Regulation of nuclear transport and degradation of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1

The regulation of the vertebrate cell cycle is controlled by the function of cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors. The Xenopus laevis kinase inhibitor, p27(Xic1) (Xic1) is a member of the p21(Cip1)/p27(Kip1)/p57(Kip2) CDK inhibitor family and inhibits CDK2-cyclin E in vitro as well as DNA replication in Xenopus egg extracts. Xic1 is targeted for degradation in interphase extracts in a manner dependent on both the ubiquitin conjugating enzyme, Cdc34, and nuclei. Here we show that ubiquitination of Xic1 occurs exclusively in the nucleus and that nuclear localization of Xic1 is necessary for its degradation. We find that Xic1 nuclear localization is independently mediated by binding to CDK2-cyclin E and by nuclear localization sequences within the C terminus of Xic1. Our results also indicate that binding of Xic1 to CDK2-cyclin E is dispensable for Xic1 ubiquitination and degradation. Moreover, we show that amino acids 180-183 of Xic1 are critical determinants of Xic1 degradation. This region of Xic1 may define a motif of Xic1 essential for recognition by the ubiquitin conjugation machinery or for binding an alternate protein required for degradation.     

Ubiquitination of p27Kip1 requires physical interaction with cyclin E and probable phosphate recognition by SKP2

p27Kip1 is an essential cell cycle inhibitor of Cyclin-dependent kinases. Ubiquitin-mediated proteolysis of p27Kip1 is an important mechanism for activation of Cyclin E-Cdk2 and facilitates G1/S transition. Ubiquitination of p27 is primarily catalyzed by a multisubunit E3 ubiquitin ligase, SCF(Skp2), and requires an adapter protein Cks1. In addition, phosphorylation of p27 at Thr187 by Cyclin E and Cdk2 is also essential for triggering substrate ubiquitination. Here we investigate the molecular mechanism of p27 ubiquitination. We show that Cyclin E-Cdk2 is essential for targeting the p27 substrate to SCF(Skp2). Direct physical contact between Cyclin E but not Cdk2 and p27 is required for p27 recruitment to SCF(Skp2). In a search for positively charged amino acid residues that may be involved in recognition of the Thr187 phosphate group, we found that Arg306 of Skp2 is required for association and ubiquitination of phosphorylated p27 but dispensable for ubiquitination of unphosphorylated p21. Thus, our data unravel the molecular organization of the ubiquitination complex that catalyzes p27 ubiquitination and provide unique insights into the specificity of substrate recognition by SCF(Skp2).     

Cytoplasmic ubiquitin ligase KPC regulates proteolysis of p27(Kip1) at G1 phase

The cyclin-dependent kinase inhibitor p27(Kip1) is degraded at the G0-G1 transition of the cell cycle by the ubiquitin-proteasome pathway. Although the nuclear ubiquitin ligase (E3) SCF(Skp2) is implicated in p27(Kip1) degradation, proteolysis of p27(Kip1) at the G0-G1 transition proceeds normally in Skp2(-/-) cells. Moreover, p27(Kip1) is exported from the nucleus to the cytoplasm at G0-G1 (refs 9-11). These data suggest the existence of a Skp2-independent pathway for the degradation of p27(Kip1) at G1 phase. We now describe a previously unidentified E3 complex: KPC (Kip1 ubiquitination-promoting complex), consisting of KPC1 and KPC2. KPC1 contains a RING-finger domain, and KPC2 contains a ubiquitin-like domain and two ubiquitin-associated domains. KPC interacts with and ubiquitinates p27(Kip1) and is localized to the cytoplasm. Overexpression of KPC promoted the degradation of p27(Kip1), whereas a dominant-negative mutant of KPC1 delayed p27(Kip1) degradation. The nuclear export of p27(Kip1) by CRM1 seems to be necessary for KPC-mediated proteolysis. Depletion of KPC1 by RNA interference also inhibited p27(Kip1) degradation. KPC thus probably controls degradation of p27(Kip1) in G1 phase after export of the latter from the nucleus.     

A high-throughput screen measuring ubiquitination of p53 by human mdm2

Tumor suppressor p53 is typically maintained at low levels in normal cells. In response to cellular stresses, such as DNA damage, p53 is stabilized and can stimulate responses leading to cell cycle arrest or apoptosis. Corresponding to its central role in preventing propagation of damaged cells, mutation or deletion of p53 is found in nearly 50% of all human tumors. Mdm2 (mouse-d-minute 2) and its human ortholog (hmdm2 or hdm2) catalyze the ubiquitination of p53, targeting it for degradation via the proteosome. Thus, the activity of mdm2 is inversely correlated with p53 levels. Based on this, inhibition of human mdm2 activity by a small-molecule therapeutic will lead to net stabilization of p53 and be the basis for development of a novel cancer therapeutic. Previous high-throughput screening assays of mdm2 measured the autoubiquitination activity of mdm2, which occurs in the absence of an acceptor substrate such as p53. The major drawback to this approach is that inhibitors of mdm2 autoubiquitination may lead to a net stabilization of mdm2 and thus have the opposite effect of inhibitors that interfere with p53 ubiquitination. The authors describe the development, validation, and execution of a high-throughput screening measuring the ubiquitination of p53 by mdm2, with p53 labeled with europium and the other substrate (Ub-UbcH5b) labeled with a Cy5 on the ubiquitin. After confirming that known inhibitors are detected with this assay, it was successfully automated and used to query >600,000 compounds from the GlaxoSmithKline collection for mdm2 inhibitors.     

A negatively charged amino acid in Skp2 is required for Skp2-Cks1 interaction and ubiquitination of p27Kip1

Proteolysis of cyclin-dependent kinase inhibitor p27 occurs predominantly in the late G1 phase of the cell cycle through a ubiquitin-mediated protein degradation pathway. Ubiquitination of p27 requires the SCFSkp2 ubiquitin ligase and Skp2 F-box binding protein Cks1. The mechanisms by which Skp2 recognizes Cks1 to ubiquitylate p27 remain obscure. Here we show that Asp-331 in the carboxyl terminus of Skp2 is required for its association with Cks1 and ubiquitination of p27. Mutation of Asp-331 to Ala disrupts the interaction between Skp2 and Cks1. Although Asp-331 mutation negates the ability of the Skp1-Cullin-F-box protein (SCF) complex to ubiquitylate p27, such a mutation has no effect on Skp2 self-ubiquitination. A conservative change from Asp to Glu at position 331 of Skp2 does not affect Skp2-Cks1 interaction. Our results revealed a unique requirement for a negatively charged residue in the carboxyl-terminal region of Skp2 in recognition of Cks1 and ubiquitination of p27.     

The C-terminal domain of the Xenopus cyclin-dependent kinase inhibitor, p27Xic1, is both necessary and sufficient for phosphorylation-independent proteolysis

Cell cycle progression is regulated by cyclin-dependent kinases (CDKs), cyclins, and CDK inhibitors. In the frog, Xenopus laevis, the CDK inhibitor p27(Xic1) (Xic1) inhibits DNA synthesis by negatively regulating CDK2-cyclin E. Using the frog egg extract as a model system for the study of Xic1, studies have demonstrated that Xic1 protein levels are regulated by nuclear ubiquitination and proteolysis. To characterize the molecular mechanism that regulates Xic1 turnover, we have identified the minimal sequences of Xic1 that are necessary and sufficient for its nuclear ubiquitination and degradation. Using deletion mutagenesis, our studies indicated that the C-terminal 50 amino acids of Xic1 are critical for its proteolysis beyond a role in nuclear transport. Replacement of the Xic1 C terminus with the SV40 nuclear localization sequence resulted in the nuclear localization of Xic1 but not its ubiquitination or degradation. Our deletion studies also indicated that the CDK2-cyclin binding domain of Xic1 is important for its efficient retention in the nucleus. Further deletion analyses identified at least 3 lysine residues within the Xic1 C terminus that are targeted for specific ubiquitination. Importantly, our studies demonstrated that the Xic1 C-terminal 50 amino acids can serve as a nuclear degradation signal when fused to a stable heterologous nuclear protein. Moreover, a 30-amino-acid region within the C terminus of Xic1 can serve as a nuclear ubiquitination signal. To address the role of phosphorylation on Xic1 turnover, all the potential phosphorylation sites within the C-terminal 50 amino acids of Xic1 were mutated to alanine to prevent possible phosphorylation. This resulted in a Xic1 protein that was nevertheless degraded in a manner similar to wild-type Xic1, suggesting that phosphorylation of Xic1 is not critical for its nuclear ubiquitination or proteolysis.     

Largazole and its derivatives selectively inhibit ubiquitin activating enzyme (e1)

Protein ubiquitination plays an important role in the regulation of almost every aspect of eukaryotic cellular function; therefore, its destabilization is often observed in most human diseases and cancers. Consequently, developing inhibitors of the ubiquitination system for the treatment of cancer has been a recent area of interest. Currently, only a few classes of compounds have been discovered to inhibit the ubiquitin-activating enzyme (E1) and only one class is relatively selective in E1 inhibition in cells. We now report that Largazole and its ester and ketone analogs selectively inhibit ubiquitin conjugation to p27(Kip1) and TRF1 in vitro. The inhibitory activity of these small molecules on ubiquitin conjugation has been traced to their inhibition of the ubiquitin E1 enzyme. To further dissect the mechanism of E1 inhibition, we analyzed the effects of these inhibitors on each of the two steps of E1 activation. We show that Largazole and its derivatives specifically inhibit the adenylation step of the E1 reaction while having no effect on thioester bond formation between ubiquitin and E1. E1 inhibition appears to be specific to human E1 as Largazole ketone fails to inhibit the activation of Uba1p, a homolog of E1 in Schizosaccharomyces pombe. Moreover, Largazole analogs do not significantly inhibit SUMO E1. Thus, Largazole and select analogs are a novel class of ubiquitin E1 inhibitors and valuable tools for studying ubiquitination in vitro. This class of compounds could be further developed and potentially be a useful tool in cells.     

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.     

Targeting the Ubiquitin E3 Ligase MuRF1 to Inhibit Muscle Atrophy

Progressive muscle wasting, also known as myopathy or muscle atrophy is a debilitating and life-threatening disorder. Myopathy is a pathological condition of many diseases including cancer, diabetes, COPD, and AIDS and is a natural consequence of inactivity and aging (sarcopenia). Muscle atrophy occurs when there is a net loss of muscle mass resulting in a change in the balance between protein synthesis and protein degradation. The ubiquitin pathway and specific ubiquitin pathway enzymes have been directly implicated in the progression of atrophy. The ubiquitin E3 ligase Muscle-specific RING Finger E3 ligase (MuRF1) is upregulated and increases protein degradation and muscle wasting in numerous muscle atrophy models. The inhibition of MuRF1 could be a novel mechanism to prevent or reverse muscle wasting associated with various pathologies. We screened a small molecule library for inhibitors to MuRF1 activity and identified P013222, an inhibitor of MuRF1 autoubiquitylation. Further, P013222 was shown to inhibit MuRF1-dependent substrate ubiquitylation, and was active in inhibiting MuRF1 in a cellular atrophy model. Thus MuRF1 can be targeted in a specific manner and produce positive results in cellular atrophy models.     

PTEN regulates the ubiquitin-dependent degradation of the CDK inhibitor p27(KIP1) through the ubiquitin E3 ligase SCF(SKP2)

The PTEN tumor suppressor acts as a phosphatase for phosphatidylinositol-3,4,5-trisphosphate (PIP3) [1, 2]. We have shown previously that PTEN negatively controls the G1/S cell cycle transition and regulates the levels of p27(KIP1), a CDK inhibitor [3, 4]. Recently, we and others have identified an ubiquitin E3 ligase, the SCF(SKP2) complex, that mediates p27 ubiquitin-dependent proteolysis [5-7]. Here we report that PTEN and the PI 3-kinase pathway regulate p27 protein stability. PTEN-deficiency in mouse embryonic stem (ES) cells causes a decrease of p27 levels with concomitant increase of SKP2, a key component of the SCF(SKP2) complex. Conversely, in human glioblastoma cells, ectopic PTEN expression leads to p27 accumulation, which is accompanied by a reduction of SKP2. We found that ectopic expression of SKP2 alone is sufficient to reverse PTEN-induced p27 accumulation, restore the kinase activity of cyclin E/CDK2, and partially overcome the PTEN-induced G1 cell cycle arrest. Consistently, recombinant SCF(SKP2) complex or SKP2 protein alone can rescue the defect in p27 ubiquitination in extracts prepared from cells treated with a PI 3-kinase inhibitor. Our findings suggest that SKP2 functions as a critical component in the PTEN/PI 3-kinase pathway for the regulation of p27(KIP1) and cell proliferation.     

Regulation of the cell cycle at the G1-S transition by proteolysis of cyclin E and p27Kip1

The transition from G1 phase to S phase of the mammalian cell cycle is controlled by many positive and negative regulators, among which cyclin E and p27Kip1, respectively, undergo the most marked changes in concentration at this transition. The abundance of both cyclin E and p27Kip1 is regulated predominantly by posttranslational mechanisms, in particular by proteolysis mediated by the ubiquitin-proteasome pathway. Cyclin E and p27Kip1 each bind to and undergo polyubiquitination by the same ubiquitin ligase, known as SCF(Skp2). The degradation of cyclin E and p27Kip1 is greatly impaired in Skp2-deficient mice, resulting in intracellular accumulation of these proteins. In this article, recent progress in characterization of the molecular mechanisms that control the proteolysis of cyclin E and p27Kip1 is reviewed.     

TPR proteins required for anaphase progression mediate ubiquitination of mitotic B-type cyclins in yeast.

The abundance of B-type cyclin-CDK complexes is determined by regulated synthesis and degradation of cyclin subunits. Cyclin proteolysis is required for the final exit from mitosis and for the initiation of a new cell cycle. In extracts from frog or clam eggs, degradation is accompanied by ubiquitination of cyclin. Three genes, CDC16, CDC23, and CSE1 have recently been shown to be required specifically for cyclin B proteolysis in yeast. To test whether these genes are required for cyclin ubiquitination, we prepared extracts from G1-arrested yeast cells capable of conjugating ubiquitin to the B-type cyclin Clb2. The ubiquitination activity was cell cycle regulated, required Clb2's destruction box, and was low if not absent in cdc16, cdc23, cdc27, and cse1 mutants. Furthermore all these mutants were also defective in ubiquitination of another mitotic B-type cyclin, Clb3. The Cdc16, Cdc23, and Cdc27 proteins all contain several copies of the tetratricopeptide repeat and are subunits of a complex that is required for the onset of anaphase. The finding that gene products that are required for ubiquitination of Clb2 and Clb3 are also required for cyclin proteolysis in vivo provides the best evidence so far that cyclin B is degraded via the ubiquitin pathway in living cells. Xenopus homologues of Cdc16 and Cdc27 have meanwhile been shown to be associated with a 20S particle that appears to function as a cell cycle-regulated ubiquitin-protein ligase.     

Stability of homologue of Slimb F-box protein is regulated by availability of its substrate

The homologue of Slimb (HOS) F-box protein is a receptor of the Skp1-Cullin1-F-box protein (SCF(HOS)) E3 ubiquitin ligase, which mediates ubiquitination and degradation of beta-catenin and the inhibitor of NFkappaB, IkappaB. We found that HOS itself is an unstable protein that undergoes ubiquitination and degradation in a 26 S proteasome-dependent manner. A HOS mutant lacking the F-box that is deficient in binding to the core SCF components underwent ubiquitination less efficiently and was more stable than the wild type protein. Furthermore, ubiquitination and degradation of HOS was impaired in ts41 cells, in which the activities of Cullin-based ligases were decreased because the NEDD8 pathway was abrogated. Whereas HOS was directly ubiquitinated within the SCF(HOS) complex in vitro, the addition of phosphorylated IkappaBalpha inhibited this ubiquitination. Increasing cellular levels of HOS substrate (phosphorylated IkappaBalpha) by activating IkappaB kinase inhibited HOS ubiquitination and led to stabilization of HOS, indicating that interaction between HOS and its substrate might protect HOS from proteolysis. Taken together, our data suggest that proteolysis of HOS depends on its interaction with active components of the SCF complex and that HOS stability is regulated by a bound substrate. These findings may define a mechanism for maintaining activities of specific SCF complexes based on availability of a particular substrate.     

Ubiquitin-protein ligases in muscle wasting

Muscle wasting occurs when rates of protein degradation outstrip rates of protein synthesis. Accelerated rates of protein degradation develop in atrophying muscle largely through activation of the ubiquitin-proteasome pathway. The complexity of the ubiquitination process, however, has hampered our understanding of how this pathway is activated in atrophying muscles and which enzymes of the ubiquitin conjugation system are responsible. Recent studies demonstrate that two ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MuRF1 are critical in the development of muscle atrophy. Other experiments implicate E2(14k) and E3alpha, of the N-end rule pathway, as important players in the process. It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as to develop inhibitors of, these E3s.     

Distinct functions of the ubiquitin-proteasome pathway influence nucleotide excision repair

The Rad23/Rad4 nucleotide excision repair (NER) protein complex functions at an early stage of the NER reaction, possibly promoting the recognition of damaged DNA. Here we show that Rad4 protein is ubiquitinated and degraded in response to ultraviolet (UV) radiation, and identify a novel cullin-based E3 ubiquitin ligase required for this process. We also show that this novel ubiquitin ligase is required for optimal NER. Our results demonstrate that optimal NER correlates with the ubiquitination of Rad4 following UV radiation, but not its subsequent degradation. Furthermore, we show that the ubiquitin-proteasome pathway (UPP) regulates NER via two distinct mechanisms. The first occurs independently of de novo protein synthesis, and requires Rad23 and a nonproteolytic function of the 19S regulatory complex of the 26S proteasome. The second requires de novo protein synthesis, and relies on the activity of the newly identified E3 ubiquitin ligase. These studies reveal that, following UV radiation, NER is mediated by nonproteolytic activities of the UPP, via the ubiquitin-like domain of Rad23 and UV radiation-induced ubiquitination of Rad4.     

Ras binding triggers ubiquitination of the Ras exchange factor Ras-GRF2

Ras is a small GTPase that is activated by upstream guanine nucleotide exchange factors, one of which is Ras-GRF2. GRF2 is a widely expressed protein with several recognizable sequence motifs, including a Ras exchanger motif (REM), a PEST region containing a destruction box (DB), and a Cdc25 domain. The Cdc25 domain possesses guanine nucleotide exchange factor activity and interacts with Ras. Herein we examine if the DB motif in GRF2 results in proteolysis via the ubiquitin pathway. Based on the solved structure of the REM and Cdc25 regions of the Son-of-sevenless (Sos) protein, the REM may stabilize the Cdc25 domain during Ras binding. The DB motif of GRF2 is situated between the REM and the Cdc25 domains, tempting speculation that it may be exposed to ubiquitination machinery upon Ras binding. GRF2 protein levels decrease dramatically upon activation of GRF2, and dominant-negative Ras induces degradation of GRF2, demonstrating that signaling downstream of Ras is not required for the destruction of GRF2 and that binding to Ras is important for degradation. GRF2 is ubiquitinated in vivo, and this can be detected using mass spectrometry. In the presence of proteasome inhibitors, Ras-GRF2 accumulates as a high-molecular-weight conjugate, suggesting that GRF2 is destroyed by the 26S proteasome. Deleting the DB reduces the ubiquitination of GRF2. GRF2 lacking the Cdc25 domain is not ubiquitinated, suggesting that a protein that cannot bind Ras cannot be properly targeted for destruction. Point mutations within the Cdc25 domain that eliminate Ras binding also eliminate ubiquitination, demonstrating that binding to Ras is necessary for ubiquitination of GRF2. We conclude that conformational changes induced by GTPase binding expose the DB and thereby target GRF2 for destruction.     

Ubiquitin ligase Cbl-b and inhibitory Cblin peptides

This review focuses on the Cbl-b muscle atrophy-associated ubiquitin ligase and its inhibitors. Herein, the role of E3 ubiquitin ligase-associated muscle atrophy genes (atrogenes), including MAFbx-1/agrogin-1 and MuRF-1, as well as another ubiquitin ligase, Cbl-b and its inhibitors, is discussed. Cbl-b plays an important role in unloading muscle atrophy caused by spaceflight and in bedridden patients: Cbl-b ubiquitinated and induced the degradation of IRS-1, a key intermediate in the IGF-1 signaling. Furthermore, a pentapetpide (DGpYMP), inhibited Cbl-b-mediated IRS-1 ubiquitination. This peptide-based Cbl-b inhibitor Cblin and its homologous peptides in foods presumably affect muscle atrophy under such conditions.