We have established an in vitro assay system in which the regulation of stress-activated MAP3Ks can be assayed. Our results point to a comparatively simple model for the regulation of MEKK1 by elements coupled to the TNFRs. In this model, GCK and MEKK1 exist as an inactive complex in resting cells, and within this complex MEKK1 is either monomeric or at a stoichiometry inconsistent with activation. Recruitment to the membrane by upstream activators such as TRAF2 creates a higher-order aggregate that activates GCK and alters the conformation of the components in the complex, forcing MEKK1 into an oligomeric state that fosters its activation. Inasmuch as autophosphorylation within the kinase activation loop at T1381 and T1393 is required for MEKK1 activity, and forced oligomerization or coincubation with GCK enhances MEKK1 autophosphorylation, it is reasonable to hypothesize that autophosphorylation at T1381 and T1393 is a consequence of MEKK1 oligomerization and a prerequisite for activation. This model is illustrated in Fig. . In an overexpression context, GCK spontaneously oligomerizes and activates, resulting in oligomerization and activation of any associated MEKK1 or other immediate downstream components.
Model for TNF and agonist activation of MEKK1 by GCK and TRAF2. Details are discussed in the text. P, phosphorylation of the MEKK1 polypeptide.
The position of some GCK group kinases as effectors for TRAFs is becoming clear. Thus, GCK-related enzyme (GCKR), which is closely similar to GCK, also binds TRAF2 and is activated in vivo by TRAF2 (33
). Moreover, two hybrid screens that employed the Drosophila
GCK group kinase Misshapen identified a Drosophila
TRAF as a Misshapen interactor (25
). Inasmuch as purified TRAF2 can activate MEKK1 in vivo and in vitro in the absence of GCK, it is possible that TRAF2 can activate MEKK1 in vivo either indirectly through GCK or directly, independently of GCK. It will be important to determine the contribution of this direct MEKK1 activation relative to indirect activation. Although TRAF2 and GCK interact in vivo, we have thus far been unable to generate assay conditions in which activation of GCK by TRAF2 can be detected in vitro. Further work will be necessary to establish whether TRAF2 can directly activate GCK, stimulating its ability to activate MEKK1. It is also likely that stimuli other than TRAF2 could recruit the GCK-MEKK1 complex and trigger MEKK1 activation. Of note, the structure of the GCK CTD includes several putative SH3 binding motifs (aa 428 to 434 and 462 to 466) (15
) suggesting that GCK may be regulated by SH3 containing adapter proteins.
In support of the model in Fig. , we find that endogenous GCK and MEKK1 exist as a preformed complex in cells, and recombinant TRAF2 and GCK (2
; this study) can bind and activate MEKK1 in vivo. MEKK1 activation by TRAF2 or GCK is direct inasmuch as this activation can be reproduced in vitro with purified proteins. In vivo and in vitro, GCK activation of MEKK1 coincides with an increase in MEKK1 phosphate incorporation that is essentially indistinguishable from that incurred upon autophosphorylation in vitro. Kinase-inactive mutants of MEKK1 do not undergo appreciable phosphorylation upon incubation with GCK in vitro; however, incubation of wild-type MEKK1 with GCK, catalytically inactive GCK(K44M), or the free GCK CTD in vitro stimulates MEKK1 activation. Thus, GCK’s kinase function is dispensable for MEKK1 activation. T1381 and T1393, residues in the MEKK1 kinase domain activation loop, undergo autophosphorylation in vitro and in vivo, when truncated constitutively active MEKK1 constructs are assayed (4
). We find that for full-length MEKK1, stimulation of autophosphorylation and activation in vitro by GCK is drastically reduced in T1381A or T1393A MEKK1, suggesting that autophosphorylation at these residues is a prerequisite for MEKK1 activation. It should be noted, however, that while MEKK1 activation is always accompanied by an increase in autophosphorylation (Fig. to ), there is not always a 1:1 correspondence between the degree of MEKK1 autophosphorylation and the degree of activation by GCK of MEKK1’s SEK1 kinase activity. Examination of the tryptic phosphopeptide maps of MEKK1 phosphorylated in vivo and in vitro reveal a highly complex pattern of phosphopeptides. Although it is evident that T1381 and T1393 are important for maintenance of MEKK1 activity, it is likely that MEKK1 undergoes autophosphorylation at many sites, a substantial portion of which may be trivial, undergoing phosphorylation in a manner that does not correlate with MEKK1 activity. These trivial autophosphorylation events may obscure more pronounced changes in MEKK1 autophosphorylation at sites such as T1381 and T1393 that are relevant to activation.
Consistent with previous results indicating that TRAF2 oligomerization can activate SAPK/JNK (2
), we find that oligomerization of MEKK1 also activates SAPK/JNK and that coexpression with MEKK1 of GCK, which is constitutively active when overexpressed, increases MEKK1 oligomerization in vivo. Finally, GCK’s effectors are not limited to MEKK1. We also observe that GCK can bind MLK3 in vivo and activate MLK3 in vitro.
GCK is a member of the STE20
family of protein kinases (20
). An emerging body of evidence indicates that Ste20p-like kinases and other upstream components activate MAP3Ks either by direct phosphorylation or by fostering MAP3K activating autophosphorylation. In the mating pheromone pathway of S. cerevisiae
, Ste20p contributes to activation of the MAP3K Ste11p by direct phosphorylation at S302 and/or Ser306 within the Ste11p kinase activation loop, residues that correspond to T1381 and T1393 of MEKK1 (39
). In mammals, p21-activated kinases phosphorylate the mitogenic MAP3K Raf1 at S338/339 at the amino-terminal end of the kinase domain (17
). However, although MEKK1 requires regulatory phosphorylation for activation in response to an upstream signal from GCK, this phosphorylation is self catalyzed and the kinase activity of GCK is dispensable for MEKK1 activation. This finding is similar to results for the transforming growth factor β- and interleukin 1-activated MAP3K transforming growth factor β-activated kinase-1 (TAK1) and for the MLK family MAP3K dual leucine zipper kinase (DLK) (18
). Upon ectopic expression with its activating subunit TAK1 binding protein 1, TAK1 is activated in vivo by autophosphorylation at S192 within the kinase activation loop. Dimerization of DLK, mediated by the DLK leucine zipper, also coincides with autophosphorylation within the kinase domain activation loop. This autophosphorylation appears to be necessary for activation (28
As mentioned above, MEKK1 autophosphorylation is complex and does not tightly correlate with the degree of MEKK1 activation. We do not know if additional (auto)phosphorylation events are critical to MEKK1 activation. MEKK1 binds proteins of the 14-3-3 family (14-3-3
). Binding of 14-3-3 proteins has been mapped to the amino-terminal 393 aa of MEKK1 (9
). 14-3-3 proteins bind to the consensus sequence R-S-X-Sp-X-P (X = any amino acid, Sp = phosphoserine) (27
). S242 of MEKK1 lies within a rough consensus 14-3-3 binding motif—S242 is, in fact, the only residue in the MEKK1 polypeptide that lies within a potential 14-3-3 binding motif. Mutagenesis of S242 to Ala has no effect on MEKK1 activity, GCK binding, or regulation (data not shown). Thus, either 14-3-3 proteins bind to structural motifs on MEKK1 yet to be identified, or 14-3-3 proteins do not regulate MEKK1 catalytic activity but may instead regulate MEKK1 localization, turnover, etc.
Oligomerization is emerging as a theme in MAP3K regulation. Thus, ASK1, Raf1, and the MLKs are activated, in part, by oligomerization (10
). Our findings (Fig. ) suggest that active MEKK1 exists as an oligomer and that forced oligomerization of MEKK1 is sufficient to engender activation in vivo and in vitro. We find that GCK enhances MEKK1 oligomerization, as determined by coimmunoprecipitation of heterologously tagged MEKK1 constructs and by sucrose density gradient centrifugation. MEKK1 oligomerization may relieve autoinhibition mediated by the MEKK1 amino-terminal domain insofar as full-length MEKK1, when expressed transiently, is less active in vivo than the free MEKK1 catalytic domain (42
). Oligomerization also increases MEKK1 phosphorylation in vivo; thus, oligomerization may trigger MEKK1, activating autophosphorylation.
Given that the kinase domain of GCK is dispensable for MEKK1 activation, what is the function of the GCK kinase domain? Data from our previous studies (48
) indicate that, in vivo, wild-type GCK is the most potent activator of coexpressed SAPK/JNK, while kinase-dead GCK(K44M) and the free GCK CTD are comparatively modest activators of coexpressed SAPK/JNK (29
). By contrast, the free GCK CTD interacts with MEKK1 more stably than wild-type GCK or GCK(K44M) (48
), supporting the idea that activation of GCK’s kinase activity enables the GCK CTD to trigger MEKK1 oligomerization, autophosphorylation, and activation, as well as, possibly, turnover of activated MEKK1. On the other hand, we find (Fig. and ) that in vitro GCK, GCK(K44M), and the free GCK CTD activate MEKK1 to comparable extents, highlighting the lack of a role for the GCK kinase domain in the actual process of activation of GCK effectors but not ruling out the possibility that activation of GCK may regulate access to GCK effectors. We also observe that endogenous GCK and MEKK1 interact constitutively in cells (Fig. ). It is possible that GCK’s kinase function is evolutionarily vestigial. However, our in vitro studies employed GCK from transfected cells, conditions under which even catalytically inactive forms of the enzyme may display substantial basal activity. Thus, while our data clearly indicate that GCK’s kinase activity is not necessary for activation of MEKK1, further studies will be necessary to establish if the ability of GCK to trigger MEKK1 autoactivation can be linked to activation GCK’s kinase function.
The comparatively simple mechanism of MEKK1 activation described herein belies the size and complexity of the MEKK1 polypeptide. MEKK1 possesses plekstrin homology domains (aa 439 to 455 and 643 to 750) and proline-rich motifs (aa 74 to 149 and 233 to 291) that may mediate binding to proteins that possess SH3 domains (23
). Accordingly, MEKK1 is probably regulated by multiple upstream signals, in addition to those from TRAFs and GCKs, and MEKK1 may, in turn, have diverse functions over and above activation of the SAPKs/JNKs. Consistent with these ideas, MEKK1 interacts with Ras superfamily GTPases (Ras, Rac, and Cdc42) in a GTP-dependent manner. These interactions are mediated by the MEKK1 kinase domain (8
). In addition, the extreme amino terminus of MEKK1 binds SAPKs/JNKs and Raf-1 (aa 30 to 221 and 221 to 370, respectively) (13
). A small segment of the MEKK1 amino-terminal domain (aa 30 to 200) also associates with the HTLV1 Tax protein, coupling Tax to activation of NF-κB (47
). Thus, MEKK1 may perform a scaffold function regulating or at least nucleating additional pathways, besides the SAPK/JNK pathway, including NF-κB and, through Raf-1, the ERK pathway. GCK, on the other hand, is apparently a quite selective regulator of the SAPKs/JNKs and does not activate ERK or NF-κB (20
). It will be important to determine if, in addition to activating MEKK1, GCK sequesters MEKK1 from other signaling pathways, directing it to specific activation of SAPK/JNK.
With regard to the putative SH3 binding domains in the GCK CTD, it is noteworthy that MLK3, which contains an SH3 domain, is also activated by GCK but not by TRAF2 (at least not directly, as is the case for MEKK1). It is plausible to speculate that the GCK-MLK3 interaction involves one or more of GCK’s SH3 binding domains and the SH3 domain of MLK3. In support of this idea, the MLK3 SH3 domain interacts with the carboxyl-terminal two of four polyproline SH3 binding motifs in the GCK group kinase HPK (16
), and a similar interaction may mediate the observed association between MLK3 and another GCK group kinase, GCKR (40
). The structures of MLK3 and other members of the MLK family are also suggestive of complex regulation and function. Accordingly, MLK3 can associate in vivo with the SAPK/JNK scaffold proteins JNK-interacting protein 1 (JIP-1) and JIP-2 through the JIP SH3 domains. This interaction may serve to nucleate a signaling complex containing MKK7 and SAPK/JNK (5
). The interaction between GCKR and MLK3 through the MLK3 SH3 domain allows the incorporation of GCKR into the MLK3-MKK7-SAPK/JNK-JIP complex (40
). It will be important to determine if the binding of MLK3 to JIP proteins or other scaffolds also directs GCK-mediated activation of MLK3 by upstream components other than TRAFs.
Thus, GCK may integrate multiple signaling pathways to recruit and activate at least two MAP3Ks, likely through induced proximity and/or oligomerization-dependent autophosphorylation. In addition, TRAF2 can directly activate MEKK1, probably in a similar manner (Fig. ). Future work will identify the pathways that couple GCK to MLK3 as well as the kinetics and relative contributions of GCK-dependent and -independent activation of MEKK1 and the SAPKs by TNF.