Recognition of phosphodegron motifs in human cyclin E by the SCF(Fbw7) ubiquitin ligase

Turnover of cyclin E is controlled by SCF(Fbw7). Three isoforms of Fbw7 are produced by alternative splicing. Whereas Fbw7alpha and -gamma are nuclear and the beta-isoform is cytoplasmic in 293T cells, all three isoforms induce cyclin E destruction in an in vivo degradation assay. Cyclin E is phosphorylated on Thr(62), Ser(88), Ser(372), Thr(380), and Ser(384) in vivo. To examine the roles of phosphorylation in cyclin E turnover, a series of alanine point mutations in each of these sites were analyzed for Fbw7-driven degradation. As expected, mutation of the previously characterized residue Thr(380) to alanine led to profound defects of cyclin E turnover, and largely abolished association with Fbw7. Mutation of Thr(62) to alanine led to a dramatic reduction in the extent of Thr(380) phosphorylation, suggesting an indirect effect of this mutation on cyclin E turnover. Nevertheless, phosphopeptides centered at Thr(62) associated with Fbw7, and residual binding of cyclin E(T380A) to Fbw7 was abolished upon mutation of Thr(62), suggesting a minor role for this residue in direct association with Fbw7. Mutation of Ser(384) to alanine also rendered cyclin E resistant to degradation by Fbw7, with the largest effects being observed with Fbw7beta. Cyclin E(S384A) associated more weakly with Fbw7alpha and -beta isoforms but was not defective in Thr(380) phosphorylation. Analysis of the localization of cyclin E mutant proteins indicated selective accumulation of cyclin E(S384A) in the nucleus, which may contribute to the inability of cytoplasmic Fbw7beta to promote turnover of this cyclin E mutant protein.     

CD44 modulates Smad1 activation in the BMP-7 signaling pathway

Bone morphogenetic protein 7 (BMP-7) regulates cellular metabolism in embryonic and adult tissues. Signal transduction occurs through the activation of intracellular Smad proteins. In this paper, using a yeast two-hybrid screen, Smad1 was found to interact with the cytoplasmic domain of CD44, a receptor for the extracellular matrix macromolecule hyaluronan. Coimmunoprecipitation experiments confirmed the interaction of Smad1 with full-length CD44-interactions that did not occur when CD44 receptors truncated within the cytoplasmic domain were tested. Chondrocytes overexpressing a truncated CD44 on a background of endogenous full-length CD44 no longer exhibited Smad1 nuclear translocation upon BMP-7 stimulation. Further, pretreatment of chondrocytes with Streptomyces hyaluronidase to disrupt extracellular hyaluronan-cell interactions inhibited BMP-7-mediated Smad1 phosphorylation, nuclear translocation of Smad1 or Smad4, and SBE4-luciferase reporter activation. These results support a functional link between the BMP signaling cascade and CD44. Thus, changes in hyaluronan-cell interactions may serve as a means to modulate cellular responsiveness to BMP.     

The anaphase-promoting complex mediates TGF-beta signaling by targeting SnoN for destruction.

Degradation of SnoN is thought to play an important role in the transactivation of TGF-beta responsive genes. We demonstrate that the anaphase-promoting complex (APC) is a ubiquitin ligase required for the destruction of SnoN and that the APC pathway is regulated by TGF-beta. The destruction box of SnoN is required for its degradation in response to TGF-beta signaling. Furthermore, the APC activator CDH1 and Smad3 synergistically regulate SnoN degradation. Under these circumstances, CDH1 forms a quaternary complex with SnoN, Smad3, and APC. These results suggest that APC(CDH1) and SnoN play central roles in regulating growth through the TGF-beta signaling system.     

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.     

Regulation of APCCdh1 E3 ligase activity by the Fbw7/cyclin E signaling axis contributes to the tumor suppressor function of Fbw7

Fbw7 and Cdh1 are substrate-recognition subunits of the SCF- and APC-type E3 ubiquitin ligases, respectively. There is emerging evidence suggesting that both Fbw7 and Cdh1 function as tumor suppressors by targeting oncoproteins for destruction. Loss of Fbw7, but not Cdh1, is frequently observed in various human tumors. However, it remains largely unknown how Fbw7 mechanistically functions as a tumor suppressor and whether there is a signaling crosstalk between Fbw7 and Cdh1. Here, we report that Fbw7-deficient cells not only display elevated expression levels of SCF^Fbw7 substrates, including cyclin E, but also have increased expression of various APC^Cdh1 substrates. We further defined cyclin E as the critical signaling link by which Fbw7 governs APC^Cdh1 activity, as depletion of cyclin E in Fbw7-deficient cells results in decreased expression of APC^Cdh1 substrates to levels comparable to those in wild-type (WT) cells. Conversely, ectopic expression of cyclin E recapitulates the aberrant APC^Cdh1 substrate expression observed in Fbw7-deficient cells. More importantly, 4A-Cdh1 that is resistant to Cdk2/cyclin E-mediated phosphorylation, but not WT-Cdh1, reversed the elevated expression of various APC^Cdh1 substrates in Fbw7-deficient cells. Overexpression of 4A-Cdh1 also resulted in retarded cell growth and decreased anchorage-independent colony formation. Altogether, we have identified a novel regulatory mechanism by which Fbw7 governs Cdh1 activity in a cyclin E-dependent manner. As a result, loss of Fbw7 can lead to aberrant increase in the expression of both SCF^Fbw7 and APC^Cdh1 substrates. Our study provides a better understanding of the tumor suppressor function of Fbw7, and suggests that Cdk2/cyclin E inhibitors could serve as effective therapeutic agents for treating Fbw7-deficient tumors.     

Phosphorylation-dependent ubiquitination of cyclin D1 by the SCF(FBX4-alphaB crystallin) complex

Growth factor-dependent accumulation of the cyclin D1 proto-oncogene is balanced by its rapid phosphorylation-dependent proteolysis. Degradation is triggered by threonine 286 phosphorylation, which promotes its ubiquitination by an unknown E3 ligase. We demonstrate that Thr286-phosphorylated cyclin D1 is recognized by a Skp1-Cul1-F box (SCF) ubiquitin ligase where FBX4 and alphaB crystallin govern substrate specificity. Overexpression of FBX4 and alphaB crystallin triggered cyclin D1 ubiquitination and increased cyclin D1 turnover. Impairment of SCF(FBX4-alphaB crystallin) function attenuated cyclin D1 ubiquitination, promoting cyclin D1 overexpression and accelerated cell-cycle progression. Purified SCF(FBX4-alphaB crystallin) catalyzed polyubiquitination of cyclin D1 in vitro. Consistent with a putative role for a cyclin D1 E3 ligase in tumorigenesis, FBX4 and alphaB crystallin expression was reduced in tumor-derived cell lines and a subset of primary human cancers that overexpress cyclin D1. We conclude that SCF(FBX4-alphaB crystallin) is an E3 ubiquitin ligase that promotes ubiquitin-dependent degradation of Thr286-phosphorylated cyclin D1.     

Regulation of the Wnt signaling pathway by disabled-2 (Dab2)

The adaptor molecule Disabled-2 (Dab2) has been shown to link cell surface receptors to downstream signaling pathways. Using a small-pool cDNA screening strategy, we identify that the N-terminal domain of Dab2 interacts with Dishevelled-3 (Dvl-3), a signaling mediator of the Wnt pathway. Ectopic expression of Dab2 in NIH-3T3 mouse fibroblasts attenuates canonical Wnt/beta-catenin-mediated signaling, including accumulation of beta-catenin, activation of beta-catenin/T-cell-specific factor/lymphoid enhancer-binding factor 1-dependent reporter constructs, and endogenous cyclin D1 induction. Wnt stimulation leads to a time-dependent dissociation of endogenous Dab2-Dvl-3 and Dvl-3-axin interactions in NIH-3T3 cells, while Dab2 overexpression leads to maintenance of Dab2-Dvl-3 association and subsequent loss of Dvl-3-axin interactions. In addition, we find that Dab2 can associate with axin in vitro and stabilize axin expression in vivo. Mouse embryo fibroblasts which lack Dab2 exhibit constitutive Wnt signaling as evidenced by increased levels of nuclear beta-catenin and cyclin D1 protein levels. Based on these results, we propose that Dab2 functions as a negative regulator of canonical Wnt signaling by stabilizing the beta-catenin degradation complex, which may contribute to its proposed role as a tumor suppressor.     

p27(Kip1) ubiquitination and degradation is regulated by the SCF(Skp2) complex through phosphorylated Thr187 in p27.

Many tumorigenic processes affect cell-cycle progression by their effects on the levels of the cyclin-dependent kinase inhibitor p27(Kip1) [1,2]. The phosphorylation- and ubiquitination-dependent proteolysis of p27 is implicated in control of the G1-S transition in the cell cycle [3-6]. To determine the factors that control p27 stability, we established a cell-free extract assay that recapitulates the degradation of p27. Phosphorylation of p27 at Thr187 was essential for its degradation. Degradation was also dependent on SCF(Skp2), a protein complex implicated in targeting phosphorylated proteins for ubiquitination [7-10]. Immunodepletion of components of the complex - Cul-1, Skp1, or Skp2 - from the extract abolished p27 degradation, while addition of purified SCF(Skp2) to Skp2- depleted extract restored the capacity to degrade p27. A specific association was observed between Skp2 and a p27 carboxy-terminal peptide containing phosphorylated Thr187, but not between Skp2 and the non-phosphorylated peptide. Skp2-dependent associations between Skp1 or Cul-1 and the p27 phosphopeptide were also detected. Isolated SCF(Skp2) contained an E3 ubiquitin ligase activity towards p27. Our data thus suggest that SCF(Skp2) specifically targets p27 for degradation during cell-cycle progression.     

Protein targeting by the bacterial twin-arginine translocation (Tat) pathway

The Tat (twin-arginine translocation) protein export system is found in the cytoplasmic membrane of most prokaryotes and is dedicated to the transport of folded proteins. The Tat system is now known to be essential for many bacterial processes including energy metabolism, cell wall biosynthesis, the nitrogen-fixing symbiosis and bacterial pathogenesis. Recent studies demonstrate that substrate-specific accessory proteins prevent improperly assembled substrates from interacting with the Tat transporter. During the transport cycle itself substrate proteins bind to a receptor complex in the membrane which then recruits a protein-translocating channel to carry out the transport reaction.     

Isoform- and cell cycle-dependent substrate degradation by the Fbw7 ubiquitin ligase

The SCF(FBW7) ubiquitin ligase degrades proteins involved in cell division, growth, and differentiation and is commonly mutated in cancers. The Fbw7 locus encodes three protein isoforms that occupy distinct subcellular localizations, suggesting that each has unique functions. We used gene targeting to create isoform-specific Fbw7-null mutations in human cells and found that the nucleoplasmic Fbw7alpha isoform accounts for almost all Fbw7 activity toward cyclin E, c-Myc, and sterol regulatory element binding protein 1. Cyclin E sensitivity to Fbw7 varies during the cell cycle, and this correlates with changes in cyclin E-cyclin-dependent kinase 2 (CDK2)-specific activity, cyclin E autophosphorylation, and CDK2 inhibitory phosphorylation. These data suggest that oscillations in cyclin E-CDK2-specific activity during the cell cycle regulate the timing of cyclin E degradation. Moreover, they highlight the utility of adeno-associated virus-mediated gene targeting in functional analyses of complex loci.     

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.