of ERK toward its substrates, one of which is the transcription factor Elk1. In addition, we showed that PKB reduces the amount of Elk1 protein. Both of these events apparently result in efficient down-regulation of SRE-dependent transcription. Our current model for PKB-mediated regulation of c-fos induction is summarized in Fig. 8.
We show that co-transfection of PKB inhibits the activity of ERK and, thus, its ability to phosphorylate Elk (Fig. 5A). Constant levels of TEY phosphorylation in the activation loop (Fig.  5A) rule out the likelihood that PKB signals through a phosphatase and, thus, inactivates ERK. Our data indicate that PKB negatively regulates ERK activity either by direct phosphorylation at a site distinct from TEY in the activation loop or indirectly, by phosphorylating a yet unidentified inhibitor of ERK. Results from in vitro kinase assays (Fig. 5A) exclude the possibility that PKB activates a phosphatase that dephosphorylates Elk. Regulation of ERK activation is rather complex, and it depends on two phosphorylations in the activation loop and subsequent re-ordering of the N-and C-terminal domains of the kinase (36). It is known that ERKs are phosphorylated on tyrosine before threonine in two separate reactions, both performed by MEK (37). The first phosphorylation of Tyr-185 (in ERK2) induces a conformational change that forms the Cterminal part of the substrate binding site (38,39). Once this is completed, the second phosphorylation on Thr-183 occurs, leading to the orientation of N-terminal part of the active site (38,39). Thus, ERK becomes activated only upon completion of both phosphorylation events. Our time course experiments (Fig. 5B) with the endogenous kinases suggest that PKB delays the activation of ERK without affecting the TEY phosphorylation. This could reflect the different rates of phosphorylation of Tyr-185 and Thr-183, suggesting that phosphorylation of Tyr-185 happens relatively fast (37) and could already be detected by the polyclonal phosphospecific antibody, but it is not sufficient for the enzymatic activity of ERK toward its substrates. Further experiments are required to elucidate the precise mechanism for ERK down-regulation by PKB.
PKB-mediated decrease in ERK activity would lead to lower levels of Elk-1 phosphorylation, resulting in a reduction in transcriptional activity. Phosphorylation regulates protein targeting for degradation. In some cases, phosphorylation is required before ubiquitination (IB, ␤-catenin), whereas in others, phosphorylation protects proteins from degradation (c-Jun, p53, ATF2) (for review, see Ref. 30). Stress-activated kinases regulate stability of transcription factors, the best example being JNK/c-Jun (31). Thus, phosphorylation on Ser-73 by JNK protects c-Jun from degradation, whereas unphosphorylated c-Jun is efficiently ubiquitinated upon binding to JNK (31). It is tempting to speculate that phosphorylation protects Elk from degradation. To our knowledge, little is known about the regulation of Elk stability. In contrast to c-Jun, JNK is able to phosphorylate Elk, but it neither associates with nor promotes Elk1 ubiquitination (40). Our preliminary data show that decrease of Elk protein was not proteasome-dependent (data not shown) but relies rather on the activity of other cellular proteases. On the other hand, reduction of Elk protein levels may be indirect and due to activation of another enzyme or a specific protease by PKB. For this reason, we cannot exclude the possibility that the two events (decrease in ERK activity and decrease in Elk protein levels) that lead to the inhibition of SRE-dependent transcription are actually uncoupled. We did not investigate whether PKB is also able to regulate the activity of other MAP kinases but, rather, focused on the consequences of lowered ERK activity on down-stream signaling. Nevertheless, because Elk represents a convergence point for all three MAPK pathways, the observed decreased Elk protein could alone account for the inhibition of SRE-dependent transcription.
By its action on Elk, PKB drastically decreases levels of immediate-early gene transcription. Based on several reports suggesting that continuous c-fos expression precedes apoptosis in vivo, at least in the tissues that require de novo protein synthesis for programmed cell death (for review, see Ref. 41), the antiapoptotic role of PKB becomes apparent at this level. Moreover, under some conditions, Elk is very efficient in causing cell death (42). In addition, by regulating the levels of Elk, PKB controls not only transcription of c-fos gene but also reduces the level of AP1 transcription factor. This may lead to down-regulation of other genes containing AP-binding sites within their promoters that are preferably occupied by a Fos-containing heterodimers. Moreover, TCFs can act independently of SRF via direct binding to PEA3 elements in some promoters (43). Therefore, our data ( Fig.  4A) might also indicate a role for PKB in the expression of those SRE/AP1-independent genes.
The mechanism (Fig. 8) we propose here is consistent with the anti-apoptotic role of PKB. PKB promotes cell survival by inhibition of an intrinsic cell death machinery in the cytoplasm (BAD, caspase 9) as well as in the nucleus (Forkhead transcription factors, NFB, p53) (for review, see Ref. 4). Although it seems contradictory at first glance that PKB inhibits Rasinduced MAPK activity, this can be explained given the dual FIG. 8. Proposed mechanism for down-regulation of SRE-dependent transcription by PKB. A, in response to Ras signaling, ERK is activated by MEK-mediated dual phosphorylation (P) in the activation loop. Active ERK phosphorylates its substrates, one of which is the transcription factor Elk1. Phosphorylated Elk1 accumulates in the nucleus and strongly promotes transcription from SRE-regulated genes, such as c-fos. B, active PKB reduces ERK activity either directly by phosphorylating ERK at a site distinct from TEY in the activation loop or by phosphorylating a yet unidentified inhibitor of ERK (1). Low ERK activity results in decreased phosphorylation and shifts the equilibrium toward unphosphorylated Elk1 (2). Unphosphorylated Elk1 is inactive, unstable, and prone to degradation (3). It is also possible that reduction of ERK activity (1) and decline of Elk1 protein levels (3) involve distinct mechanisms, both regulated by PKB. However, each of these events could separately account for down-regulation of SRE-dependent transcription (4). role of Ras in apoptosis. Namely, regarding its oncogenic potential, Ras is generally considered in the context of activation of cellular proliferation and suppression of apoptosis. However, this is only partially correct because Ras also induces apoptosis (for review, see Ref. 44). Generally, Ras signals mostly through Raf-MEK-ERK but also via the PI3K/PKB pathway. This differential signaling seems to mirror opposing effects of Rasinduced pathways on apoptosis (for review, see Ref. 44). Although signaling through PI3K/PKB always promotes cell survival, proapoptotic Ras signals exclusively through the Raf-MEK-ERK cascade (for review, see Ref. 44). In this context, Elk is not the only transcription factor differentially regulated by Ras and PKB. It was shown that transformation of fibroblasts by Ras causes inhibition of NFB transcriptional activity, which leads to apoptosis (45,46). In contrast, PKB regulates the levels of NFB inhibitor IB through activation of IK (7). When phosphorylated by IK, IB is targeted for degradation, which enables NFB to translocate to the nucleus and promote transcription of anti-apoptotic genes. Thus Ras-transformed cells require activation of PKB to survive (46). However, the role of the Raf-MEK-ERK pathway in apoptosis can be completely opposite depending on the particular cellular setting. This has to be taken into account when investigating the interplay between the two pathways. Based on data from many reports addressing these issues it can be concluded that the specific physiological outcome results from a number of parameters including concentration of the particular component, nature and duration of the signal, specific interaction between signaling molecules, differentiation status, and distinct cellular machinery in a particular tissue. Therefore, the type of SRE regulation by PKB that we observed in HEK 293 cells and confirmed in NIH 3T3 and COS-1 cells (data not shown) may not be extended to all cell lines and tissues.
It is very well established that PKB activity can be regulated by PTEN, a tumor-suppressor frequently mutated in cancers (for review, see Ref. 32). Recently, we discovered another PKB inhibitor, which we named C-terminal modulator protein (CTMP). Like PTEN, CTMP is also able to revert transforming phenotypes of PKB and, thus, acts as a potential tumor suppressor (21). Here, we demonstrate that CTMP can revert the effect of PKB on c-fos transcription (Fig. 7A). Moreover, our data suggest that the proposed regulation of SRE-dependent transcription by PKB might occur in vivo. We reported previously the differential expression of CTMP in glioblastoma multiforme cell lines with different PTEN status (21). Thus, in cells with compromised PTEN function, CTMP is also expressed at very low levels, which explains why PKB is constitutively active in these cells. Accordingly, in cells with normal PTEN status, the level of CTMP is also higher (21). Moreover, in addition to its activity toward PI3K, PTEN is also able to down-regulate the MAPK pathway (for review, see Ref. 33). Consequently, one would expect that both the PI3K/PKB and MAPK pathways are active in PTEN-negative cells. However, we demonstrate here in U87MG and U343MG cells, which are both PTEN-negative and have high PKB activity, that the ERK pathway was apparently down-regulated, as measured by the low levels of Fos protein.
In summary, we have identified two new points of convergence in the cross-talk between the MAPK and PKB pathways, (i) PKB reduces the activity of ERK and (ii) indirectly decreases the amount of the transcription factor Elk1. In combination these regulatory responses lead to drastic inhibition of SREdependent transcription. Our findings suggest a novel role for PKB in the fine-tuning of the signals between the cell surface and nucleus, leading to differential regulation of gene expression. This shows how the same or similar stimuli induce dif-ferential responses in certain cells/tissues to regulate various biological processes. Because PKB is a major mediator of insulin and IGF-1 signaling, the identification of new PKB effectors in cell survival may provide more specific targets for therapy of malignancies without affecting metabolism.