RIP4 (DIK/PKK), a novel member of the RIP kinase family, activates NF-kappa B and is processed during apoptosis

Abstract

RIP1 and its homologs, RIP2 and RIP3, form part of a family of Ser/Thr kinases that regulate signal transduction processes leading to NF-kappa B activation. Here, we identify RIP4 (DIK/PKK) as a novel member of the RIP kinase family. RIP4 contains an N-terminal RIP-like kinase domain and a C-terminal region characterized by the presence of 11 ankyrin repeats. Overexpression of RIP4 leads to activation of NF-kappa B and JNK. Kinase inactive RIP4 or a truncated version containing the ankyrin repeats have a dominant negative (DN) effect on NF-kappa B induction by multiple stimuli. RIP4 binds to several members of the TRAF protein family, and DN versions of TRAF1, TRAF3 and TRAF6 inhibit RIP4-induced NF-kappa B activation. Moreover, RIP4 is cleaved after Asp340 and Asp378 during Fas-induced apoptosis. These data suggest that RIP4 is involved in NF-kappa B and JNK signaling and that caspase-dependent processing of RIP4 may negatively regulate NF-kappa B-dependent pro-survival or pro-inflammatory signals.

Figure 1

RIP4 is a novel member of the RIP family of kinases. (A) Domain organization of RIP family members. All members share a homologous N-terminal kinase domain. RIP1 and RIP2 have C-terminal DD and CARD motifs, respectively, whereas the C-terminus of RIP3 lacks obvious sequence homology to known proteins in public databases. RIP4 has C-terminal ankyrin repeats. (B) Sequence alignment of the kinase domains of RIPs. Black and gray shading indicate 75% amino acid sequence identity and similarity, respectively. I–XI represent conserved modules (Hanks and Hunter, 1995). Indicated are residues conserved in 95% of the kinases (©), residues important for ATP binding (filled circle) and the Gly residue present in RIP3 (open circle) that is conserved in 95% of all kinases but is substituted into Ala in RIP1, RIP2 and RIP4. (C) Phylogenic tree of the kinase domain of human RIPs and IRAKs.

Figure 2

The kinase activity of RIP4 is critical for NF-κB and JNK activation. (A) 293T cells were transfected with an NF-κB reporter plasmid, together with mock or MyD88 plasmids (1 μg) or the indicated amounts of a RIP4 construct, and analyzed for NF-κB-dependent luciferase activity. Data shown are mean values ± standard deviations from one representative out of three independent experiments, each done in triplicate. (B) 293T cells were co-transfected with IκB and mock, RIP4 or MyD88 constructs, as indicated, and cell lysates were analyzed for the presence of phosphorylated IκB (P-IκB) by western blot. (C) 293T cells were transfected with the indicated RIP4 constructs, and anti-VSV immunoprecipitates (IP) were analyzed by in vitro kinase assay and autoradiography (upper) or by anti-VSV western blot (lower) The arrowhead indicates phosphorylated RIP4. (D) 293T cells transfected with the indicated RIP4 constructs were analyzed as in (A). The structure and expression level of the various RIP4 constructs used is shown. (E) 293T cells transfected with IκB and the indicated RIP4 constructs were analyzed for IκB phosphorylation as in (B). (F–H) 293T cells were co-transfected with FLAG-JNK, HA-p38 or HA-ERK constructs, respectively, together with the indicated RIP4 constructs, and cell lysates were assessed for MAPK activation by western blot.

Figure 3

DN versions of RIP4 inhibit NF-κB activation by multiple pathways. (A) 293T cells were transfected with an NF-κB reporter plasmid and RIP4, in combination with mock plasmid (1 μg) or the indicated RIP4 constructs, DN-IKKβ or DN-IκBα, and analyzed for NF-κB-dependent luciferase activity. The expression level of the various transfected constructs is shown. (B) 293T cells were transfected with the indicated RIP4 constructs, and anti-VSV immunoprecipitates (IP) and cell extracts were analyzed by western blot. (C) 293T cells were transfected with an NF-κB reporter plasmid and the indicated constructs, together with the RIP4 ankyrin domain (+) or mock plasmid (−). (D) HeLa cells were transfected with an NF-κB reporter plasmid together with RIP4 K51R (+) or mock plasmid (−) and analyzed for TNF- and IL-1β-induced NF-κB activation.

Figure 4

RIP4 interacts with TRAF proteins. (A) 293T cells were transfected with a VSV-tagged RIP4 construct together with the indicated TRAF constructs, and anti-FLAG immunoprecipitates (IP) and cell extracts were analyzed by western blot. (B) 293T cells were transfected with an NF-κB reporter plasmid together with RIP4 and the indicated DN-TRAF constructs, and lysates were analyzed for NF-κB-dependent luciferase activity.

Figure 5

RIP4 is cleaved after Asp340 and Asp378 during FasL-dependent induction of apoptosis. (A) The ID of RIP4 contains two consensus caspase cleavage sites, SQLD₃₄₀ and SSVD₃₇₈. (B and C) 293T cells were transfected with the indicated N-terminally VSV-tagged RIP4 constructs. Twenty-four hours after transfection, cells were treated with recombinant FasL in the presence or absence of the caspase inhibitor zVAD, and cell lysates were analyzed for the presence of RIP4 cleavage products by western blot using anti-VSV and anti-RIP4 antibodies, as indicated. The arrowheads in (B) indicate full-length and cleaved forms of RIP4. (D) 293T cells were transfected with wild-type (wt) RIP4, the double D340/D378-Glu (DD/EE) or the respective single mutants, together with an NF-κB reporter plasmid, and NF-κB activation was analyzed by luciferase assay.