MicroRNA-223 Regulates Cyclin E Activity by Modulating Expression of F-box and WD-40 Domain Protein 7

F-box and WD-40 domain protein 7 (Fbw7) provides substrate specificity for the Skp1-Cullin1-F-box protein (SCF) ubiquitin ligase complex that targets multiple oncoproteins for degradation, including cyclin E, c-Myc, c-Jun, Notch, and mammalian target of rapamycin (mTOR). Fbw7 is a bona fide tumor suppressor, and loss-of-function mutations in FBXW7 have been identified in diverse human tumors. Although much is known about targets of the Fbw7 ubiquitin ligase pathway, relatively little is known about the regulation of Fbw7 expression. We identified a panel of candidate microRNA regulators of Fbw7 expression within a study of gene expression alterations in primary erythroblasts obtained from cyclin E^{T74A T393A} knock-in mice, which have markedly dysregulated cyclin E expression. We found that overexpression of miR-223, in particular, significantly reduces FBXW7 mRNA levels, increases endogenous cyclin E protein and activity levels, and increases genomic instability. We next confirmed that miR-223 targets the FBXW7 3′-untranslated region. We then found that reduced miR-223 expression in primary mouse embryonic fibroblasts leads to increased Fbw7 expression and decreased cyclin E activity. Finally, we found that miR-223 expression is responsive to acute alterations in cyclin E regulation by the Fbw7 pathway. Together, our data indicate that miR-223 regulates Fbw7 expression and provide the first evidence that activity of the SCF^Fbw7 ubiquitin ligase can be modulated directly by the microRNA pathway.     

The Stomatin-Like Protein SLP-1 and Cdk2 Interact with the F-Box Protein Fbw7-γ

Control of cellular proliferation is critical to cell viability. The F-box protein Fbw7 (hAgo/hCdc4/FBXW7) functions as a specificity factor for the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex and targets several proteins required for cellular proliferation for ubiquitin-mediated destruction. Fbw7 exists as three splice variants but the mechanistic role of each is not entirely clear. We examined the regulation of the Fbw7-γ isoform, which has been implicated in the degradation of c-Myc. We show here that Fbw7-γ is an unstable protein and that its turnover is proteasome-dependent in transformed cells. Using a two-hybrid screen, we identified a novel interaction partner, SLP-1, which binds the N-terminal domain of Fbw7-γ. Overexpression of SLP-1 inhibits the degradation of Fbw7-γ, suggesting that this interaction can happen in vivo. When Fbw7-γ is stabilized by overexpression of SLP-1, c-Myc protein abundance decreases, suggesting that the SCF^Fbw7-γ complex maintains activity. We demonstrate that Cdk2 also binds the N-terminal domain of Fbw7-γ as well as SLP-1. Interestingly, co-expression of Cdk2 and SLP-1 does not inhibit Fbw7-γ degradation, suggesting that Cdk2 and SLP-1 may have opposing functions.     

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.     

USP2 is an SKP2 deubiquitylase that stabilizes both SKP2 and its substrates

The stability of a protein is regulated by a balance between its ubiquitylation and deubiquitylation. S-phase kinase-associated protein 2 (SKP2) is an oncogenic F-box protein that recognizes tumor suppressor substrates for targeted ubiquitylation by the E3 ligase SKP1-Cullin1-F-box and degradation by proteasome. SKP2 is itself ubiquitylated by the E3 ligases APC/C^CDH1 and SCF^FBXW2, and deubiquitylated by deubiquitylases (DUBs) USP10 and USP13. Given the biological significance of SKP2, it is likely that the other E3s or DUBs may also regulate its stability. Here, we report the identification and characterization of USP2 as a new DUB. We first screened a panel of DUBs and found that both USP2 and USP21 bound to endogenous SKP2, but only USP2 deubiquitylated and stabilized SKP2 protein. USP2 inactivation via siRNA knockdown or small-molecule inhibitor treatment remarkably shortened SKP2 protein half-life by enhancing its ubiquitylation and subsequent degradation. Unexpectedly, USP2-stabilized SKP2 did not destabilize its substrates p21 and p27. Mechanistically, USP2 bound to SKP2 via the leucine-rich repeat substrate-binding domain on SKP2 to disrupt the SKP2-substrate binding, leading to stabilization of both SKP2 and these substrates. Biologically, growth suppression induced by USP2 knockdown or USP2 inhibitor is partially mediated via modulation of SKP2 and its substrates. Our study revealed a new mechanism of the cross-talk among the E3–DUB substrates and its potential implication in targeting the USP2–SKP2 axis for cancer therapy.     

The Canonical Wnt Signal Restricts the Glycogen Synthase Kinase 3/Fbw7-Dependent Ubiquitination and Degradation of Eya1 Phosphatase

Haploinsufficiency of Eya1 causes the branchio-oto-renal (BOR) syndrome, and abnormally high levels of Eya1 are linked to breast cancer progression and poor prognosis. Therefore, regulation of Eya1 activity is key to its tissue-specific functions and oncogenic activities. Here, we show that Eya1 is posttranslationally modified by ubiquitin and that its ubiquitination level is self-limited to prevent premature degradation. Eya1 has an evolutionarily conserved CDC4 phosphodegron (CPD) signal, a target site of glycogen synthase kinase 3 (GSK3) kinase and Fbw7 ubiquitin ligase, which is required for Eya1 ubiquitination. Genetic deletion of Fbw7 and pharmacological inhibition of GSK3 significantly decrease Eya1 ubiquitination. Conversely, activation of the phosphatidylinositol 3-kinase (PI3K)/Akt and the canonical Wnt signal suppresses Eya1 ubiquitination. Compound Eya1^+/−; Wnt9b^+/− mutants exhibit an increased penetrance of renal defect, indicating that they function in the same genetic pathway in vivo. Together, these findings reveal that the canonical Wnt and PI3K/Akt signal pathways restrain the GSK3/Fbw7-dependent Eya1 ubiquitination, and they further suggest that dysregulation of this novel axis contributes to tumorigenesis.     

Negative regulation of the stability and tumor suppressor function of Fbw7 by the Pin1 prolyl isomerase

Fbw7 is the substrate recognition component of the Skp1-Cullin-F-box (SCF)-type E3 ligase complex and a well-characterized tumor suppressor that targets numerous oncoproteins for destruction. Genomic deletion or mutation of FBW7 has been frequently found in various types of human cancers; however, little is known about the upstream signaling pathway(s) governing Fbw7 stability and cellular functions. Here we report that Fbw7 protein destruction and tumor suppressor function are negatively regulated by the prolyl isomerase Pin1. Pin1 interacts with Fbw7 in a phoshorylation-dependent manner and promotes Fbw7 self-ubiquitination and protein degradation by disrupting Fbw7 dimerization. Consequently, overexpressing Pin1 reduces Fbw7 abundance and suppresses Fbw7's ability to inhibit proliferation and transformation. By contrast, depletion of Pin1 in cancer cells leads to elevated Fbw7 expression, which subsequently reduces Mcl-1 abundance, sensitizing cancer cells to Taxol. Thus, Pin1-mediated inhibition of Fbw7 contributes to oncogenesis, and Pin1 may be a promising drug target for anticancer therapy.     

Parkin-Dependent Degradation of the F-Box Protein Fbw7β Promotes Neuronal Survival in Response to Oxidative Stress by Stabilizing Mcl-1

Parkinson''s disease (PD) is characterized by progressive loss of midbrain dopaminergic neurons resulting in motor dysfunction. While most PD is sporadic in nature, a significant subset can be linked to either dominant or recessive germ line mutations. PARK2, encoding the ubiquitin ligase parkin, is the most frequently mutated gene in hereditary Parkinson''s disease. Here, we present evidence for a neuronal ubiquitin ligase cascade involving parkin and the multisubunit ubiquitin ligase SCF^Fbw7β. Specifically, parkin targets the SCF substrate adapter Fbw7β for proteasomal degradation. Furthermore, we show that the physiological role of parkin-mediated regulation of Fbw7β levels is the stabilization of the mitochondrial prosurvival factor Mcl-1, an SCF^Fbw7β target in neurons. We show that neurons depleted of parkin become acutely sensitive to oxidative stress due to an inability to maintain adequate levels of Mcl-1. Therefore, loss of parkin function through biallelic mutation of PARK2 may lead to death of dopaminergic neurons through unregulated SCF^Fbw7β-mediated ubiquitylation-dependent proteolysis of Mcl-1.     

Chromosome Instability Underlies Hematopoietic Stem Cell Dysfunction and Lymphoid Neoplasia Associated with Impaired Fbw7-Mediated Cyclin E Regulation

The Fbw7 ubiquitin ligase critically regulates hematopoietic stem cell (HSC) function, though the precise contribution of individual substrate ubiquitination pathways to HSC homeostasis is unknown. In the work reported here, we used a mouse model in which we introduced two knock-in mutations (T74A and T393A [changes of T to A at positions 74 and 393]) to disrupt Fbw7-dependent regulation of cyclin E, its prototypic substrate, and to examine the consequences of cyclin E dysregulation for HSC function. Serial transplantation revealed that cyclin E^{T74A T393A} HSCs self-renewed normally; however, we identified defects in their multilineage reconstituting capacity. By inducing hematologic stress, we exposed an impaired self-renewal phenotype in cyclin E knock-in HSCs that was associated with defective cell cycle exit and the emergence of chromosome instability (CIN). Importantly, p53 deletion induced both defects in self-renewal and multilineage reconstitution in cyclin E knock-in HSCs with serial transplantation and CIN in hematopoietic stem and progenitor cells. Moreover, CIN was a feature of fatal T-cell malignancies that ultimately developed in recipients of cyclin E^{T74A T393A}; p53-null HSCs. Together, our findings demonstrate the importance of Fbw7-dependent cyclin E control to the hematopoietic system and highlight CIN as a characteristic feature of HSC dysfunction and malignancy induced by deregulated cyclin E.     

An integrated view of cyclin E function and regulation

Cancers of diverse cell lineages express high levels of cyclin E, and in various studies, cyclin E overexpression correlates with increased tumor aggression. One way that normal control of cyclin E expression is disabled in cancer cells is via loss-of-function mutations sustained by FBXW7. This gene encodes the Fbw7 tumor suppressor protein that provides substrate specificity for a ubiquitin ligase complex that targets multiple oncoproteins for degradation. Numerous other mechanisms besides Fbw7 mutations can deregulate cyclin E expression and activity in cancer cells. Recent reports demonstrate that inappropriate cyclin E expression may have far-reaching biological consequences for cell physiology, including altering gene expression programs governing proliferation, differentiation, survival and senescence. In this Perspective, we discuss the function of mammalian cyclin E in the context of these new data as well as the complex network that connects cyclin E functions to the cellular controls regulating its expression and activity.Key words: cell cycle, cyclin E, Cdk2, Fbw7, E2F, p21, p27, regulatory network     

The F-box protein Fbw7 is required for cerebellar development

The F-box protein Fbw7 (also known as Fbxw7, hCdc4 and Sel-10) functions as a substrate recognition component of a SCF-type E3 ubiquitin ligase. SCF(Fbw7) facilitates polyubiquitination and subsequent degradation of various proteins such as Notch, cyclin E, c-Myc and c-Jun. Fbw7 is highly expressed in the nervous system and controls neural stem cell differentiation and apoptosis via Notch and c-Jun during embryonic development (Hoeck et al., 2010). Fbw7 deletion in the neural lineage is perinatal lethal and thus prohibits studying the role of Fbw7 in the adult nervous system. fbw7 mRNA is highly expressed in the postnatal brain and to gain insights into the function of Fbw7 in postnatal neurogenesis we analysed Fbw7 function in the cerebellum. We generated conditional Fbw7-knockout mice (fbw7^?Cb) by inactivating Fbw7 specifically in the cerebellar anlage. This resulted in decreased cerebellar size, reduced Purkinje cell number and defects in axonal arborisation. Moreover, Fbw7-deficient cerebella showed supranumeral fissures and aberrant progenitor cell migration. Protein levels of the Fbw7 substrates Notch1 and N-terminally phosphorylated c-Jun were upregulated in fbw7^?Cb mice. Concomitant deletion of c-Jun, and also the junAA knock-in mutation which specifically abrogates c-Jun N-terminal phosphorylation, rescued Purkinje cell numbers and arborisation in the fbw7^?Cb background. Taken together these data demonstrate that Fbw7 is essential during cerebellar development, and identify N-terminally phosphorylated c-Jun as an important substrate of SCF(Fbw7) during neurogenesis.     

Phosphorylation-dependent degradation of c-Myc is mediated by the F-box protein Fbw7

The F-box protein Skp2 mediates c-Myc ubiquitylation by binding to the MB2 domain. However, the turnover of c-Myc is largely dependent on phosphorylation of threonine-58 and serine-62 in MB1, residues that are often mutated in cancer. We now show that the F-box protein Fbw7 interacts with and thereby destabilizes c-Myc in a manner dependent on phosphorylation of MB1. Whereas wild-type Fbw7 promoted c-Myc turnover in cells, an Fbw7 mutant lacking the F-box domain delayed it. Furthermore, depletion of Fbw7 by RNA interference increased both the abundance and transactivation activity of c-Myc. Accumulation of c-Myc was also apparent in mouse Fbw7-/- embryonic stem cells. These observations suggest that two F-box proteins, Fbw7 and Skp2, differentially regulate c-Myc stability by targeting MB1 and MB2, respectively.