A wide range of cellular functions are therefore affected by the UPP, including proliferation, differentiation, programmed cell death and antigen presentation (Ciechanover et al., 2000). Most polyubiquitinated proteins are degraded by the 26S proteasome, an ATP-dependent, multicatalytic, multisubunit protease (Voges et al., 1999). One such pathway is the endoplasmic reticulum (ER)-associated degradation (ERAD) pathway, which targets proteins misfolded in the ER for retrograde transport into the cytosol and degradation through the UPP ( Lord et al., 2000 ).
The prototype HECT domain protein (homologous to the carboxyl terminus of E6-AP) is E6-AP, a cellular protein that is recruited by the human papillomavirus E6 oncoprotein for ubiquitination of the tumor suppressor p53 (Scheffner et al., 1993). HECT domain-containing ubiquitin ligases are the only E3s known to form a thioester bond with ubiquitin (via a conserved cysteine in the HECT domain) prior to transfer of ubiquitin to the substrate protein (Huibregtse et al., 1995). HECT E3 domains contain divergent amino-terminal domains that function in binding the respective substrate (Huibregtse et al., 1995).
PP2A is recruited to Mid1 through binding of the PP2A α4 regulatory subunit to the first of two B-boxes in Mid1 (Trockenbacher et al., 2001). Roc1/Rbx1/Hrt1 and Apc11 are RING finger domain-containing proteins, many of which have recently been implicated in the ubiquitination process (see above) (Lorick et al., 1999).
The destruction signal in the substrate consists of either a nine-residue destruction box (D-box) (Glotzer et al., 1991). However, the exact mechanisms of epitope generation by the immunoproteasome remain uncertain and the precise immunological role(s) of the immunoproteasome remain unclear (Groettrup et al., 2001). Mutations in the UBP family member Fat facets (Faf) resulted in elevated numbers of photoreceptors in each facet of the Drosophila compound eye (Fischer-Vize et al., 1992).
The mouse homologue of Faf, Fam, has been shown to bind and stabilize b-catenin (Taya et al., 1999). Furthermore, unlike the case with lqf, kano mutations do not act as strong dominant enhancers of the faf mutant phenotype (Cadavid et al., 2000). Another group has described a ubiquitin-aldehyde-sensitive isopeptidase editing activity present in the 19S regulatory complex of the proteasome (Lam et al., 1997;
Mutation of the active site cysteine to serine (C60S), aspartic acid to asparagine (D133N), or histidine to glutamine (H298Q). As is the case for Dub-1, Dub-2 and Dub-2A each have deubiquitinating activity that is abolished by mutation of the active site cysteine to serine (C60S) ( Baek et al., 2001 ). Third, catalytically inactive versions of IKKs inhibit cytokine-induced NF-kB activation in a dominant-negative manner ( Mercurio et al., 1997 ).
It has been shown that NF-kB regulates the G1/S transition in several cell types (Joyce et al., 2001).
L32 GAPDH
Immunoanalyses of the anti-HA immune complexes using an antibody against the carboxyl terminus of Dub-1 revealed multiple high-molecular-weight proteins, consistent with the appearance of a Dub-1 poly HA-ubiquitin “ladder”. Collectively, these experiments indicated that Dub-1 polyubiquitinates and is degraded by the UPP. The observation that one member of the Dub-1 family (Dub-1) but not another member (Dub-2) is degraded by the UPP was unexpected, given the amino acid similarities of the Dub-1 and Dub- 2 proteins (Figure) 1.5). Since mutation of the active site cysteine of Dub-1 to serine (C60S) abolishes Dub-1 deubiquitinating activity (Zhu et al., 1996), I tested whether Dub-1 (C60S) is transported through the UPP in HEK-293T cells degraded using the same experimental strategy used to demonstrate degradation of wild type Dub-1 by the UPP.
These studies use a riboprobe corresponding to the 3' end of the dub-1 ORF and an antibody directed against the carboxyl terminus of Dub-1. The overlapping expression patterns of dub-1 and c-rel mRNA in the developing limb bud, as well as the correlation between Dub-1 expression and IkBa stability in FL5.12 cells after IL-3 starvation, are consistent with a possible role for Dub- 1 in the regulation of the NF-kB signal transduction pathway. An examination of a potential role for Dub proteins in regulation of the NF-kB pathway is presented in CHAPTER IV.
Deubiquitination of proteins occurs via hydrolysis of the isopeptide bond at the carboxyl terminus of ubiquitin (D'Andrea and Pellman, 1998; Wilkinson, 2000). These observations include i) the relatively large number of deubiquitinating enzymes compared to components of the ubiquitination machinery, suggesting distinct roles for individual deubiquitinating enzymes. The protein concentration of clarified lysates was determined using the DC Protein Assay kit (Bio-Rad Laboratories, Hercules, CA).
FWD1/b-TrCP binds directly to IκBα phosphorylated at serine 32 and is a component of the ubiquitin ligase IκBα. A similar pattern of protein expression was observed on these blots (Figure 3.1 - right panel) as on the reciprocal immunoprecipitation blots (left and middle panels). The product of HA immunoprecipitations (Figure 3.1 - lane 3) was used as a substrate of an in vitro assay to monitor the deubiquitination activity of IkBa present in M12 cell lysates.
DTT was included in the lysis and reaction buffers to maintain a reduced state of cysteine in the active site of the deubiquitination enzymes. After a one-hour incubation at 37 °C, the deubiquitination of T7-IkBa was monitored by Western analysis of the reaction products using the IkBa carboxyl end antibody (Figure 3.2). Analysis of the products of the deubiquitination reaction (Figure 3.2, lanes 5–7) showed the presence of mouse IkBa (from M12 cell lysates) and poly HA-.
SUBSTRATE M1 2
Tag-Dub-1 and -2
Dub-1 and Dub-2 are members of the ubiquitin-processing protease (UBP) family of deubiquitinating enzymes. Together, these results provide strong evidence that Dub-1 and Dub-2 do not serve as positive or negative regulators of the NF-kB signaling pathway. Despite these alternative interpretations, the data presented in this chapter collectively provide strong evidence that Dub-1 and Dub-2 do not play a central role in the regulation of the NF-kB signaling pathway.
Inhibition of NF-kappaB activity leads to disruption of the apical ectodermal ridge and aberrant limb morphogenesis [see comments]. Activation of the IkappaB kinase complex by TRAF6 requires a dimeric ubiquitin-conjugating enzyme complex and a unique polyubiquitin chain. A subcomplex of the proteasome regulatory particle required for ubiquitin-conjugated degradation and related to the COP9-.
Regulation of Hedgehog and Wingless signaling pathways by the F-box/WD40-repeat protein Slimb. The role of Rel/NF-kappaB transcription factors during vertebrate limb growth [see comments]. Construction and Analysis of Mouse Strains Lacking the Ubiquitin Ligase UBR1 (E3alpha) of the N-End Rule Pathway.
The multiubiquitin chain-binding protein Mcb1 is a component of the 26S proteasome in Saccharomyces cerevisiae and plays a. Genetic analysis of the role of the drosophila fat-facet gene in the ubiquitin pathway.
