A Novel Zinc Finger Protein Interacts with Receptor-interacting Protein (RIP) and Inhibits Tumor Necrosis Factor (TNF)- and IL1-induced NF-κB Activation

Abstract

Receptor-interacting protein (RIP) is a serine/threonine protein kinase that is critically involved in tumor necrosis factor receptor-1 (TNF-R1)-induced NF-B activation. In a yeast two-hybrid screening for potential RIP-interacting proteins, we identified ZIN (zinc finger protein inhibiting NF-B), a novel protein that specifically interacts with RIP. ZIN contains four RING-like zinc finger domains at the middle and a proline-rich domain at the C terminus. Overexpression of ZIN inhibits RIP-, IKK␤-, TNF-, and IL1-induced NF-B activation in a dose-dependent manner in 293 cells. Domain mapping experiments indicate that the RING-like zinc finger domains of ZIN are required for its interaction with RIP and inhibition of RIP-mediated NF-B activation. Overexpression of ZIN also potentiates RIP-and TNF-induced apoptosis. Moreover, immunofluorescent staining indicates that ZIN is a cytoplasmic protein and that it colocalizes with RIP. Our findings suggest that ZIN is an inhibitor of TNF-and IL1-induced NF-B activation pathways. Tumor necrosis factor receptor 1 (TNF-R1)1 is a prototypical member of the TNF receptor family (1). TNF stimulation of TNF-R1 simultaneously induces three divergent effects: apoptosis, activation of the transcription factor NF-B, and the serine/threonine protein kinase JNK (1). TNF-R1 contains a death domain, which interacts with the cytoplasmic death domain-containing protein TRADD in a TNF-dependent process (2-5). Once TRADD is recruited to TNF-R1, it functions as an adapter protein to recruit several structurally and functionally divergent proteins, including FADD, RIP, TRAF2, and cellular inhibitor of apoptosis protein (cIAP) (2, 4). The interaction of TRADD with FADD leads to apoptosis through activation of a caspase cascade, which is initiated by the interaction of FADD with caspase-8 (2, 6 -10). The interaction of TRADD with TRAF2 and RIP activates a downstream IB kinase complex called IKK, which contains two catalytic subunits, IKK␣ and IKK␤, and a regulatory subunit, IKK␥/NEMO (11-19). The activated IKK phosphorylates IBs, leading to their degradation and subsequent activation of NF-B (11-19).RIP is a unique signal transducer in the TNF-R1-mediated NF-B activation pathway. RIP was first identified as a Fasinteracting protein by the yeast two-hybrid system (20). It was later demonstrated that RIP is a component of the TNF-R1 signaling complex (4, 21). Gene knock-out experiments suggest that RIP is required for TNF-R1-mediated NF-B activation but is not required for Fas-and TNF-R1-mediated apoptosis (22,23). RIP is a serine/threonine kinase that contains three domains, including an N-terminal kinase domain, an intermediate domain, and a C-terminal death domain (4,20). RIP interacts with TRADD through their respective death domains. The intermediate domain of RIP interacts with the RING finger domain of TRAF2, and this interaction is required for RIP-mediated NF-B activation (21). Recently, it has been suggested that RIP directly interacts with IKK␥ and therefore recruits IKK to the TNF-R1 c...

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