Nedd8 Modification of Cul-1 Activates SCFβTrCP-Dependent Ubiquitination of IκBα

摘要: NF-κB is a transcription factor required for inducible expression of number proinflammatory mediators including cytokines, chemokines, and leukocyte adhesion molecules (6). In addition, regulates the survival genes which prevent cell death in response to tumor necrosis alpha (TNF-α) (7, 37, 59, 62). member Rel family proteins typically heterodimer composed p50 p65 subunits. quiescent cells, retained cytosol bound IκB, inhibitory mask nuclear localization DNA binding sequences on (5, 22). Stimulation these cells with various lipopolysaccharide, viruses, antigens, or oxidants triggers signaling events that ultimately lead phosphorylation degradation allowing translocate into nucleus activate target (3, 21, 38, 54). Phosphorylation Ser32 Ser36 has been shown IκB ubiquitination subsequent proteolysis by ubiquitin-proteasome pathway (UPP) protein (2, 8, 45, 49). The UPP principal intracellular turnover, regulatory (9). Protein substrates enter are first marked covalent ligation polyubiquitin chains mediated cascade enzymes called E1 (ubiquitin activation enzyme), E2 (ubiquitin-conjugating E3 ligase) reaction requiring ATP, ubiquitin activated charged onto an through thioester formed between active-site cysteine residue C-terminal glycine ubiquitin. then directs transfer from lysine residues within specific substrate proteins, resulting formation ubiquitin-protein conjugate. Polyubiquitinated recognized degraded 26S proteasome complex yield small peptides monomeric ubiquitin. Recently, receptor component was identified as βTrCP (beta-transducin repeat-containing protein) E3RSIκB (39, 53, 63, 65) HOS (11). much larger F-box domain containing form SCF complexes. core components complexes include Skp-1, interacts domain, Cul-1, linked via Skp-1 (4, 10, 35, 46, 47, 51). At least two additional have described: (i) Rbx1, thought stabilize interaction Cul-1 E2s, Cdc34, Ubc5 (25, 26, 43, 50, 52, 56), (ii) Sgt1, (27). were initially described yeast function ligases variety phosphorylated cycle regulator, Sic1 (10, addition also contains WD40 repeat specifically recognizes IκBα only when Ser36. Similarly, at other phosphorylation-dependent manner, β-catenin (16, 31, 36, 63) human immunodeficiency virus type 1 Vpu (40). deleted (ΔF-βTrCP) retains its specificity but fails interact no longer supports reaction. Thus, essential function. SCFβTrCP alone, however, not sufficient support (53, 65). Additional components, supplied crude cellular extracts (63) recombinant (including UbcH5 [43, 65], Cdc34 [56], Rbx1 [56]), activity, suggesting and/or modifications existing needed SCFβTrCP. To date, characterized. In effort understand requirements SCFβTrCP-mediated IκBα, we examined associate well IκBα. Remarkably, observed endogenous associated exclusively singly modified ubiquitin-like Nedd8. Along this line, found optimal vitro presence Nedd8 Nedd8-conjugating enzyme, Ubc12, ubiquitin-conjugating enzymes, UbcH5A Cdc34. Moreover, point mutant ability lacks site modification, showed greatly reduced ubiquitinate vitro. It established percentage related cullin conjugates single mammalian (30, 44, 60), homologue, Rub1, genetically (30) plants (14). However, functional role any process demonstrated. Here show modification necessary SCFβTrCP, linking pathways regulation targeted degradation.