MEK5-ERK5 enhanced PPARγ1 transcriptional activity in ECs.
To test the hypothesis that ERK5 regulates PPARγ1 transcriptional activity, we examined the effect of CA-MEK5α and ERK5 on PPARγ1 transcriptional activity. We coexpressed PPARγ1, CA-MEK5α, and ERK5 in HUVECs and examined PPARγ1-mediated transcriptional activity, as assayed by a luciferase reporter gene driven by three copies of a PPAR response element (PPRE) linked to a thymidine kinase (tk) promoter.
As shown in Fig. , addition of CA-MEK5α significantly increased full-length PPARγ1 transcriptional activity (1.24 ± 0.34 versus 2.86 ± 0.31; P < 0.05) (lane 1 versus lane 3). Interestingly, cotransfection of CA-MEK5α and wild-type ERK5 (ERK5a) significantly enhanced PPARγ transcriptional activity to a greater extent than CA-MEK5α transfection alone (2.86 ± 0.31 versus 4.04 ± 0.34; P < 0.05) (lane 3 versus lane 2). We also found that CA-MEK5α expression enhanced transcriptional activity of a PPARγ1 ligand binding domain-truncated mutant (aa 162 to 475) in a ligand-dependent manner, suggesting that the NH2 terminal of the PPARγ1 region is likely not involved in the MEK5-ERK5-mediated effect on PPARγ1 activity (data not shown). PPARγ expression levels were not significantly different among the samples based on Western blot analyses (data not shown).
FIG. 1. MEK5-ERK5 activation increases PPARγ1-mediated transactivation of the (PPRE)3-tk-luciferase reporter construct in HUVECs, which is independent of PPARγ1 S82 phosphorylation. (A and B) MEK5-ERK5 activation induced PPARγ1 transcriptional (more ...)
Because transfection of ERK5a increased PPARγ1 activity, we examined the role of ERK5 kinase activity in PPARγ1 transcriptional activation. We cotransfected a dominant negative form of ERK5a (DN-ERK5 [dual phosphorylation site mutant T219A/Y221P]) or ERK5b (ATP binding site deleted, alternative splicing form [aa 78 to 806 of ERK5a]) (33
) with CA-MEK5α and measured ciglitazone activation of PPARγ (Fig. ). Compared with cotransfection of CA-MEK5α and ERK5a, cotransfection of DN-ERK5 or ERK5b with CA-MEK5α significantly reduced ligand-mediated PPARγ1 activity (Fig. , lane 2 versus lanes 4 and 5). These data suggested that ERK5 kinase activity is required for full stimulation of PPARγ1 transcriptional activity. However, DN-ERK5 or ERK5b did not inhibit CA-MEK5α-induced PPARγ1 activity (Fig. , lane 3 versus lanes 4 and 5), suggesting that an ERK5 function besides endogenous kinase activity may be important. The scaffold function of ERK5 has previously been reported for MEF2, because the association of ERK5 with MEF2, but not MEF2 phosphorylation by ERK5, was regulated by MEF2 transcriptional activity (15
). Therefore, association of ERK5 with PPARγ in addition to ERK5 kinase activity may regulate PPARγ1 transcriptional activity.
In contrast to ERK5, it has been reported that ERK1/2 and JNK inhibit PPARγ transcriptional activity through phosphorylation of Ser82 on PPARγ1 (6
). Therefore, we investigated whether this inhibition by ERK1/2 and JNK could be observed in our cell system. For this purpose, we cotransfected full-length PPARγ1 and the luciferase reporter gene containing PPRE with or without CA-MEKK1 (as an upstream activator of ERK1/2 and JNK). We found that ciglitazone-induced PPARγ1 transcriptional activity was significantly inhibited by CA-MEKK1 transfection (Fig. ), demonstrating that ERK1/2 and JNK behaved as anticipated. We confirmed that CA-MEKK1 induced ERK1/2 and the JNK signaling pathway (8
) (data not shown). These data support our finding that ERK5 differs from ERK1/2 and JNK with respect to PPARγ transcriptional activity.
ERK5 kinase did not phosphorylate PPARγ1 in vitro.
Since activation of ERK5 regulated PPARγ activity, as shown in Fig. , we asked whether ERK5 could phosphorylate PPARγ in vitro. We cotransfected CA-MEK5α and Xpress-tagged ERK5a in Cos7 cells to activate ERK5a constitutively. Activated ERK5a was immunoprecipitated with an anti-ERK5 antibody, and an in vitro kinase assay was performed with GST, GST-PPARγ-AF-1 (aa 1 to 110, including Ser82, which is the ERK1/2 and JNK phosphorylation site), GST-PPARγ-DBD(aa 109-175), and GST-PPARγ-LBD(aa 163-475) as substrates. We did not use GST-PPARγ1 wild type, containing the complete sequence, because it was difficult to dissolve in lysis buffer and was easily degraded. As shown in Fig. , transfection of CA-MEK5α activated ERK5 kinase, as shown by ERK5 autophosphorylation (Fig. , bottom). However, ERK5 did not phosphorylate any PPARγ substrate (Fig. , top).
Since ERK5, ERK1/2, and JNK phosphorylate similar proline-targeted consensus sequences, we mutated Ser82, which is the ERK1/2 and JNK phosphorylation site in PPARγ1, to alanine (S82A) or aspartate (S82D) and determined the effect of CA-MEK5α on PPARγ1 activity. As shown in Fig. , we did not find any significant differences in ciglitazone-stimulated and/or CA-MEK5α-stimulated PPARγ1 activity between the wild type and PPARγ1 mutants. These data further suggest that the regulation of PPARγ1 activity by ERK5 is different from that of ERK1/2 and JNK.
Endogenous ERK5 associates with endogenous PPARγ in ECs.
To investigate the potential interaction between ERK5 and PPARγ1, we analyzed their interaction by using coimmunoprecipitation. Since PPARγ is a nuclear receptor and ERK5 needs to be activated for its nuclear translocation, as our investigators previously described (33
), we stimulated the cells with 10% serum for 30 min and immunoprecipitated with an anti-PPARγ antibody or rabbit immunoglobulin G (IgG) as a control. We found that endogenous PPARγ coimmunoprecipitated with endogenous ERK5 in ECs, but control rabbit IgG did not (Fig. ).
FIG. 2. Endogenous ERK5 associates with endogenous PPARγ at the hinge-helix 1 region of PPARγ1, and the hinge-helix 1 region of the PPARγ1 fragment inhibited the ERK5-PPARγ interaction and CA-MEK5α-mediated PPARγ (more ...)
To investigate the binding site of PPARγ1 with ERK5, we utilized a mammalian two-hybrid assay. A plasmid expressing the GAL4-DBD and the PPARγ (full-length or deletion mutants) was constructed by inserting PPARγ (including mutants) isolated from pSG5-PPARγ1 in frame into the pBIND vector. The plasmid expressing VP16-ERK5 (including mutants) was constructed by inserting the fragment of ERK5 into the VP16 activation domain containing plasmid pACT vector. As shown in Fig. , hinge-helix 1 (aa 202 to 231) was required for the ERK5a-PPARγ1 interaction. To confirm the role of hinge-helix 1 region in the ERK5a-PPARγ1 interaction, we generated a truncated mutant form of PPARγ1 (Δaa202-231) and the PPARγ1(aa 195-227) fragment. As shown in Fig. , deletion of hinge-helix 1 region completely inhibited the ERK5a-PPARγ1 interaction, but PPARγ1(aa 195-227) could associate with ERK5a, suggesting the critical role of hinge-helix 1 regions for the ERK5-PPARγ1 interaction.
Disruption of the ERK5-PPARγ1 interaction induced by the hinge-helix 1 fragment inhibited CA-MEK5α-induced PPARγ1 activity.
It is possible that the deletion mutant of the hinge-helix 1 region of PPARγ1 may change the tertiary structure of PPARγ1. To demonstrate the critical role of the hinge-helix 1 region for the ERK5-PPARγ1 interaction without mutating and destroying PPARγ1 structure, we determined whether the hinge-helix 1 fragment, which is the binding site of ERK5a, could disrupt the association of wild-type ERK5a and wild-type PPARγ. For this purpose, we generated six different hinge-helix 1 fragments. Our experimental approach was to fuse these peptide fragments with the VP16 active domain (which contains 46 aa). Since the VP16 active domain has a nuclear localization signal, fragments are able to translocate to the nucleus efficiently with this domain, and the fusion with the VP16 active domain will prevent degradation of these small peptide fragments in the cells. In addition, evaluation of the expression of small peptide fragments by Western blotting analysis is difficult, but we could easily detect the fused proteins by immunostaining with anti-VP16 antibody. We cotransfected cells with pcDNA-CA-MEK5α, pcDNA-ERK5a, or pSG5-PPARγ1 with empty VP16 construct. Cell lysates were tested for the effects of six different hinge-helix 1 fragments on ERK5 coprecipitation, and we immunoprecipitated with rabbit IgG or anti-rabbit PPARγ antibody and immunoblotted with anti-ERK5 antibody. As shown in Fig. , ERK5 was coimmunoprecipitated by the anti-PPARγ antibody, but not by IgG. We found that cotransfection of the VP16-PPARγ1(aa 195-227) fragment, but not VP16 alone, significantly inhibited the coimmunoprecipitation of ERK5 with PPARγ. Among the tested fragments, the VP16-PPARγ1(aa 195-227) fragment had the most significant disrupting effect on the ERK5a-PPARγ1 interaction (data not shown). The expression levels of ERK5 and PPARγ were equal among the samples (Fig. , lower). Since the VP16-PPARγ1(aa 195-227) fragment is too small to be detected by Western blotting, we confirmed the expression of the VP16 and VP16-PPARγ1(aa 195-227) fragment by immunostaining with anti-VP16 antibody (data not shown). We did not see any inhibitory effect with this VP16-PPARγ1(aa 195-227) fragment on the MEK5-ERK5a interaction, also suggesting the specific inhibitory effect of this fragment on PPARγ-ERK5 association (data not shown). These data support our findings from the mammalian two-hybrid assay that the hinge-helix 1 region of PPARγ is critical for ERK5a-PPARγ interaction.
Next, to demonstrate the critical role of the ERK5-PPARγ1 interaction for PPARγ1 activity, we determined whether the VP16-PPARγ1(aa 195-227) fragment could inhibit CA-MEK5α-ciglitazone-induced PPARγ1 activation. As shown in Fig. , we found that VP16-fused PPARγ1(aa 195-227), but not VP16 alone or PPARγ1(aa 195-227) alone (data not shown), inhibited ciglitazone and/or CA-MEK5α-induced PPARγ1 activation. Of note, we did not observe significant inhibition of ciglitazone (alone)-induced PPARγ1 activation with the VP16-fused PPARγ1(aa 195-227) fragment, suggesting the specific effect of this fragment on the ERK5a-PPARγ interaction. To determine whether the inhibitory effect of VP16-PPARγ1(aa 195-227) is specific for CA-MEK5α-induced PPARγ1 activation, we investigated the effect of the VP16-PPARγ1(aa 195-227) fragment on CA-MEK5α-induced MEF2 activation. CA-MEK5α significantly induced MEF2 activation. In contrast to PPARγ1 activity, the VP16-PPARγ1(aa 195-227) fragment did not inhibit CA-MEK5α-induced MEF2 activation (Fig. ), suggesting the specific effect of VP16-PPARγ1(aa 195-227) on CA-MEK5α-induced PPARγ1 activation.
Flow enhances ciglitazone-induced PPARγ transcriptional activity via association of ERK5a kinase with the hinge-helix 1 region of PPARγ1.
In conduit arteries, steady laminar flow and physiological shear stress are atheroprotective, whereas turbulent flow and low shear stress are atherogenic (32
). Previously, our group found that 20 min of steady flow significantly increased ERK5 activation in ECs (34
Since PPARγ activation has been reported to be atheroprotective (19
), we studied the effect of flow on PPARγ transcriptional activity to determine the physiological relevance of the ERK5a-PPARγ1 interaction. We transfected (PPRE)3
-tk-luciferase, pSG5-PPARγ, and vector to provide equal amounts of transfected DNA in HUVECs. As shown in Fig. , flow (20 min) enhanced ciglitazone-induced PPARγ1 transcriptional activity (Fig. ), and cotransfection of the VP16-PPARγ1(aa 195-227) fragment significantly inhibited flow- and ciglitazone-induced PPARγ 1 transcriptional activity (Fig. ). To confirm the importance of ERK5, we utilized DN-MEK5β to inhibit ERK5 activation (5
). Of note, DN-MEK5β inhibits ERK5 kinase activation but does not change ERK5 expression, which is different from that with DN-ERK5 or the ERK5b construct (5
). As shown in Fig. , DN-MEK5β could not inhibit ciglitazone-induced PPARγ activation but significantly inhibited flow-enhanced ciglitazone-mediated PPARγ activation. Furthermore, we found that 9 h of flow and the combination of flow and ciglitazone significantly increased PPARγ1 transcriptional activity in HUVECs. We did not find any significant change in PPARγ expression induced by flow (data not shown). Cotransfection of the VP16-PPARγ1(aa 195-227) fragment significantly inhibited ciglitazone- and flow-induced PPARγ1 transcriptional activity (Fig. ), similar to the effects of 20 min of flow described in Fig. . In addition, 9 h of flow significantly increased ERK5 activation (2.0-fold [±0.4] increase; P
< 0.01), as assayed with the Gal4-ERK5a construct in the one-hybrid mammalian assay (Fig. ). These data show a critical role for ERK5a association with PPARγ1 in flow-regulated PPARγ1 transcriptional activity and support the physiological significance of ERK5a and PPARγ1 interaction in ECs.
FIG. 3. Flow-induced PPARγ1 transcriptional activation by the ERK5-PPARγ interaction and ERK5 activation. (A to C) Effect of short-term flow on PPARγ1 activity. At 24 h after transfection, growth-arrested HUVECs were stimulated by ciglitazone (more ...) ERK5 activation is critical for the inhibitory effect of flow on TNF-α-mediated NF-κB activation.
Previously, we and others found that both flow and PPARγ ligands inhibited TNF-α-mediated NF-κB activation (18
). To show a physiological role for ERK5/PPARγ activation by flow, we studied the role of ERK5 activation in the inhibitory effect of flow on TNF-α-mediated NF-κB activation. Since DN-MEK5β significantly inhibited flow (short-term flow) and PPARγ ligand-mediated PPARγ activation (Fig. ), we investigated whether DN-MEK5β could prevent the inhibitory effect of flow on TNF-α-induced NF-κB activation. As shown in Fig. , DN-MEK5β significantly inhibited the ability of flow (6 h) to decrease TNF-α-mediated NF-κB activation. These data suggest that the inhibitory effect of flow on NF-κB activation is, at least partially, due to the activation of ERK5 and subsequent PPARγ activation.
FIG. 4. Flow-inhibited TNF-α-mediated NF-κB activation and VCAM-1 expression by ERK5 and PPARγ activation. (A) HUVECs were transfected with pFR-Luc plasmid and pNF-kBLuc-plasmid. To control transfection efficiency, pRL-TK was transfected (more ...) ERK5 and PPARγ activation is critical for the inhibitory effect of flow on TNF-α-mediated VCAM-1 expression.
It is well documented that VCAM-1 and E-selectin expression is regulated by NF-κB activation (11
). Since we determined the role of ERK5 for the inhibitory effect of flow on TNF-α-mediated NF-κB activation (Fig. ), first we investigated whether flow can inhibit TNF-α-mediated VCAM-1 and E-selectin mRNA expression via activation of ERK5. We used BLMECs in these particular experiments, because BLMECs have high transfection efficiency, as our investigators have previously described (24
). As shown in Fig. (left), TNF-α induced VCAM-1 and E-selectin mRNA expression after 4 h of stimulation (lane 3), and flow significantly inhibited VCAM-1 and E-selectin mRNA induction (lane 5), as previously described (9
). We found that DN-MEK5β significantly blocked flow-mediated inhibition of TNF-α-mediated VCAM-1 and E-selectin mRNA expression (lane 6), suggesting a critical role for ERK5 activation in flow-mediated inhibition of VCAM-1 and E-selectin mRNA expression. To confirm that this mRNA regulation correlated with regulation at the protein level, we also determined whether flow could inhibit TNF-α-mediated VCAM-1 protein expression induction after 6 h of stimulation. As shown in Fig. (right), consistent with the mRNA expression data (left), we found that DN-MEK5β significantly inhibited flow-mediated inhibition of VCAM-1 protein expression (Fig. , right, lane 6).
Furthermore, to investigate the involvement of PPARγ activation in ERK5-mediated inhibition of VCAM-1 and E-selectin expression, we utilized a dominant negative form of PPARγ1 (DN-PPARγ1, L438A/E441A) and CA-MEK5α. As shown in Fig. (left), TNF-α increased VCAM-1 and E-selectin mRNA expression (lane 2), and CA-MEK5α (lane 4) and ciglitazone (lane 8) significantly inhibited TNF-α-mediated VCAM-1 and E-selectin mRNA expression. Transfection of DN-PPARγ1 significantly decreased the inhibitory effect of ERK5 activation on VCAM-1 and E-selectin mRNA expression (lane 5), suggesting that the inhibitory effect of ERK5 activation is due to activation of PPARγ. As shown in Fig. (right), consistent with the mRNA expression data (left), we also found that CA-MEK5α significantly inhibited TNF-α-mediated VCAM-1 protein expression (lane 4), and DN-PPARγ1 significantly recovered this inhibitory effect of ERK5 activation on VCAM-1 protein expression (Fig. , right, lane 5).
Hinge-helix 1 region is critical for PPARγ1 transcriptional activity via its regulation of the association of SMRT with PPARγ1.
To determine the role of hinge-helix 1 in regulation of PPARγ1 transcriptional activity (Fig. ), we generated a hinge-helix 1 deletion mutant in PPARγ-LBD(aa 162-475). To measure PPARγ-LBD transcriptional activity, we used a mammalian one-hybrid assay (Fig. ). HUVECs were cotransfected with Gal4-PPARγ-LBD(aa 162-475) or Gal4-PPARγ-LBDΔ202-231 and pG5-luc with or without CA-MEK5α or ERK5a, and PPARγ-LBD transcriptional activities in response to ciglitazone and ERK5 were measured. As shown in Fig. , CA-MEK5α/ERK5 significantly increased PPARγ1-LBD transcriptional activity, and deletion of hinge-helix 1 region from the LBD completely abolished PPARγ1-LBD transcriptional activity in ECs.
FIG. 5. Activated ERK5 disrupts the association of corepressor SMRT with PPARγ1. (A) Scheme of the hinge-helix 1 region of PPARγ1. (B) The hinge-helix 1 region of PPARγ1 is critical for CA-MEK5α and ciglitazone-induced PPARγ1-mediated (more ...)
Previous studies have demonstrated that PPARγ interaction with the corepressor SMRT inhibits its activation. Helices 3 to 5 and 12 in the LBD are important for interaction with corepressors (14
). Since we found that the hinge-helix 1 domain is involved in the ERK5-PPARγ1 interaction, we investigated whether the interaction of activated ERK5 and PPARγ1 alters binding of SMRT to PPARγ1. We determined the expression of nuclear corepressor (N-CoR1) and SMRT mRNA in HUVECs by RT-PCR (data not shown). A dominant negative form of SMRT increased full-length PPARγ1 activity, suggesting a functional role for SMRT in HUVECs (Fig. ). As shown in Fig. , two-hybrid analysis using Gal4-SMRT and VP16-wild-type PPARγ1-LBD(aa 173-475) indicated interaction of PPARγ1 and SMRT (lane 3), and ciglitazone inhibited this interaction (lane 4). Interestingly, cotransfection with ERK5a (lane 5), CA-MEK5α (lane 6), or ERK5a and CA-MEK5α (lane 7) significantly inhibited the interaction between corepressor SMRT and PPARγ1-LBD.
To confirm that ERK5 activation disrupted the SMRT-PPARγ interaction, we transfected Cos7 cells with CA-MEK5α. As shown in Fig. , we found that transfection of CA-MEK5α significantly inhibited the SMRT-PPARγ interaction, as measured by coimmunoprecipitation. When ERK5 is phosphorylated by CA-MEK5α, it can inhibit SMRT binding to PPARγ. Since the deletion mutant of hinge-helix 1 region in PPARγ1 had significantly reduced transcriptional activity, we examined whether the deletion of hinge-helix 1 interferes with the disruption of SMRT and PPARγ1 induced by ERK5 binding. As shown in Fig. , the deletion mutant of hinge-helix 1 domain of PPARγ1 (VP16-PPARγΔaa202-231) associated with Gal4-SMRT (lane 3). Although ciglitazone disrupted the interaction of PPARγ1 wild type and SMRT as described previously (Fig. ), the deletion of hinge-helix 1 domain abolished ciglitazone-induced disruption of PPARγ1 and SMRT. In addition, activation of ERK5 was required for full interaction of ERK5a and PPARγ1, as shown in Fig. , and we found that even this activated ERK5 could not interfere with the binding of SMRT and PPARγ1 (Fig. , lanes 5 to 7).
Finally, to determine whether this SMRT-PPARγ disruption induced by activation of ERK5 is specific, we determined the effect of activated ERK5 on the interaction of PPARγ1 and coactivator SRC-1. As previously reported (21
), the association of coactivator SRC-1 with PPARγ1 was induced by ciglitazone. In contrast to SMRT, we did not find any effect of CA-MEK5α/ERK5 on this interaction (data not shown), suggesting a specific effect of ERK5 on the PPARγ1 hinge-helix 1 region via binding of SMRT and PPARγ1.
PPARγ1 binding site of ERK5.
To clarify the role of PPARγ1 association with ERK5, we determined the binding site of PPARγ1 on ERK5. A plasmid expressing VP16-ERK5a was constructed by inserting the fragment of ERK5a into the VP16 active domain plasmid pact as measured by pG5-luc activity (Fig. ). We found that cotransfection of CA-MEK5α induced association with PPARγ1-LBD(aa 202-475). Dominant negative forms of ERK5 (dual phosphorylation site mutants [ERK5a T219A/Y221F] and ERK5b) exhibited reduced PPARγ1 association as measured with pG5-luc. However, a truncated mutant of ERK5(aa 1-418) did not associate with PPARγ1, suggesting that the COOH-terminal region of ERK5a is required for association with PPARγ1 (Fig. ). Since dominant negative forms of ERK5 partially reduced the ERK5-PPARγ1 association and the ERK5 with the COOH terminal deleted did not associate with PPARγ1, we speculated that autophosphorylation of ERK5 was required for ERK5-PPARγ1 interaction. Therefore, we mutated several putative autophosphorylation sites in the COOH-terminal region of ERK5 and determined the ERK5-PPARγ1 interaction. However, every ERK5 putative autophosphorylation site mutant that we examined (S433A, S697A, T723A, and S793A) associated with PPARγ1 (data not shown). These data suggest that autophosphorylation of the ERK5 COOH-terminal region may not be required for PPARγ1-ERK5 interaction.
FIG. 6. ERK5a binding site of PPARγ1. (A and B) Requirement of ERK5a kinase activity and the COOH-terminal region of ERK5 for the ERK5a-PPARγ1 interaction. (A) Cos7 cells were transfected with plasmids expressing wild-type Gal4-PPARγ1-LBD(aa (more ...)
We next generated several deletion mutants of ERK5 to define the domains required for PPARγ association. ERK5 deletion mutants were cloned into the VP16 active domain plasmid pACT, and the interaction with Gal4-PPARγ1-LBD was determined in a two-hybrid mammalian assay. As shown in Fig. , the deletion mutant ERK5a(aa 1-577), but not ERK5a(aa 1-418), associated with PPARγ1, suggesting that aa 419 to 577 contain the ERK5 binding domain for PPARγ1. To rule out the possibility of another binding site in COOH-terminal region, we also generated several small fragments of the COOH-terminal region of ERK5 and performed a two-hybrid mammalian assay. We found that ERK5 aa 412 to 806 associated with PPARγ1, but not ERK5 aa 571 to 806 and aa 684 to 806, suggesting that ERK5 aa 412 to 570 contains the only binding site of ERK5a for PPARγ1.
The COOH-terminal region of ERK5 has two transactivation domains.
Because ERK5 associates with PPARγ1 and enhances its activity, we investigated whether ERK5 itself exhibits any transcriptional activity, as reported previously (15
). To evaluate ERK5 as an activator of transcription, we determined its effect on the transcriptional activation of a reporter gene in a one-hybrid mammalian assay. For this purpose, Cos7 cells were cotransfected with Gal4-ERK5a and its mutants with or without CA-MEK5α, and the effect on basal transcriptional activity was determined. As shown in Fig. , ERK5a exhibited a very low transcriptional activity without CA-MEK5α transfection, but cotransfection of CA-MEK5α dramatically increased transcriptional activity. Dominant negative forms of ERK5 (DN-ERK5a and -ERK5b) significantly reduced transcriptional activity, suggesting that activated ERK5 is required for an active coactivator function. Analysis of COOH-terminal ERK5 deletion mutants showed that the coactivator function was associated with the COOH-terminal region of ERK5a (Fig. , lower).
FIG. 7. Transcriptional activation domains of ERK5a. (A) Cos7 cells were transfected with Gal4-dependent (Gal4-luc) reporter constructs with dominant negative forms of Gal4-DN-ERK5 or ERK5b (upper) and Gal4-ERK5 COOH-terminal deletion mutants (lower). Luciferase (more ...)
To identify the ERK5 transcriptional activation domain and the role of the NH2-terminal region, we also generated several COOH-terminal-truncated mutants of ERK5 and determined transcriptional activities in a mammalian one-hybrid assay. We found that the ERK5 COOH-terminal tail (aa 684 to 806) had very high transcriptional activity even without CA-MEK5α transfection (Fig. , upper). The middle region of ERK5 (aa 412 to 577) also had a small but significant transcriptional activity (Fig. , lower). These data suggest that the COOH-terminal ERK5 region has two transcriptional activator domains. Since full-length ERK5a required cotransfection of CA-MEK5α for full activation but the transcriptional activation domain fragments did not require CA-MEK5α cotransfection, our results suggest that NH2-terminal ERK5 may act as a negative regulator of these transactivation domains.
The COOH terminus of ERK5 transactivation domains is required for full activation of PPARγ1.
To determine the role of the two ERK5 transactivation domains on PPARγ1 activation, we generated several VP16-fused COOH-terminal ERK5 deletion mutants. As shown in Fig. , CA-MEK5α induced PPARγ1 activity in HUVECs, and cotransfection of wild-type VP16-ERK5a enhanced its activity. Progressive deletion of COOH-terminal ERK5a gradually reduced ciglitazone- and CA-MEK5α-induced PPARγ1 activity. Complete deletion of the COOH-terminal region of ERK5 (ERK5[aa 1-418]) totally abolished the enhancing effect of ciglitazone and CA-MEK5α/ERK5a on PPARγ1 activation. Furthermore, as shown in Fig. , we found that the entire COOH-terminal ERK5 (aa 412 to 806) and middle region of ERK5 (aa 412 to 577), which contains both the PPARγ1 binding site and transactivation domain, enhanced ciglitazone-induced PPARγ1 activation. However, the transactivation domain at the COOH-terminal tail of ERK5 (aa 684 to 806) alone could not induce PPARγ activity, suggesting a critical role for the middle region of ERK5 (aa 412 to 577) as a binding site of ERK5a with PPARγ1.
FIG. 8. Both transcriptional domains and the PPARγ1 binding site in the COOH-terminal region of ERK5 are critical to fully activate PPARγ1. (A and B) Transfection medium contained 1 μg of (PPRE)3-tk-luciferase, 0.5 μg of pSG5-PPARγ1, (more ...)