In this study, we have investigated the sensitivity of Mxi2 to mitogens. We report that Mxi2 immunoprecipitates harbor a kinase activity that is stimulated by EGF. Compared to ERK activation, this kinase activity is weaker in magnitude but is more sustained in time, as 40% of its intensity remains 30 min after stimulation. On the other hand, we show that activation of p38 by EGF is minimal, in agreement with previous results (33
). p38 can be activated by other mitogenic stimuli, like fibroblast growth factor (26
), and plays a relevant role in diverse mitogenic processes, such as myocyte hypertrophy (44
) and myeloid (13
) and T-cell (6
) proliferation. Thus, the existence of a p38 isoform with a marked function under mitogenic stimulation would be in line with the role of p38 in some proliferative situations.
Focusing on the components of the cascade that conveys the mitogenic signal to Mxi2, we show that the EGF-induced activity detected in Mxi2 precipitates is regulated mainly by Ras and not by Rho, Rac, or Cdc42. Although Rac can activate Mxi2, the fact that Mxi2 stimulation by EGF is unaffected by a Rac inhibitory mutant form suggests that Rac may have a role in Mxi2 activation, probably by stress stimuli, by analogy with other p38 members. On the other hand, Ras can mediate the activation of p38 by diverse mitogenic (4
) and stress (31
) stimuli. The fact that, under some circumstances, p38 can be activated by Ras supports the notion of an isoform specially responsive to Ras-mediated signals. While the mechanisms whereby Ras activates p38 remain elusive, in the case of the kinase activity present in Mxi2 immunoprecipitates, we show that it is activated through the canonical ERK pathway comprising Ras, Raf, and MEK.
It is known that p38 isoforms are mainly activated by MKK3, MKK6, and MKK4 (32
). The kinase activity harbored in Mxi2 is responsive to these three MAPKKs, resembling the behavior of p38. In addition, our results indicate that Mxi2 can be activated by MEK1 and -2. However, our data obtained with the inhibitory mutant forms of these MAPKKs indicate that the stimulation of Mxi2 by EGF is essentially conveyed through MEK, although an MKK6-regulated minor component is also apparent. This may indicate that MKK3, -4 and -6 mediate the activation of Mxi2 by the stress-stimulated p38 MAPK module. In light of these results, it could be anticipated that MEK should be the kinase responsible for phosphorylating Mxi2. However, we show that MEK does not coimmunoprecipitate with Mxi2. Even though stable association between MEK and its potential substrates may not be an essential requisite, since ERK mutant forms defective for MEK binding can still be phosphorylated by it (9
), this result speaks against the idea that Mxi2 is a good substrate for MEK. This concept is strengthened by our data showing that MEK1 failed to phosphorylate Mxi2 in vitro. On the other hand, in our 32
P labeling experiments, phosphorylated Mxi2 appeared in response to EGF and MEK1 stimulation. In the case of EGF, this could be attributed mainly to the MKK6 component, which is capable of phosphorylating Mxi2 in vitro. The fact that MEK1 can also phosphorylate Mxi2 opens the possibility that, in vivo, some unidentified scaffold protein could make low-affinity phosphorylation of Mxi2 possible. This situation would not be unprecedented, as MEK can activate p38 under certain circumstances (4
). In any case, the levels of Mxi2 phosphorylation induced by MEK are 10-fold weaker than those detected on the phosphoprotein coimmunoprecipitating with Mxi2, which we have identified as ERK1/2, further demonstrating that Mxi2 is a poor substrate for MEK.
We make evident that ERK1 and -2 can directly associate with Mxi2. Importantly, we have found endogenous Mxi2 and ERKs to coimmunoprecipitate in a physiologically relevant environment like the kidney. This situation is reflected by ectopic expression, as we have detected ERK1/2 in Mxi2 immunoprecipitates and vice versa. Furthermore, ERK-Mxi2 interaction is independent of the activation status of ERKs, since it is equally detected under quiescent and EGF or MEK-stimulated conditions. This association does not imply transphosphorylation between the two kinases, as we demonstrate that, in vitro, Mxi2 is not a substrate for ERK2. Likewise, Mxi2 cannot catalyze phosphotransfer onto ERK2 (our unpublished results). The interaction with Mxi2 appears to be specific for ERKs, as neither JNK nor p38 coimmunoprecipitated with Mxi2. Moreover, our results suggest that Mxi2 binds to ERKs with more efficiency than its splice isoform p38. In our experimental setting, we have not detected any ERKs coimmunoprecipitating with p38. This may seem to contradict a recent report in which an association between p38 and ERK1/2 was shown (50
). However, by using the experimental conditions used in that study, a low-stringency lysis buffer, we have been able to reproduce the reported association between p38 and ERKs (unpublished results). This could indicate that the interaction between Mxi2 and ERKs is of a greater affinity than that between p38 and ERKs. Indeed, our in vitro binding experiments demonstrate that Mxi2 binds to ERK2 with fourfold greater affinity than does p38, thus implying that the determinant(s) for high-affinity binding must reside within the unique C terminus of Mxi2. In agreement, we show that deletion or distortion of the C terminus of Mxi2 severely compromises the ability of Mxi2 to bind to ERKs. In this respect, it is noteworthy that the C terminus of Mxi2 exhibits 46% homology to residues 292 to 304 of ERK2 (49
). A detailed study of the determinant(s) within the C terminus of Mxi2 responsible for binding to ERKs is under investigation.
Having Mxi2 and ERK in association raises the question of which of the two kinases responds to which stimuli. Since MEK cannot efficiently phosphorylate Mxi2, it is likely that the kinase activity induced by mitogenic stimuli, driven through the Ras-Raf-MEK pathway, is mainly due to ERK function and not to Mxi2. On the other hand, stimuli such as anisomycin and the components of stress-stimulated MAPK modules would preferentially induce Mxi2 activity but not ERKs. Indeed, in vitro, we have shown MKK6 to potently phosphorylate Mxi2. In support of this assumption, our findings from in-gel kinase assays indicate that stimulation with MEK results in myelin basic protein (MBP) phosphorylation at the level of ERK1/2 but none colocalizing with Mxi2. Conversely, upon stimulation with MKK6, phosphorylated MBP is detected comigrating with Mxi2 and not with ERKs (our unpublished results).
We report that interaction of Mxi2 with ERK1/2 has profound consequences for ERK activation. Mxi2 potentiates ERK2 stimulation brought about by activated MEK, but this effect cannot be explained by an augmentation of ERK phosphorylation levels, as Mxi2 does not significantly affect the phosphorylation of ERKs upon stimulation with EGF. On the other hand, it is in agreement with our results indicating that Mxi2 down-regulates the ERK inactivation rate, sustaining the ERK phosphorylated state for >5 h, an effect consistent with the long-lasting kinase activity detected in Mxi2 immunoprecipitates after EGF stimulation that, in light of our results, should be attributed to the function of the associated ERKs. Rather than fixing ERKs in a perpetually phosphorylated state, constitutively active MEK maintains ERK in constant activity by keeping it in a continuous phosphorylation-dephosphorylation cycle. Thus, it is conceivable that a reduction of the rate of the dephosphorylation process, brought about by Mxi2, results in an overall enhancement of ERK activation. Interestingly, we have found that the effect of Mxi2 on ERK dephosphorylation is independent of its kinase activity, as kinase-inactive Mxi2 is identically capable of protecting against ERK deactivation. Moreover, we show that binding of Mxi2 to ERKs is critical to the mechanism by which ERK phosphorylation levels are sustained. Furthermore, this effect is isoform specific, since p38 fails to counteract ERK dephosphorylation. Once again, this may reflect the lower binding affinity of p38 for ERKs.
It could be hypothesized that to exert its effect against ERK dephosphorylation, Mxi2 would interfere with the phosphatase(s) responsible for deactivating ERKs. Binding of Mxi2 to ERKs could pose a steric hindrance, impairing ERK recognition by phosphatases. Since Mxi2 does not affect the phosphorylation of ERKs by MEK or the interaction of ERKs with its substrate Elk1, this would eliminate Mxi2 interference with the binding to a docking domain common to all ERK-interacting proteins, like the CD domain (42
). Thus, Mxi2 could impede contacts specific for ERK-phosphatases interactions. In this respect, we demonstrate that Mxi2 enhances ERK2 basal activity, something that speaks in favor of Mxi2 interfering with the dephosphorylation processes that function to maintain ERKs in an inactive state, even under conditions of minimal stimulation. A second possibility is based on our observation that MEK is not phosphorylated by Mxi2 immunoprecipitates, despite the presence of active ERKs. It is conceivable that Mxi2 could somehow interfere with MEK retrophosphorylation by ERKs, something that would hinder the negative feedback control of MEK exerted by ERKs (2
), resulting in prolonged MEK activity. These two scenarios are not mutually exclusive and could both contribute to some extent to the maintenance of ERK2 phosphorylation levels, something that is currently under investigation.
Mxi2 binding to ERKs has remarkable consequences for ERK functions. We demonstrate that Mxi2 dramatically up-regulates the transactivation of an Elk1-responsive gene induced by MEK and by EGF. This outcome is independent of Mxi2 kinase activity, as Mxi2 K52R has identical effects, consistent with our results described above. Furthermore, the effect of Mxi2 on Elk1 transactivation is mediated through the ERK pathway and is entirely dependent on ERK being functional. Mxi2 fails to significantly activate Elk1 transcriptional activity when acting alone or in combination with MKK6. In agreement, our previous results demonstrate that Elk1 is a poor substrate for Mxi2 (39
). Moreover, an ERK2 inhibitory mutant form completely blocks the synergistic stimulation of Elk1 by MEK-Mxi2. The enhanced Elk1-dependent transcription cannot be primarily due to an incremental increase in the levels of phosphorylated Elk1, as we show that effects of Mxi2 on Elk1 phosphorylation induced by MEK or EGF are, at most, small. Thus, the most likely explanation is that the sustained activity of ERKs caused by Mxi2 results in a more persistent activation of Elk1. In fact, we demonstrate that, upon stimulation with EGF, the time interval in which Elk1 is found in a phosphorylated state is significantly prolonged in the presence of Mxi2. We also demonstrate that a synergistic up-regulation of its transcriptional activity is not a unique response of Elk1 to Mxi2, since another ERK-responsive transcription factor, HIF1α, responds in an identical fashion to the collective action of Mxi2 and MEK. Surprisingly, the potentiating effect of Mxi2 on ERK functions is restricted to nuclear events such as the activation of transcription factors. In line with this, we demonstrate that the presence of Mxi2 has no consequences for ERK-mediated cytoplasmic episodes like the activation of RSK2 or of cPLA2
induced by both MEK and EGF. This is consistent with our observations, to be reported elsewhere, that a considerable proportion of Mxi2 has a nuclear localization.
In summary, our results identify an isoform with unique functions among p38 MAPKs. It is well documented that ERKs and p38 MAPKs have opposing effects in diverse biological (32
) and biochemical (50
) processes. By contrast, we demonstrate that Mxi2 is capable of potentiating ERK functions. More interestingly, the effects of Mxi2 are site specific, apparently restricted to nuclear events. In light of our findings, it is conceivable that Mxi2, by enhancing ERK functions in the nucleus, could alter the relative contributions of the signals generated by ERK at the different cellular sites. Furthermore, it is well known that sustained versus transient ERK signals have completely different biochemical and biological responses (27
). Thus, it can be anticipated that, as a result of its ability to prolong the activation of ERKs in the nucleus, Mxi2 could cause profound changes in ERK-mediated processes.
The biological results of stimuli that switch on multiple MAPK pathways are a consequence of the integration of the signals transduced by each individual route. One way to fine tune such a complex process is through regulatory cross talk between the different MAPK modules. Many such interactions have been described thus far, most of which take place among components upstream of MAPKs (32
). Herein, we describe a novel type of regulatory cross talk that occurs through the direct interaction between two MAPKs. A similar interaction has been previously described for p38 (50
), albeit with opposing effects. It is intriguing how two almost identical molecules can exert antagonistic effects upon binding to ERKs. One possible explanation is that p38 binds to ERKs in such a way that it sterically blocks ERK phosphorylation by MEK1, as discussed by Zhang et al. (50
). On the other hand, binding of the unique high-affinity C terminus of Mxi2 could reposition the bulk of the molecule so that it no longer hinders MEK but instead impedes ERK interaction with phosphatases. Since Mxi2 and p38 are splice variants of the same gene, this opens an attractive scenario in which, by switching the splicing taking place at the p38 locus, the signaling machinery could be programmed to up- or down-regulate ERK activity, with its subsequent alterations of the biological outcomes resulting from ERK activation, an exciting hypothesis that merits some attention.