In this study we show that TNF activates Etk specifically through TNFR2, as demonstrated by studies using a specific agonist to TNFR2, overexpression of TNFR2, and TNFR2-deficient cells. Etk binds to a non-TRAF2-binding motif in TNFR2 intracellular domains and is activated by TNF in a TRAF2-independent manner. Moreover, we show that Etk is a critical mediator in TNF-induced angiogenesis by in vitro models of EC migration and EC tube formation. Our findings demonstrate that Etk is the first kinase to associate specifically with TNFR2 (but not with TNFR1).
EC are among the restricted cell types that express TNFR2 (6
). The role of TNFR2 in TNF signaling in EC is not clear. Here we show that TNF through TNFR2, but not TNFR1, activates an EC-expressed kinase, Etk. This is supported by several lines of evidence. First, an agonist to TNFR2, but not to TNFR1, activates Etk in EC. Second, overexpression of TNFR2, but not TNFR1, in EC activates Etk. Third, TNF fails to activate Etk in TNFR2-deficient cells, but Etk activation by TNF is still present in TNFR1-deficient cells.
It is generally accepted that TNF utilizes TNFR1 and TNFR2 to trigger distinct signaling and to exert diverse biological functions in a context-dependent manner. TNFR1 is believed to mediate cell death, whereas TNFR2 serves to promote cell activation, migration, growth, or proliferation (9
). In T cells, TNF through TNFR2 promotes T-cell proliferation at an early stage of activation (36
). It has been shown that TNFR2, but not TNFR1, is critical in TNF-induced proliferation of oligodendrocyte progenitors and remyelination (1
). However, the mechanisms by which TNF through TNFR2 exerts these functions have not been defined. The reason, in part, is that specific proteins recruited by TNFR2 have not been identified. TRAF2 can be recruited to both TNFR1 and TNFR2 signaling complexes (46
) and is the only signaling molecule found to associate with TNFR2. Several factors, including cellular inhibitor of apoptosis and caveolin-1, are recruited to TNFR2 through TRAF2 (16
). Thus, TRAF2 and TRAF2-associated factors have been implicated in TNFR2-induced antiapoptotic or apoptotic responses.
In contrast to these findings, we show that TNF-induced activation of Etk through TNFR2 is independent of TRAF2. The interactions between Etk and TNFR2 are dependent on the N-terminal portion (the PH, TH, and SH3 domains) of Etk and the last 16 amino acids at the C-terminal intracellular domain of TNFR2. This Etk-binding sequence is distinct from the TRAF2-binding motif. Specifically, the TNFR2(−16) deletion mutant lacking the last 16 residues retains TRAF2 binding but fails to associate with and activate Etk. Also, WT-TRAF2 does not activate Etk, and DN-TRAF2 does not block TNF-induced Etk activation.
An interaction between the Btk family and the TNFR family has been demonstrated previously (59
). Similar to Etk-TNFR2 interaction, Btk interacts with Fas, a member of the TNFR family involved in apoptosis in a ligand-independent manner (59
). However, the association of Btk and Fas is dependent on the PH and kinase domains but not on the SH3 domain of Btk. This interaction disrupts the association of FADD with Fas, leading to inhibited Fas-induced apoptosis. Consistent with our finding, Btk does not interact with TNFR1 signaling complex molecules such as TRADD, FADD, and FLICE (59
The mechanism by which Etk is activated is not clear. On the basis of data from Etk activation by focal adhesion kinase (FAK), it has been proposed that integrin-induced binding of the PH domain of Etk to the FERM domain of FAK leads to conformational change of the PH domain and phosphorylation of Y40 concomitant with the membrane translocation of Etk (10
). This process resembles the effects of phospholipid binding to the PH domains of Btk family kinases in response to growth factors or cytokines. Membrane targeting of Etk will open up the closed conformation of inactive Etk and will allow the kinase to be phosphorylated by Src family kinases at the highly conserved tyrosine residue Y566 in the catalytic domain, which is originally masked by the PH domain. Tyrosine phosphorylation activates the Etk kinase, leading to autophosphorylation and activation. We show that, unlike what is shown by this two-step model, Etk forms a preexisting complex with TNFR2 located in the cytoplasm membrane. This association is independent of the phospholipid-binding PH domain of Etk, as the phospholipid-binding deficient mutant (Etk-DN) still binds to TNFR2. This membrane-bound Etk should still be in a closed inactive form. TNFR2 has no kinase activity, and it is not clear how Etk is activated in response to TNF. It has been shown that TNFR superfamily can form preassembled trimers, and ligand induces a conformation change (8
). One possibility for Etk activation by TNFR2 is that TNF-induced TNFR2 conformational change triggers a change in Etk conformation to open up the closed conformation (Fig. ). Alternatively, TNF-induced recruitment of additional factor(s) (e.g., a kinase) to a TNFR2/Etk complex, which phosphorylates Etk to open up the closed conformation, resembling phosphorylation of Y40 in the PH domain by FAK (10
). The second step of Etk activation in response to TNF has not been determined. The tyrosine phosphorylation of the kinase-inactive Etk (Etk-DK) is undetectable in response to TNF/TNFR2, indicating that Etk activation induced by TNF/TNFR2 is largely due to autokinase activity. This is similar to Etk activation by interleukin-6 (40
FIG. 7. A model for Etk activation by TNF/TNFR2 in EC angiogenesis. Etk forms a preexisting complex with TNFR2, and Etk remains in a closed inactive form. TNF induces trimerization or conformational change of TNFR2, leading to an open conformation of Etk followed (more ...)
Our data demonstrate that Etk plays an important role in TNF-induced EC migration and tube formation by in vitro models, suggesting that Etk is a critical mediator in TNF-induced angiogenesis. The roles of Etk in cell activation, proliferation, and migration have been demonstrated in other cell types, including tumor cell lines and epithelial cells. Recently it has been shown that expression of Etk is much higher in metastatic carcinoma cells than in nonmetastatic carcinoma cells (10
). Mechanistic studies suggest that interactions of Etk and FAK synergistically promote migratory potential of carcinoma cells. Etk is highly expressed in cultured EC (HUVEC and BAEC) (10
and our data) and in vivo endothelium (13
). However, the role of Etk in angiogenesis has not been elucidated. Recent studies show that although Etk-deficient mice have no obvious defect in vasculogenesis, Etk is activated by EC growth factor receptor tyrosine kinases VEGFR1 and Tie-2. These data led the authors to postulate that Etk has a redundant function downstream of receptors for angiogenic factor (41
). Angiogenesis under pathological conditions (e.g., inflammation) has not been examined with mice. Our data demonstrate that Etk mediates TNF-induced EC migration and tube formation in vitro, implying that Etk may play a critical role in inflammatory angiogenesis, such as occurs with ischemia, atherosclerosis, and rheumatoid arthritis. Our studies suggest that Etk might be a target for treatment of the inflammatory angiogenesis-dependent disease.