This report provides evidence for a novel regulatory mechanism of a MAP3K, i.e., Cot, which is modulated by its phosphorylation by the PIP3-regulated kinase Akt. Our studies with cells with or without the negative regulator PTEN indicate that the PI3K/Akt pathway is important for NF-κB induction by Cot. Furthermore, Akt and Cot can physically associate through the amino terminus of Cot. Akt can also mediate the phosphorylation of Cot, as demonstrated both in vitro and in intact cells. Although two sites of serine phosphorylation in Cot were mapped, only one of these sites appears to be critical for Cot induction of NF-κB-dependent transcription. However, neither site was required for Cot induction of AP-1 or NFAT, consistent with the dispensability of the carboxy terminus for activating these reporters. Our data provide evidence for a pool of Akt complexed with Cot, probably maintained in the cytoplasm, since Akt lacking a PH domain can still associate with Cot (Fig. and data not shown). Upon PI3K activation this complex is likely recruited to the membrane through the Akt PH domain, which binds the phospholipid PIP3. Akt is then activated by the kinase PDK-1 and perhaps another intermediate kinase (data not shown). This in turn allows Akt to phosphorylate Cot at two sites in the carboxy-terminal domain, at least one of which may promote binding of substrates or coactivators to Cot, or alternatively may relieve binding of a negative regulator. Strikingly, this mechanism for regulation of Cot does not affect Cot kinase activity or induction of AP-1-dependent transcription by Cot. What it may regulate, however, is release of Cot from the IKK complex, allowing each Cot molecule to serially activate many such complexes (not depicted in the model shown in Fig. ).
Model for regulation of Cot by Akt. See the text for details.
In total our data demonstrate that Akt and Cot can induce NF-κB-dependent transcription through activation of the IKK complex and subsequent nuclear translocation of NF-κB factors, such as p65/RelA (16
) (Fig. and ). Recently it has been suggested that the major effect of Akt in this pathway occurs through phosphorylation-dependent increases in the transactivation potential of p65 (24
). We have investigated whether this may occur in T cells, but we have been unable to detect Akt-mediated increases in p65 transactivation, as measured with a p65/gal4 transcriptional reporter (L. P. Kane and A. Weiss, unpublished data). This discrepancy may be due to cell type differences in the coupling of upstream signaling pathways to NF-κB. In addition, we have noted that a rapidly inducible, estrogen-regulated Akt fusion protein is capable of upregulating NF-κB-dependent transcription in conjunction with PMA (L. P. Kane and A. Weiss, unpublished data). This is also consistent with a relatively direct effect on the NF-κB nuclear translocation pathway, as discussed above.
Two sites of apparent Akt-dependent serine phosphorylation in the C-terminal tail of Cot were identified in this study (Fig. ). Phosphorylation of these sites is probably not carried out in a cooperative manner, as mutation of either one only partially decreased phosphorylation of Cot in vitro or in intact cells (Fig. and data not shown). Only one of these sites (S413) conforms to the established consensus for phosphorylation by Akt (1
). The sequence around the S400 site (QPRCQSL) partially fulfills the Akt consensus phosphorylation site (RXRXXS/TΦ). It is possible but unlikely that the high concentrations of proteins used in the in vitro and intact cell experiments resulted in spurious phosphorylation of S400 by Akt. It also seems unlikely that an associated kinase was carrying out this phosphorylation, as use of a kinase-inactive Akt resulted in no increase in Cot phosphorylation (Fig. and ). However, it is formally possible that Akt itself activates another kinase, which then carries out the phosphorylation of S400. Another possibility is that binding to Cot somehow alters the specificity of Akt, allowing it to directly phosphorylate this site. Regardless of the precise mechanism, either perturbation of the Akt pathway (Fig. ) or mutation of S400 (Fig. ) has profound consequences for Cot induction of NF-κB.
Regulatory phosphorylation of kinases is a common theme. Phosphorylation by Akt negatively regulates other kinases, such as Raf and GSK3 (8
), by allowing complex formation with 14-3-3 proteins, which sequester the kinases. Src family tyrosine kinases are both positively and negatively regulated by phosphorylation, as is the ZAP-70 tyrosine kinase (15
). These tyrosine kinases appear to be regulated at the level of protein tertiary structure, which controls access to the catalytic domain. These models probably do not account for the importance of serine 400 in Cot, which is dispensable for catalytic activity and some downstream effects. Other molecules regulated by serine phosphorylation include the tyrosine kinase Btk (17
) and the transcription factor STAT1 (20
). In the former case, serine phosphorylation negatively regulates the kinase, possibly at the level of membrane recruitment (17
). The case of STAT1 regulation is more similar to the effects noted here with Cot, in that only a subset of downstream target genes are affected by mutation of S727 in STAT1 (20
). This specificity may be achieved through serine-dependent interactions with gene-specific coactivators (31
). Such a protein-protein interaction model is appealing for the function of S400 in Cot and will be tested.
Although Cot may affect NF-κB coactivators, it is clear that there is a role for S400 in the activation of the IKK complex and nuclear translocation of p65. The precise mechanism by which phosphorylation at S400 influences Cot activation of the IKK complex is not yet clear. We propose that efficient activation of the IKK complex by Cot requires serial interaction of Cot with multiple complexes. As Cot interacts with and activates each complex, phosphorylation at S400 by Akt may cause the release of Cot, followed by dephosphorylation and association with a different IKK complex. Cot mutated at S400 may therefore be unable to efficiently disengage from complexes with which it is associated. How would this type of mechanism operate even in a system where Cot is overexpressed, as it is in these experiments? While we noted no effect of the S400 mutation on total Cot kinase activity (Fig. ), it remains possible that local activation of Cot, perhaps by another protein associated with the IKK complex, is affected by this mutation. In this regard, our results are consistent with those of Lin et al. (22
), who noted that Cot appears not to associate directly with the IKK complex, as demonstrated by the inability of Cot SA(2) to inhibit NIK-induced IKK activation (Fig. ). Furthermore, NF-κB induction by some inducers (CD3/CD28) is affected more than others (TNF) by this form of Cot. We have investigated whether the mutation in S400 affects the association of Cot with NIK but have not detected any difference in the efficiency of this association (data not shown). Thus, additional proteins may be required for the effects of Cot on the IKK complex.
Cot/Tpl-2 is capable of activating classical NF-κB transcription and the NF-κB-like RE/AP element in the IL-2 promoter, both of which are targets of TCR/CD28 signal integration. Our results with the S400 mutation in Cot also suggest that Cot participates in the TCR/CD28, but not the TNF, pathway leading to NF-κB-dependent transcription. However, there are discrepancies regarding the mechanism by which this occurs. Lin et al. provided evidence that the IKK complex is targeted by Cot, possibly through the intermediate kinase NIK (22
). Consistent with this evidence is the fact that T cells from aly
mice, which express a mutant NIK protein, have a cell-autonomous defect in IL-2 production (41
). Belich et al. demonstrated an interaction of Tpl-2 with p105, while these investigators failed to see an effect on IκB degradation (5
). The requirement for phosphorylation of the cytoplasmic tail of Cot that we have uncovered seems unlikely to regulate interactions with p105, since Belich et al. were able to observe interactions between Tpl-2 and p105 even when the proteins were translated in vitro. It should be pointed out that although IKK activation is thought to lie upstream of RE/AP induction, this pathway might differ from classical NF-κB-dependent transcription (18
). Previous studies are consistent with the RE/AP element being regulated by c-Rel rather than the classical RelA/p50 heterodimer (40
). Clearly, the pathway lying between activation of Akt and Cot and inducible transcription regulated by the RE/AP element requires further investigation.
Recently the relevance of Cot for NF-κB induction has been called into question by the analysis of Cot-deficient mice (9
). Although macrophages from these mice display decreased TNF-α production, no defect in NF-κB gel shift activity was seen, either in macrophages or in T cells. What could account for this discrepancy with the cell line experiments? It is likely that this is the result of redundancy with other MAP3Ks, which comprise a rather large family of homologous kinases (38
). The kinase domain of Cot is highly related to those of other members of this family, especially MEKK1 and ASK1 (MEKK5). Indeed, recent studies indicate that Akt can complex with and phosphorylate ASK1 (19
). At least two other members of the MAP3K family, MEKK1 and MLK3, have been implicated in NF-κB induction associated with T-cell activation (12
). Intriguingly, although MEKK1 to -3 are all capable of inducing NF-κB in HeLa cells, MLK3 is not (50
), suggesting that there is cell type specificity in the pathways activated by various MAP3Ks. Finally, recent genetic data from mice and flies demonstrate that at least two members of the MAP3K family, MEKK3 and TAK1, are required for induction of NF-κB by certain stimulatory receptors (46
Regardless of the possible redundancy within this family, functions ascribed to overexpressed MAP3Ks may be biologically relevant for an additional reason. In the case of Cot, cellular transformation is associated with not only truncated forms of the protein that have increased kinase activity but also with overexpression (7
). In this regard it may be interesting to determine how the mutation of serine 400 in Cot affects the ability of the kinase to effect transformation. To date, no analysis of transformation by Cot has focused on the roles of NF-κB versus those of MAP kinases in this process.