HIV-1 Vpu interacts with CD4 (186940) in the endoplasmic reticulum and triggers CD4 degradation, presumably by proteasomes. Margottin et al. (1998) identified a human beta-transducin repeat-containing protein (BTRCP), consisting of 569 amino acids, that interacts with Vpu and connects CD4 to the proteolytic machinery. The human BTRCP is homologous to Xenopus bTrCP1, yeast Met30, and Neurospora Scon2 proteins. CD4-Vpu-BTRCP complexes have been detected by coimmunoprecipitation. BTRCP-binding to Vpu and its recruitment to membranes require 2 phosphoserine residues (ser52 and ser56) in Vpu essential for CD4 degradation. BTRCP contains 7 WD repeats at the C terminus which mediate binding to Vpu, and an F box near the N terminus which is involved in interaction with SKP1 (601434), a targeting factor for ubiquitin-mediated proteolysis. An F-box deletion mutant of BTRCP had a dominant-negative effect on Vpu-mediated CD4 degradation. These data suggest that BTRCP and SKP1 represent components of an ER-associated protein degradation pathway that mediates CD4 proteolysis.

Activation of nuclear factor kappa-B (NFKB; see 164011) requires phosphorylation of the NFKB inhibitor IKB-alpha (IKBA, or NFKBIA; 164008) at N-terminal serines 32 and 36 by IKB kinase-alpha (IKBKA; 600664) or IKB kinase-beta (IKBKB; 603258). Phosphorylated IKBA (pIKBA) is then targeted for ubiquitination and destruction in the 26S proteasome. Using affinity purification, Yaron et al. (1998) isolated a ligase, which they denoted pIKBA-E3, from HeLa cells that binds strongly to pIKBA and attaches ubiquitin to lysines 21 and 22. By peptide sequencing, they identified the component of the ligase that recognizes and ubiquitinates pIKBA as an F-box/WD40 domain from BTRC, which they called E3RS-IKB (E3 receptor subunit for IKB). An F-box deletion mutant of BTRC bound to but did not ubiquitinate pIKBA and acted as a dominant-negative molecule, i.e., NFKB activation was inhibited, in stimulated Jurkat cells in vivo.

Using SKP1 as bait in a yeast 2-hybrid screen and by searching DNA databases, Cenciarelli et al. (1999) identified several genes encoding F-box proteins, including FBW1A. Northern blot analysis detected variable expression of 7.5-, 4.4-, and 2.5-kb FBW1A transcripts in nearly all tissues examined. Epitope-tagged FBW1A distributed to both the cytoplasm and nucleus of transfected HeLa cells.

Winston et al. (1999) determined by biochemical dissection and expression of assembled recombinants that BTRC, and not other F-box proteins, is the F protein in the SCF (SKP1, CUL1 (603134), and F protein) complex. They showed that the SCF complex recognizes, in a phosphorylation-dependent manner, a 21-amino acid destruction motif (residues 21 to 41) in IKBA, as well as a conserved destruction motif in beta-catenin (CTNNB1; 116806), which is also regulated by phosphorylation-dependent ubiquitination. Winston et al. (1999) noted that CTNNB1 is a component of the WNT (see WNT1; 164820) signaling pathway, and its levels are regulated by AXIN (603816) and GSK3B (605004). Whole-mount in situ hybridization analysis revealed that Btrc is expressed throughout the mouse embryo at day 11.5 postcoitum, with highest levels in brain, lung, and liver. Immunofluorescence analysis demonstrated that BTRC is expressed largely, if not exclusively, in the cytoplasm, where pIKBA ubiquitination is thought to occur.

Spencer et al. (1999) pointed out that BTRC is the vertebrate homolog of the Drosophila Slimb protein. They found that BTRC ubiquitinates pIKBA at specific lysines in the presence of ubiquitin-activating and -conjugating enzymes (see 603890).

Maniatis (1999) reviewed the roles of BTRC and the SCF complex in the NFKB, WNT, and hedgehog (see 600725) signaling pathways.

Cenciarelli et al. (1999) showed that in vitro translated FBW1A interacted with SKP1 in an in vitro pull-down assay. The F box of FBW1A was required for this interaction.

Neish et al. (2000) observed abrogation of ubiquitination of phosphorylated CTNNB1 and IKBA, but not of other proteins, in a model epithelial system with nonpathogenic Salmonella, suggesting that the effect of the nonpathogenic bacteria is specific to the SCF complex substrates CTNNB1 and IKBA.

The Drosophila circadian clock is driven by daily fluctuations of the proteins Period (Per; 602260) and Timeless (Tim; 603887), which associate in a complex and negatively regulate the transcription of their own genes. Protein phosphorylation has a central role in this feedback loop, by controlling Per stability in both cytoplasmic and nuclear compartments, as well as Per/Tim nuclear transfer. Grima et al. (2002) demonstrated that the product of the slimb (slmb) gene is an essential component of the Drosophila circadian clock. slmb mutants are behaviorally arrhythmic, and can be rescued by targeted expression of slmb in the clock neurons. In constant darkness, highly phosphorylated forms of the Per and Tim proteins are constitutively present in the mutants, indicating that the control of their cyclic degradation is impaired. Because levels of Per and Tim oscillate in slmb mutants maintained in light:dark conditions, light- and clock-controlled degradation of Per and Tim do not rely on the same mechanisms.

Casein kinase I-epsilon (600863) has a prominent role in regulating the phosphorylation and abundance of Per proteins in animals. Using a Drosophila cell culture system, Ko et al. (2002) demonstrated that Double-time, the Drosophila homolog of CKI-epsilon, promotes the progressive phosphorylation of Per, leading to the rapid degradation of hyperphosphorylated isoforms by the ubiquitin-proteasome pathway. Slimb, an F-box/WD40-repeat protein functioning in the ubiquitin-proteasome pathway, interacts preferentially with phosphorylated Per and stimulates its degradation. Overexpression of slimb or expression in clock cells of a dominant-negative version of slimb disrupts normal rhythmic activity in flies. Ko et al. (2002) concluded that hyperphosphorylated Per is targeted to the proteasome by interactions with Slimb.

Noubissi et al. (2006) demonstrated that beta-catenin stabilizes the mRNA encoding the F-box protein BTRCP1 and identified the RNA-binding protein CRDBP (608288) as a target of beta catenin/Tcf transcription factor. CRDBP binds to the coding region of BTRCP1 mRNA. Overexpression of CRDBP stabilized BTRCP1 mRNA and elevated BTRCP1 levels both in cells and in vivo, resulting in the activation of the Skp1-Cullin1-F-box protein (SCF)-BTRCP1 E3 ubiquitin ligase and in accelerated turnover of its substrates including I-kappa-B (see 164008) and beta-catenin. CRDBP is essential for the induction of both BTRCP1 and c-Myc (190080) by beta-catenin signaling in colorectal cancer cells. Noubissi et al. (2006) concluded that high levels of CRDBP that are found in primary human colorectal tumors exhibiting active beta-catenin/Tcf signaling implicates CRDBP induction in the upregulation of BTRCP1, in the activation of dimeric transcription factor NF-kappa-B (see 164011), and in the suppression of apoptosis in these cancers.

Claspin (CLSPN; 605434) is an adaptor protein required for activation of CHK1 (CHEK1; 603078), a central component of DNA checkpoint signaling. Mailand et al. (2006) and Peschiaroli et al. (2006) found that claspin became degraded at the onset of mitosis. Claspin degradation was triggered by its interaction with and ubiquitylation by BTRC. The BTRC-claspin interaction was phosphorylation dependent and required PLK1 (602098) activity and integrity of the BTRC degradation motif in the N terminus of claspin. Mailand et al. (2006) found that uncoupling claspin from BTRC by mutating the conserved serines in the degradation motif or by BTRC knockdown stabilized claspin in mitosis, impaired CHK1 dephosphorylation, and delayed G2/M transition during recovery from cell cycle arrest imposed by DNA damage or replication stress. The inability to degrade claspin allowed partial reactivation of CHK1 in cells exposed to DNA damage after passing the G2/M transition. Peschiaroli et al. (2006) found that expression of a stable claspin mutant unable to bind BTRC prolonged activation of CHK1, thereby attenuating recovery from the DNA replication stress response and significantly delaying entry into mitosis.

Dorrello et al. (2006) found that the tumor suppressor programmed cell death protein-4 (PDCD4; 608610) inhibits the translation initiation factor EIF4A (see 602641), an RNA helicase that catalyzes the unwinding of secondary structure at the 5-prime untranslated region of mRNAs. In response to mitogens, PDCD4 was rapidly phosphorylated on ser67 by the protein kinase S6K1 (608938) and subsequently degraded via the ubiquitin ligase SCF-beta(TRCP). Expression in cultured cells of a stable PDCD4 mutant that was unable to bind beta-TRCP inhibited translation of an mRNA with a structured 5-prime untranslated region, resulted in smaller cell size, and slowed down cell cycle progression. Dorrello et al. (2006) proposed that regulated degradation of PDCD4 in response to mitogens allows efficient protein synthesis and consequently cell growth.

Using an unbiased screen, Guardavaccaro et al. (2008) demonstrated that REST (600571) is an interactor with the F-box protein beta-TRCP. REST is degraded by means of the ubiquitin ligase beta-TRCP during the G2 phase of the cell cycle to allow transcriptional derepression of Mad2 (601467), an essential component of the spindle assembly checkpoint. The expression in cultured cells of a stable REST mutant, which is unable to bind beta-TRCP, inhibited Mad2 expression and resulted in a phenotype analogous to that observed in Mad2 heterozygous cells. In particular, Guardavaccaro et al. (2008) observed defects that were consistent with faulty activation of the spindle checkpoint, such as shortened mitosis, premature sister-chromatid separation, chromosome bridges and missegregation in anaphase, tetraploidy, and a faster mitotic slippage in the presence of a spindle inhibitor. An indistinguishable phenotype was observed by expressing the oncogenic REST-FS mutant, which does not bind beta-TRCP. Thus, beta-TRCP-dependent degradation of REST during G2 permits the optimal activation of the spindle checkpoint, and consequently it is required for the fidelity of mitosis.

Westbrook et al. (2008) showed that REST is regulated by ubiquitin-mediated proteolysis, and used an RNA interference screen to identify a Skp1-Cul1-F-box protein complex containing the F-box protein beta-TRCP as an E3 ubiquitin ligase responsible for REST degradation. Beta-TRCP binds and ubiquitinates REST and controls its stability through a conserved phospho-degron. During neural differentiation, REST is degraded in a beta-TRCP-dependent manner. Beta-TRCP is required for proper neural differentiation only in the presence of REST, indicating that beta-TRCP facilitates this process through degradation of REST. Conversely, failure to degrade REST attenuates differentiation. Furthermore, Westbrook et al. (2008) found that beta-TRCP overexpression, which is common in human epithelial cancers, causes oncogenic transformation of human mammary epithelial cells and that this pathogenic function requires REST degradation. Thus, Westbrook et al. (2008) concluded that REST is a key target in beta-TRCP-driven transformation and that the beta-TRCP-REST axis is a new regulatory pathway controlling neurogenesis.

Pierce et al. (2009) developed a quantitative framework based on product distribution that predicted that the really interesting new gene (RING) E3 enzymes SCF(Cdc4) (see 606278) and SCF-beta-TrCP work with the E2 Cdc34 (116948) to build polyubiquitin chains on substrates by sequential transfers of single ubiquitins. Measurements with millisecond time resolution directly demonstrated that substrate polyubiquitylation proceeds sequentially. Pierce et al. (2009) concluded that their results presented an unprecedented glimpse into the mechanism of RING ubiquitin ligases and illuminated the quantitative parameters that underlie the rate and pattern of ubiquitin chain assembly.

De Mollerat et al. (2003) conducted mutation analysis of the dactylin gene (DAC; 608071) in 7 patients with split-hand/split-foot malformation (SHFM3; 246560) and found no point mutations. However, Southern blot, pulsed field gel electrophoresis, and dosage analyses demonstrated a complex rearrangement associated with a 0.5-Mb tandem duplication in all the patients. This duplicated region contained a disrupted extra copy of the dactylin gene and the entire LBX1 (604255), BTRC, and POLL (606343) genes. Similar to BTRC, DAC has been shown to be an F-box/WD40-repeat protein involved in NFKB signaling. The authors hypothesized that dysregulation of DAC and/or BTRC expression may be responsible for the defect in limb development seen in these patients.

Lyle et al. (2006) used FISH and quantitative PCR to narrow the SHFM3 locus to a minimal 325-kb duplication containing only the BTRC and POLL genes. Expression analysis of 13 candidate genes within and flanking the duplicated region showed that BTRC and SUFU (607035), present in 3 copies and 2 copies, respectively, were overexpressed in SHFM3 patients compared to controls. Lyle et al. (2006) suggested that SHFM3 may be caused by overexpression of BTRC and SUFU, both of which are involved in beta-catenin signaling.

By radiation hybrid analysis and FISH, Fujiwara et al. (1999) mapped the BTRC gene to chromosome 10q24-q25. Winston et al. (1999) mapped the BTRC gene to 10q24 by FISH. They noted that this region displays genetic abnormalities in a limited number of prostate, melanocyte, and neural cancers.

Jin et al. (2004) reported that the BTRC gene maps to chromosome 10q24.32 and the mouse Btrc gene maps to chromosome 19C3.

Guardavaccaro et al. (2003) found that loss of Btrc in mice did not affect viability, but it induced an impairment of spermatogenesis and reduced male fertility.

Kudo et al. (2004) found that mammary glands of female Btrc -/- mice showed reduced branching and hypoplasia. They observed no differences between mutant and wildtype mammary glands during pubertal ductal elongation, pregnancy, and lactation, the times of maximal hormonal stimulation. To further investigate the role of Btrc in mammary gland development, Kudo et al. (2004) generated transgenic mice expressing human BTRC targeted to mammary epithelia. Transgenic mammary glands displayed increased lateral ductal branching and extensive arrays of alveolus-like protuberances. The mammary epithelia of transgenic mice proliferated more and showed increased NFKB DNA-binding activity and higher nuclear levels of NFKB p65 (RELA; 164014). In addition, 38% of transgenic mice developed tumors, including mammary, ovarian, and uterine carcinomas. Targeting the BTRC gene to lymphoid organs produced no effects in these tissues.