RING-dependent tumor suppression and G2/M arrest induced by the TRC8 hereditary kidney cancer gene.

TRC8/RNF139 and von Hippel-Lindau (VHL) both encode E3 ubiquitin (Ub) ligases mutated in clear-cell renal carcinomas (ccRCC). VHL, inactivated in nearly 70% of ccRCCs, is a tumor suppressor encoding the targeting subunit for a Ub ligase complex that downregulates hypoxia-inducible factor-alpha. TRC8/RNF139 is a putative tumor suppressor containing a sterol-sensing domain and a RING-H2 motif essential for Ub ligase activity. Here we report that human kidney cells are growth inhibited by TRC8. Inhibition is manifested by G2/M arrest, decreased DNA synthesis and increased apoptosis and is dependent upon the Ub ligase activity of the RING domain. Tumor formation in a nude mouse model is inhibited by TRC8 in a RING-dependent manner. Expression of TRC8 represses genes involved in cholesterol and fatty acid biosynthesis that are transcriptionally regulated by the sterol response element binding proteins (SREBPs). Expression of activated SREBP-1a partially restores the growth of TRC8-inhibited cells. These data suggest that TRC8 modulation of SREBP activity comprises a novel regulatory link between growth control and the cholesterol/lipid homeostasis pathway.

Transcriptional coregulator SNURF (RNF4) possesses ubiquitin E3 ligase activity.

SNURF/RNF4 has been implicated in transcriptional regulation and growth inhibition in a RING finger-dependent fashion. In this work, we show that SNURF mediates its own ubiquitination in vitro in a ubiquitin-conjugating enzyme (E2)-selective manner: SNURF acts as an E3 ligase with UbcH5A and B, HHR6B (RAD6B), E2-25K, MmUbc7 and UbcH13, but not with UbcH3, UbcM4, MmUbc6 or E2-20K. In contrast, the well-characterized RING E3, AO7, functions only with members of the UbcH5 family. Furthermore, depending on the E2 used, the ubiquitin modification manifests as mono- or multi-ubiquitination. Mutation of conserved cysteine residues within the RING finger motif of SNURF abolishes the ubiquitination in vitro and in intact cells. Size fractionation of murine embryonal carcinoma F9 cell proteins shows that the majority of endogenous SNURF resides in salt-resistant > or =500-kDa complexes, suggesting that SNURF functions as a RING component in a multiprotein complex. Taken together, SNURF/RNF4 functions as an E3 ligase and this activity is closely linked to its transcription regulatory functions.

RING fingers mediate ubiquitin-conjugating enzyme (E2)-dependent ubiquitination.

A RING finger-containing protein (AO7) that binds ubiquitin-conjugating enzymes (E2s) and is a substrate for E2-dependent ubiquitination was identified. Mutations of cation-coordinating residues within AO7's RING finger abolished ubiquitination, as did chelation of zinc. Several otherwise-unrelated RING finger proteins, including BRCA1, Siah-1, TRC8, NF-X1, kf-1, and Praja1, were assessed for their ability to facilitate E2-dependent ubiquitination. In all cases, ubiquitination was observed. The RING fingers were implicated directly in this activity through mutations of metal-coordinating residues or chelation of zinc. These findings suggest that a large number of RING finger-containing proteins, with otherwise diverse structures and functions, may play previously unappreciated roles in modulating protein levels via ubiquitination.

2,3,7,8-Tetrachlorodibenzo-p-dioxin alters retinoic acid receptor function in human keratinocytes.

Actions of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) on all-trans-retinoic acid (trans-RA) binding to retinoic acid receptors (RARs) in cultured human keratinocytes (SCC-12F) was investigated. TCDD and trans-RA elicited opposing actions on the production of biologically active TGF-beta. TCDD exposure caused concentration- and time-dependent decreases in trans-RA binding to SCC-12F RARs. The apparent half-maximal effective TCDD concentration = 1 nM. TCDD exerts its action via the aryl hydrocarbon receptor (AhR). TCDBF, a partial AhR agonist, reduced trans-RA binding, indicating AhR involvement (control = 0.33; TCDBF = 0.22; TCDD = 0.142 pmol trans-RA bound/mg nuclear protein). The dissociation constant (Kd) calculated from Eadie-Hofstee analysis of equilibrium binding for trans-RA was 0.13 nM in both TCDD-exposed and control cultures. Approximately half of the trans-RA binding sites were lost in TCDD-exposed cells (control = 0.195; TCDD = 0.108 pmol trans-RA bound/mg protein). The data suggest TCDD may exert its toxic action in human keratinocytes by directly modulating RAR action.