Telomere-independent Rap1 is an IKK adaptor and regulates NF-κB-dependent gene expression

Abstract

We describe a genome-wide gain-of-function screen for regulators of NF-kappaB, and identify Rap1 (Trf2IP), as an essential modulator of NF-kappaB-mediated pathways. NF-kappaB is induced by ectopic expression of Rap1, whereas its activity is inhibited by Rap1 depletion. In addition to localizing on telomeres, mammalian Rap1 forms a complex with IKKs (IkappaB kinases), and is crucial for the ability of IKKs to be recruited to, and phosphorylate, the p65 subunit of NF-kappaB to make it transcriptionally competent. Rap1-mutant mice display defective NF-kappaB activation and are resistant to endotoxic shock. Furthermore, levels of Rap1 are positively regulated by NF-kappaB, and human breast cancers with NF-kappaB hyperactivity show elevated levels of cytoplasmic Rap1. Similar to inhibiting NF-kappaB, knockdown of Rap1 sensitizes breast cancer cells to apoptosis. These results identify the first cytoplasmic role of Rap1 and provide a mechanism through which it regulates an important signalling cascade in mammals, independent of its ability to regulate telomere function.

The human TTAGGG repeat factors 1 and 2 bind to a subset of interstitial telomeric sequences and satellite repeats

“…Emerging evidence indicates that the shelterin components have non-telomeric functions in DNA repair [19], Epstein-Barr virus replication [20], transcriptional regulation [21] and NF-κB activation [22]. These nontelomeric functions might be, at least partially, explained by their binding to ITSs.…”

Rap1 induces cytokine production in pro-inflammatory macrophages through NFκB signaling and is highly expressed in human atherosclerotic lesions

“…17 The presence of Rap1 has been positively correlated to the advancement of invasive human breast cancers. 10 The ability of Rap1 to modify NFkB signaling is conserved in vivo, as heterozygous mice lacking one functional Rap1 allele do not develop proper immune responses. 10 Collectively, the available experimental evidence suggests that Rap1 is not only a static structural component of the telomere but exerts other functions both within and outside the nucleus.…”

“…13 Unlike in the nucleus, cytoplasmic Rap1 does not interact with telomeric repeat-binding factor 2, but rather constitutively binds to I Kappa B kinase (IKK), 10 which is responsible for the phosphorylation and subsequent degradation of inhibitor of kappa b (IkB) protein, 14 the endogenous inhibitor of NFkB. 15 Cytoplasmic Rap1 ensures the efficient recruitment of IKKs and the phosphorylation of the p65 subunit of NFkB, 10,16 an essential step for rendering NFkB transcriptionally competent. 17 The presence of Rap1 has been positively correlated to the advancement of invasive human breast cancers.…”

“…9 Expression of Rap1 was initially believed to be restricted to the nucleus, however it was later found to be expressed in the cytoplasm of breast carcinoma BT476 and epithelial carcinoma HeLa S3 cell lines. 10 In these human cancer cell lines, cytoplasmic Rap1 regulates the nuclear factor kappa B (NFkB) signaling cascade, 10,11 a master transcription factor that plays fundamental roles in inflammatory, immune responses 12 and other signaling cascades. 13 Unlike in the nucleus, cytoplasmic Rap1 does not interact with telomeric repeat-binding factor 2, but rather constitutively binds to I Kappa B kinase (IKK), 10 which is responsible for the phosphorylation and subsequent degradation of inhibitor of kappa b (IkB) protein, 14 the endogenous inhibitor of NFkB.…”

“…10 In these human cancer cell lines, cytoplasmic Rap1 regulates the nuclear factor kappa B (NFkB) signaling cascade, 10,11 a master transcription factor that plays fundamental roles in inflammatory, immune responses 12 and other signaling cascades. 13 Unlike in the nucleus, cytoplasmic Rap1 does not interact with telomeric repeat-binding factor 2, but rather constitutively binds to I Kappa B kinase (IKK), 10 which is responsible for the phosphorylation and subsequent degradation of inhibitor of kappa b (IkB) protein, 14 the endogenous inhibitor of NFkB. 15 Cytoplasmic Rap1 ensures the efficient recruitment of IKKs and the phosphorylation of the p65 subunit of NFkB, 10,16 an essential step for rendering NFkB transcriptionally competent.…”