The NEDD8 pathway is essential for SCF(beta -TrCP)-mediated ubiquitination and processing of the NF-kappa B precursor p105

The p50 subunit of NF-kappaB is generated by limited processing of the precursor p105. IkappaB kinase-mediated phosphorylation of the C-terminal domain of p105 recruits the SCF(beta-TrCP) ubiquitin ligase, resulting in rapid ubiquitination and subsequent processing/degradation of p105. NEDD8 is known to activate SCF ligases following modification of their cullin component. Here we show that NEDDylation is required for conjugation and processing of p105 by SCF(beta-TrCP) following phosphorylation of the molecule. In a crude extract, a dominant negative E2 enzyme, UBC12, inhibits both conjugation and processing of p105, and inhibition is alleviated by an excess of WT- UBC12. In a reconstituted cell-free system, ubiquitination of p105 was stimulated only in the presence of all three components of the NEDD8 pathway, E1, E2, and NEDD8. A Cul-1 mutant that cannot be NEDDylated could not stimulate ubiquitination and processing of p105. Similar findings were observed also in cells. It should be noted that NEDDylation is required only for the stimulated but not for basal processing of p105. Although the mechanisms that underlie processing of p105 are largely obscure, it is clear that NEDDylation and the coordinated activity of SCF(beta-TrCP) on both p105 and IkappaBalpha serve as an important regulatory mechanism controlling NF-kappaB activity.     

Shared pathways of IkappaB kinase-induced SCF(betaTrCP)-mediated ubiquitination and degradation for the NF-kappaB precursor p105 and IkappaBalpha

p105 (NFKB1) acts in a dual way as a cytoplasmic IkappaB molecule and as the source of the NF-kappaB p50 subunit upon processing. p105 can form various heterodimers with other NF-kappaB subunits, including its own processing product, p50, and these complexes are signal responsive. Signaling through the IkappaB kinase (IKK) complex invokes p105 degradation and p50 homodimer formation, involving p105 phosphorylation at a C-terminal destruction box. We show here that IKKbeta phosphorylation of p105 is direct and does not require kinases downstream of IKK. p105 contains an IKK docking site located in a death domain, which is separate from the substrate site. The substrate residues were identified as serines 923 and 927, the latter of which was previously assumed to be a threonine. S927 is part of a conserved DSGPsi motif and is functionally most critical. The region containing both serines is homologous to the N-terminal destruction box of IkappaBalpha, -beta, and -epsilon. Upon phosphorylation by IKK, p105 attracts the SCF E3 ubiquitin ligase substrate recognition molecules betaTrCP1 and betaTrCP2, resulting in polyubiquitination and complete degradation by the proteasome. However, processing of p105 is independent of IKK signaling. In line with this and as a physiologically relevant model, lipopolysaccharide (LPS) induced degradation of endogenous p105 and p50 homodimer formation, but not processing in pre-B cells. In mutant pre-B cells lacking IKKgamma, processing was unaffected, but LPS-induced p105 degradation was abolished. Thus, a functional endogenous IKK complex is required for signal-induced p105 degradation but not for processing.     

Dual effects of IkappaB kinase beta-mediated phosphorylation on p105 Fate: SCF(beta-TrCP)-dependent degradation and SCF(beta-TrCP)-independent processing

Processing of the p105 NF-kappaB precursor to yield the p50 active subunit is a unique and rare case in which the ubiquitin system is involved in limited processing rather than in complete destruction of its target. The mechanisms involved in this process are largely unknown, although a glycine repeat in the middle of p105 has been identified as a processing stop signal. IkappaB kinase (IKK)beta-mediated phosphorylation at the C-terminal domain with subsequent recruitment of the SCF(beta-TrCP) ubiquitin ligase leads to accelerated processing and degradation of the precursor, yet the roles that the kinase and ligase play in each of these two processes have not been elucidated. Here we demonstrate that IKKbeta has two distinct functions: (i) stimulation of degradation and (ii) stimulation of processing. IKKbeta-induced degradation is dependent on SCF(beta-TrCP), which acts through multiple lysine residues in the IkappaBgamma domain. In contrast, IKKbeta-induced processing of p105 is beta-transduction repeat-containing protein (beta-TrCP) independent, as it is not affected by expression of a dominant-negative beta-TrCP or following its silencing by small inhibitory RNA. Furthermore, removal of all 30 lysine residues from IkappaBgamma results in complete inhibition of IKK-dependent degradation but has no effect on IKK-dependent processing. Yet processing still requires the activity of the ubiquitin system, as it is inhibited by dominant-negative UbcH5a. We suggest that IKKbeta mediates its two distinct effects by affecting, directly and indirectly, two different E3s.     

Epitope mapping of the phosphorylation motif of the HIV-1 protein Vpu bound to the selective monoclonal antibody using TRNOESY and STD NMR spectroscopy

The conformational preferences of a 22-amino acid peptide (LIDRLIERAEDpSGNEpSEGEISA) that mimics the phosphorylated HIV-1-encoded virus protein U (Vpu) antigen have been investigated by NMR spectroscopy. Degradation of HIV receptor CD4 by the proteasome, mediated by the HIV-1 protein Vpu, is crucial for the release of fully infectious virions. Phosphorylation of Vpu at sites Ser52 and Ser56 on the DSGXXS motif is required for the interaction of Vpu with the ubiquitin ligase SCF(beta)(-TrCP) which triggers CD4 degradation by the proteasome. This motif is conserved in several signaling proteins known to be degraded by the proteasome. The interaction of the P-Vpu(41-62) peptide with its monoclonal antibody has been studied by transferred nuclear Overhauser effect NMR spectroscopy (TRNOESY) and saturation transfer difference NMR (STD NMR) spectroscopy. The peptide was found to adopt a bend conformation upon binding to the antibody; the peptide residues (Asp51-pSer56) forming this bend are recognized by the antibody as demonstrated by STD NMR experiments. The three-dimensional structure of P-Vpu(41-62) in the bound conformation was determined by TRNOESY spectra; the peptide adopts a compact structure in the presence of mAb with formation of several bends around Leu45 and Ile46 and around Ile60 and Ser61, with a tight bend created by the DpS(52)GNEpS(56) motif. STD NMR studies provide evidence for the existence of a conformational epitope containing tandem repeats of phosphoserine motifs. The peptide's epitope is predominantly located in the large bend and in the N-terminal segment, implicating bidentale association. These findings are in excellent agreement with a recently published NMR structure required for the interaction of Vpu with the SCF(beta)(-TrCP) protein.     

DEPTOR ubiquitination and destruction by SCF(β-TrCP)

β-Transducin repeats-containing protein (β-TrCP) is the substrate recognition subunit of the SCF (SKP1, CUL1, and F-box protein)-type E3 ubiquitin ligase complex. SCF(β-TrCP) ubiquitinates specifically phosphorylated substrates to promote their subsequent destruction by the 26S proteasome and plays a critical role in various human diseases including tumorigenesis. We and others (Duan S et al. Mol Cell 44: 317-324, 2011; Gao D et al. Mol Cell 44: 290-303, 2011; Zhao Y et al. Mol Cell 44: 304-316, 2011) recently reported that SCF(β-TrCP) regulates cell growth and autophagy by controlling the ubiquitination and destruction of DEPTOR, an endogenous mammalian target of rapamycin inhibitor, in a phosphorylation-dependent manner. In this review, we discuss β-TrCP's new downstream substrate, DEPTOR, as well as summarize the novel functional aspects of β-TrCP in controlling cell growth and regulating autophagy, in part through governing the stability of DEPTOR.     

Molecular dissection of the interactions among IkappaBalpha, FWD1, and Skp1 required for ubiquitin-mediated proteolysis of IkappaBalpha.

The SCF complex containing Skp1, Cul1, and the F-box protein FWD1 (the mouse homologue of Drosophila Slimb and Xenopus beta-TrCP) functions as the ubiquitin ligase for IkappaBalpha. FWD1 associates with Skp1 through the F-box domain and also recognizes the conserved DSGXXS motif of IkappaBalpha. The structural requirements for the interactions of FWD1 with IkappaBalpha and with Skp1 have now been investigated further. The D31A mutation (but not the G33A mutation) in the DSGXXS motif of IkappaBalpha abolished the binding of IkappaBalpha to FWD1 and its subsequent ubiquitination without affecting the phosphorylation of IkappaBalpha. The IkappaBalpha mutant D31E still exhibited binding to FWD1 and underwent ubiquitination. These results suggest that, in addition to site-specific phosphorylation at Ser(32) and Ser(36), an acidic amino acid at position 31 is required for FWD1-mediated ubiquitination of IkappaBalpha. Deletion analysis of Skp1 revealed that residues 61-143 of this protein are required for binding to FWD1. On the other hand, the highly conserved residues Pro(149), Ile(160), and Leu(164) in the F-box domain of FWD1 were dispensable for binding to Skp1. Together, these data delineate the structural requirements for the interactions among IkappaBalpha, FWD1, and Skp1 that underlie substrate recognition by the SCF ubiquitin ligase complex.     

Phosphorylation-dependent structure of ATF4 peptides derived from a human ATF4 protein, a member of the family of transcription factors

ATF4 plays a crucial role in the cellular response to stress and the F-box protein β-TrCP, the receptor component of the SCF E3 ubiquitin ligase responsible for ATF4 degradation by the proteasome, binds to ATF4, and controls its stability. Association between the two proteins depends on ATF4 phosphorylation of serine residues 219 and 224 present in the context of DpSGXXXpS, which is similar but not identical to the DpSGXXpS motif found in most other substrates of β-TrCP. We used NMR spectroscopy to analyze the structure of the 23P-ATF4 peptide. The 3D structure of the ligand was determined on the basis of NOESY restraints that provide an hairpin loop structure. In contrast, no ordered structure was observed in the NMR experiments for the nonphosphorylated 23-ATF4 in solution. This structural study provides information, which could be used to study the β-TrCP receptor–ligand interaction in docking procedure. Docking studies showed that the binding epitope of the ligand, is represented by the DpSGIXXpSXE motif. 23P-ATF4 peptide fits the binding pocket of protein β-TrCP very well, considering that the DpSGIXXpSXE motif adopts an S-turning conformation contrary to the extended DpSGXXpS motif in the other known β-TrCP ligands.     

Meiotic regulation of the CDK activator RINGO/Speedy by ubiquitin-proteasome-mediated processing and degradation

Xenopus RINGO/Speedy (XRINGO) is a potent inducer of oocyte meiotic maturation that can directly activate Cdk1 and Cdk2. Here, we show that endogenous XRINGO protein accumulates transiently during meiosis I entry and then is downregulated. This tight regulation of XRINGO expression is the consequence of two interconnected mechanisms: processing and degradation. XRINGO processing involves recognition of at least three distinct phosphorylated recognition motifs by the SCF(betaTrCP) ubiquitin ligase, followed by proteasome-mediated limited degradation, resulting in an amino-terminal XRINGO fragment. XRINGO processing is directly stimulated by several kinases, including protein kinase A and glycogen synthase kinase-3beta, and may contribute to the maintenance of G2 arrest. On the other hand, XRINGO degradation after meiosis I is mediated by the ubiquitin ligase Siah-2, which probably requires phosphorylation of XRINGO on Ser 243 and may be important for the omission of S phase at the meiosis-I-meiosis-II transition in Xenopus oocytes.     

Phosphorylation of Ser72 is dispensable for Skp2 assembly into an active SCF ubiquitin ligase and its subcellular localization

F-box proteins are the substrate recognition subunits of SCF (Skp1, Cul1, F-box protein) ubiquitin ligase complexes. Skp2 is a nuclear F-box protein that targets the CDK inhibitor p27 for ubiquitin- and proteasome-dependent degradation. In G0 and during the G1 phase of the cell cycle, Skp2 is degraded via the APC/CCdh1 ubiquitin ligase to allow stabilization of p27 and inhibition of CDKs, facilitating the maintenance of the G0/G1 state. APC/CCdh1 binds Skp2 through an N-terminal domain (amino acids 46-94 in human Skp2). It has been shown that phosphorylation of Ser69 and Ser72 in this domain dissociates Skp2 from APC/C. More recently, it has instead been proposed that phosphorylation of Skp2 on Ser72 by Akt/PKB allows Skp2 binding to Skp1, promoting the assembly of an active SCFSkp2 ubiquitin ligase, and Skp2 relocalization/retention into the cytoplasm, promoting cell migration via an unknown mechanism. According to these reports, a Skp2 mutant in which Ser72 is substituted with Ala is unable to promote cell proliferation and loses its oncogenic potential. Given the contrasting reports, we revisited these results and conclude that phosphorylation of Skp2 on Ser72 does not control Skp2 binding to Skp1 and Cul1, has no influence on SCFSkp2 ubiquitin ligase activity, and does not affect the subcellular localization of Skp2.     

The ubiquitin ligase SCF(betaTrCP) regulates the degradation of the growth hormone receptor

SCF ubiquitin ligases play a pivotal role in the regulation of cell division and various signal transduction pathways, which in turn are involved in cell growth, survival, and transformation. SCF(TrCP) recognizes the double phosphorylated DSGPhiXS destruction motif in beta-catenin and IkappaB. We show that the same ligase drives endocytosis and degradation of the growth hormone receptor (GHR) in a ligand-independent fashion. The F-box protein beta-TrCP binds directly and specifically with its WD40 domain to a novel recognition motif, previously designated as the ubiquitin-dependent endocytosis motif. Receptor degradation requires an active neddylation system, implicating ubiquitin ligase activity. GHR-TrCP binding, but not GHR ubiquitination, is necessary for endocytosis. TrCP2 silencing is more effective on GHR degradation and endocytosis than TrCP1, although overexpression of either isoform restores TrCP function in silenced cells. Together, these findings provide direct evidence for a key role of the SCF(TrCP) in the endocytosis and degradation of an important factor in growth, immunity, and life span regulation.     

Role of the SCFSkp2 ubiquitin ligase in the degradation of p21Cip1 in S phase

The cyclin-dependent kinase inhibitor p21Cip1 has important roles in the control of cell proliferation, differentiation, senescence, and apoptosis. It has been observed that p21 is a highly unstable protein, but the mechanisms of its degradation remained unknown. We show here that p21 is a good substrate for an SCF (Skp1-Cullin1-F-box protein) ubiquitin ligase complex, which contains the F-box protein Skp2 (S phase kinase-associated protein 2) and the accessory protein Cks1 (cyclin kinase subunit 1). A similar ubiquitin ligase complex has been previously shown to be involved in the degradation of a related cyclin-dependent kinase inhibitor, p27Kip1. The levels of Skp2 oscillate in the cell cycle, reaching a maximum in S phase. The ubiquitylation of p21 in vitro required the supplementation of all components of the SCF complex as well as of Cks1 and Cdk2-cyclin E. The protein kinase Cdk2-cyclin E acts both by the phosphorylation of p21 on Ser-130 and by the formation of a complex with p21, which is required for its presentation to the ubiquitin ligase. As opposed to the case of p27, the phosphorylation of p21 stimulates its ubiquitylation but is not absolutely required for this process. Levels of p21 are higher in Skp2-/- mouse embryo fibroblasts than in wild-type fibroblasts in the S phase, and the rates of the degradation of p21 are slower in cells that lack Skp2. It is suggested that SCFSkp2 participates in the degradation of p21 in the S phase.     

The mRNA-stabilizing Factor HuR Protein Is Targeted by β-TrCP Protein for Degradation in Response to Glycolysis Inhibition

The mRNA-stabilizing protein HuR acts a stress response protein whose function and/or protein stability are modulated by diverse stress stimuli through posttranslational modifications. Here, we report a novel mechanism by which metabolic stress facilitates proteasomal degradation of HuR in cancer cells. In response to the glucose transporter inhibitor CG-5, HuR translocates to the cytoplasm, where it is targeted by the ubiquitin E3 ligase β-TrCP1 for degradation. The cytoplasmic localization of HuR is facilitated by PKCα-mediated phosphorylation at Ser-318 as the Ser-318 → alanine substitution abolishes the ability of the resulting HuR to bind PKCα and to undergo nuclear export. The mechanistic link between β-TrCP1 and HuR degradation was supported by the ability of ectopically expressed β-TrCP1 to mimic CG-5 to promote HuR degradation and by the protective effect of dominant negative inhibition of β-TrCP1 on HuR ubiquitination and degradation. Substrate targeting of HuR by β-TrCP1 was further verified by coimmunoprecipitation and in vitro GST pull-down assays and by the identification of a β-TrCP1 recognition site. Although HuR does not contain a DSG destruction motif, we obtained evidence that β-TrCP1 recognizes an unconventional motif, ²⁹⁶EEAMAIAS³⁰⁴, in the RNA recognition motif 3. Furthermore, mutational analysis indicates that IKKα-dependent phosphorylation at Ser-304 is crucial to the binding of HuR to β-TrCP1. Mechanistically, this HuR degradation pathway differs from that reported for heat shock and hypoxia, which underlies the complexity in the regulation of HuR turnover under different stress stimuli. The ability of glycolysis inhibitors to target the expression of oncogenic proteins through HuR degradation might foster novel strategies for cancer therapy.     

BTB/POZ domain proteins are putative substrate adaptors for cullin 3 ubiquitin ligases

Cullins (CULs) are subunits of a prominent class of RING ubiquitin ligases. Whereas the subunits and substrates of CUL1-associated SCF complexes and CUL2 ubiquitin ligases are well established, they are largely unknown for other cullin family members. We show here that S. pombe CUL3 (Pcu3p) forms a complex with the RING protein Pip1p and all three BTB/POZ domain proteins encoded in the fission yeast genome. The integrity of the BTB/POZ domain, which shows similarity to the cullin binding proteins SKP1 and elongin C, is required for this interaction. Whereas Btb1p and Btb2p are stable proteins, Btb3p is ubiquitylated and degraded in a Pcu3p-dependent manner. Btb3p degradation requires its binding to a conserved N-terminal region of Pcu3p that precisely maps to the equivalent SKP1/F box adaptor binding domain of CUL1. We propose that the BTB/POZ domain defines a recognition motif for the assembly of substrate-specific RING/cullin 3/BTB ubiquitin ligase complexes.     

Genetic evidence for the essential role of beta-transducin repeat-containing protein in the inducible processing of NF-kappa B2/p100

Processing of the nf kappa b2 gene product p100 to generate p52 is an important step in NF-kappa B regulation. This step is regulated by a nonclassical NF-kappa B signaling pathway involving the NF-kappa B-inducing kinase (NIK). NIK induces p100 processing by triggering phosphorylation of specific C-terminal serines of p100. However, the downstream molecular events leading to p100 processing remain unclear. Here we show that NIK induced the physical recruitment of beta-transducin repeat-containing protein (beta-TrCP), a component of the SCF ubiquitin ligase complex, to p100. This event required the phosphorylation sites as well as the death domain of p100. Using the RNA interference technique, we demonstrated that beta-TrCP is essential for NIK-induced p100 ubiquitination and processing. Interestingly the constitutive processing of p100 mutants was independent of beta-TrCP. These results suggest that beta-TrCP is an essential component of NIK-induced p100 processing.