Herein we demonstrate that the LIM protein Ajuba is a new component of the proinflammatory cytokines IL-1 and TNF signal transduction cascades regulating NF-κB activation.
IL-1 can activate NF-κB by interrelated signaling pathways (reviewed in references 7
). First, IL-1 interacts with a specific cell surface receptor (IL-1R) on target cells. Receptor activation leads to the recruitment of the myeloid differentiation factor (MyD88) to the receptor complex. MyD88 serves to further recruit members of the IL-1R kinase family (IRAK) to the receptor complex. IRAK plays a critical role in the activation of NF-κB through its interaction with TRAF6. In response to IL-1, TRAF-6 becomes ubiquitinated, oligomerizes, and interacts with TAB-2, the transforming growth factor-activated kinase 1 (TAK1), and TAB-1. Activated TAK1 can then activate the IκΒ kinase complex (IKK), leading to phosphorylation of IκΒ and its subsequent degradation with release of NF-κB proteins and their translocation into the nucleus, where inflammatory, apoptotic, or antiapoptotic pathways are regulated, depending upon cell type (3
Alternatively, IL-1 can activate NF-κB through a cytosolic pathway involving aPKC (13
), p62 (41
), and TRAF6 (41
). p62 has been proposed to function as a scaffold protein, in that it interacts with both PKCζ and TRAF6 in an IL-1-dependent manner. Dominant negative aPKC blocks IL-1-induced and TRAF6-induced NF-κB activation (41
), suggesting that TRAF6, when bound to p62, may activate aPKC. Furthermore, the aPKC/p62/TRAF6 complex appears to function downstream of IRAK (41
). Cells and organs from mice deficient in p62 (41
), TRAF6 (25
), or PKCζ (24
) all exhibit defective NF-κB activation in response to proinflammatory cytokines of the IL-1 family.
Primary MEFs from Ajuba null mice and the lungs from Ajuba null mice injected with IL-1 have diminished NF-κB activation following IL-1 and TNF stimulation. This defect in NF-κB activation is biologically relevant, as Ajuba null MEFs are more sensitive to TNF-induced apoptosis (data not shown), and reintroduction of Ajuba into Ajuba null MEFs rescues the IL-1- and TNF-induced NF-κB signaling defect and prevents TNF-induced cell death. Likewise, overexpression of Ajuba in primary MEFs increased NF-κB activity following IL-1 stimulation.
We propose that Ajuba influences IL-1-induced NF-κΒ activation by affecting the assembly of the PKCζ/p62/TRAF6 multiprotein complex in cells and thus IKK activity. Using purified proteins in vitro, we also found that Ajuba could be a PKCζ substrate and enhance PKCζ activity. The aPKC-interacting protein p62 was identified as interacting with Ajuba in a two-hybrid screen, and endogenous p62 coimmunoprecipitates and colocalizes with endogenous Ajuba in cells. Within the Ajuba-related LIM protein family, there was specificity in interacting with p62. Importantly, p62-noninteracting Zyxin null MEFs have no defect in IL-1-induced NF-κB activity, suggesting that Ajuba's interaction with p62 was crucial for its regulation of NF-κB activity.
In addition to interacting with p62, Ajuba also interacted with TRAF6. The interaction with TRAF6 was also specific, as Ajuba did not interact with related TRAF2. Mapping studies identified the TRAF domain of TRAF6 as mediating its interaction with Ajuba. Within the TRAF family, the TRAF domain in TRAF6 is most divergent (1
). Other proteins interacting with this domain are p62 (41
) and ECSIT (22
), and each, like Ajuba, is specific for TRAF6. The TRAF domain is responsible for oligomerization of TRAF6, which is thought to be critical for signaling by the N-terminal region (3
). Progressive overexpression of Ajuba results in increased recruitment of TRAF6 to p62 and enhanced NF-κB activity. So it is possible that Ajuba and p62 serve to “activate” TRAF6, bringing its effector domain into close proximity to PKCζ bound to p62 or its other downstream kinase, TAK1 (Fig. ).
FIG. 8. Working model for how Ajuba influences IL-1-induced NF-κB activation. PKCζ and the PKCζ binding site in p62 (AID) are in black. Ajuba and the LIM protein binding site in p62 (LB) are in white. TRAF6 and the TRAF6 binding site in (more ...)
Mapping studies identified a unique domain (LB domain) in p62 that directs its association with Ajuba. Importantly, the LB domain does not overlap with the aPKC, RIP, or TRAF6 binding domains previously identified in p62 (41
). Interestingly, the LB domain of p62 lies just N-terminal to the TRAF6 binding domain. Taken together, these observations suggest the following possibilities for how Ajuba could influence the recruitment of TRAF6 to p62: (i) following Ajuba binding to p62, TRAF6 could be recruited to p62 through an interaction with Ajuba or p62 (i.e., independent binding sites); (ii) a preformed Ajuba-TRAF6 protein complex could be recruited to p62; (iii) Ajuba could stabilize the association of TRAF6 with p62; (iv) Ajuba could also influence the activation (i.e., oligomerization and ubiquitination) of TRAF6 bound to p62 (6
); or (v) some combination of the above mechanisms. Future biochemical studies with purified protein components and genetic studies of mutant proteins expressed in cells from knockout mice should begin to address these possibilities.
We also found that Ajuba could interact with PKCζ independently of p62. In vitro, Ajuba activated PKCζ enzyme activity, and Ajuba could be a PKCζ substrate. Interestingly, the LIM region of Ajuba activates PKCζ but is not phosphorylated, whereas the PreLIM is readily phosphorylated by PKCζ but does not significantly activate PKCζ activity. That components of Ajuba are more efficient as activator and substrate than the full-length protein suggests the possibility that Ajuba may exist in an inhibited conformation, possibly with the PreLIM region folded back on the LIM regions, as has been suggested for Vinculin, WASP, and another LIM protein, Tes (9
), or may be inaccessible due to an interaction with another cellular protein. Phosphorylation of the PreLIM region of Ajuba could then possibly open up the structure or release it from a cellular complex, now allowing for the LIM region to interact with p62, TRAF6, and PKCζ. An interaction with PKCζ could further activate PKCζ activity, thereby influencing NF-κB activation.
In addition to regulating NF-κB activity, IL-1 signals also modulate the activity of the MAPKs, particularly JNK and p38. While Ajuba appeared to have a mild effect upon p38 activity, there was sustained activation of JNK in Ajuba null MEFs. JNK can be activated by TRAF6-activated TAK1, which can also activate NF-κB (3
). Whether TRAF6-regulated TAK1 activity is altered in Ajuba null cells is unknown. Alternatively, sustained JNK activity in Ajuba null cells may simply result from the inhibition of NF-κB (37
When one compares the activation of NF-κB by the IL-1 family of proinflammatory cytokines in primary cells from knockout mice, Ajuba's action overlaps with that observed for TRAF6−/−
) and PKCζ−/−
) mice. p62 knockout mice have not been fully analyzed (8
). In vivo, TRAF6−/−
mice both have defects in bone metabolism through effects upon IL-1, TNF, and RANKL signaling during osteoclast differentiation. Ajuba is present at low levels in BMDM and doesn't change during RANKL differentiation into osteoclasts (Y. Feng and G. Longmore, unpublished). Thus, we saw no change in LPS-induced NF-κB activity in Ajuba−/−
BMDM (data not shown), and bone development in Ajuba null mice was not abnormal (data not shown). Ajuba-related LIMD1 also interacts with p62. LIMD1 was originally identified as an open reading frame present on a segment of chromosome 3 deleted in some cervical cancers (20
). Little else is known about LIMD1 cell biology or function. Interestingly, LIMD1 expression, like p62, is upregulated during RANKL-induced osteoclast differentiation of BMDM (8
; Y. Feng and G. Longmore, unpublished). Therefore, it will be interesting to determine whether genetic ablation of LIMD1 affects bone metabolism through modulation of IL-1, TNF, and RANKL signaling pathways. Finally, residual NF-κB activity in Ajuba null MEFs and lungs may reflect the presence of LIMD1 in these cells and/or tissues.