CARP2 deficiency does not alter induction of NF-κB by TNFα

We found that Rififylin/Carp2-deficient knockout mice were born at expected Mendelian ratios, were healthy and displayed no overt phenotype, and exhibited normal fertility (Figure S and Table S). This contrasts with both RIP-deficient mice and mice lacking several proteins implicated in K48-and/ or K63-linked ubiquitylation and regulation of TNFR–RIP signaling [,3,6–4]. Isolated wild-type (WT) murine embryonic fibroblasts (MEFs) expressed readily detectable levels of CARP2 mRNA, while MEFs isolated from several independent Rififylin/ Carp2-deficient embryos did not (Figure S2C). Knockdown of CARP2 in 293T cells by transient transfection with CARP2-targeting shRNA was reported to enhance activation of NF-κB by TNFα [2]. Consistent with this, Liao et al. [2] also reported that overexpression of FLAG–CARP2 prevented TNFα from activating NF-κB, and this correlated with CARP2-mediated RIP degradation. In Rififylin/Carp2-deficient MEFs, however, we found that both high (00 ng/ml) and low (0 ng/ml) doses of TNFα induced phosphorylation of p65, the degradation of IκB, and the subsequent NF-κB dependent restoration of IκB with the same kinetics as in WT MEFs (Figure A,B and Figure S2D). In addition, induction of FLAG–CARP2 overexpression in D645 cells did not interfere with TNFα-induced degradation and subsequent NF-κB-dependent induction of IκB (Figure C). If endosomal-localised CARP2 acts as an E3 ligase for the minor pool of total RIP recruited to TNFR endocytic vesicles, as Liao et al. [2] suggested, one would not expect total cellular pools of cytosolic RIP to be degraded upon CARP2 overexpression. However, CARP2 overexpression depletes cellular endogenous p53 [5] (Figure S2A,B) and Liao et al. [2] demonstrated that overexpressed CARP2 resulted in a significant loss of cytosolic overexpressed RIP in 293T cells. We therefore asked whether overexpressed CARP2 could deplete endogenous RIP, and whether this would be reflected by increased RIP levels in Rififylin/Carp2-deficient MEFs. We observed that cellular RIP levels were the same in WT and TNFα can activate pathways leading to caspase-8-mediated apoptosis, as well as inflammatory pathways signaled by transcription factors. The adaptor protein RIP is a critical component for TNF receptor (TNFR)-mediated activation of NF-κB, because deletion of the gene encoding RIP in mice prevents induction of NF-κB by TNFα and causes severe runting with early postnatal lethality []. Recently, it has been proposed that caspase 8 and 0 associated RING protein-2 (CARP2, also named RIFIFYLIN/ SAKURA) binds to the TNFR complex, leading to ubiquitylation and proteasome-mediated degradation of RIP, thereby limiting the level of NF-κB activated by TNFα [2]. …