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SCF (Skp1/Cul1/F-box) ubiquitin ligases act as master regulators of cellular homeostasis by targeting key proteins for ubiquitylation. Here, we identified a hitherto uncharacterized F-box protein, FBXO28 that controls MYC-dependent transcription by non-proteolytic ubiquitylation. SCFFBXO28 activity and stability are regulated during the cell cycle by CDK1/2-mediated phosphorylation of FBXO28, which is required for its efficient ubiquitylation of MYC and downsteam enhancement of the MYC pathway. Depletion of FBXO28 or overexpression of an F-box mutant unable to support MYC ubiquitylation results in an impairment of MYC-driven transcription, transformation and tumourigenesis. Finally, in human breast cancer, high FBXO28 expression and phosphorylation are strong and independent predictors of poor outcome. In conclusion, our data suggest that SCFFBXO28 plays an important role in transmitting CDK activity to MYC function during the cell cycle, emphasizing the CDK-FBXO28-MYC axis as a potential molecular drug target in MYC-driven cancers, including breast cancer.

Cyclin E1, an activator of cyclin-dependent kinase 2 (Cdk2) that promotes replicative functions, is normally expressed periodically within the mammalian cell cycle, peaking at the G(1)-S-phase transition. This periodicity is achieved by E2F-dependent transcription in late G(1) and early S phases and by ubiquitin-mediated proteolysis. The ubiquitin ligase that targets phosphorylated cyclin E is SCF(Fbw7) (also known as SCF(Cdc4)), a member of the cullin ring ligase (CRL) family. Fbw7, a substrate adaptor subunit, is expressed as three splice-variant isoforms with different subcellular distributions: Fbw7α is nucleoplasmic but excluded from the nucleolus, Fbw7β is cytoplasmic, and Fbw7γ is nucleolar. Degradation of cyclin E in vivo requires SCF complexes containing Fbw7α and Fbw7γ, respectively. In vitro reconstitution showed that the role of SCF(Fbw7α) in cyclin E degradation, rather than ubiquitylation, is to serve as a cofactor of the prolyl cis-trans isomerase Pin1 in the isomerization of a noncanonical proline-proline bond in the cyclin E phosphodegron. This isomerization is required for subsequent binding and ubiquitylation by SCF(Fbw7γ). Here we show that Pin1-mediated isomerization of the cyclin E phosphodegron and subsequent binding to Fbw7γ drive nucleolar localization of cyclin E, where it is ubiquitylated by SCF(Fbw7γ) prior to its degradation by the proteasome. It is possible that this constitutes a mechanism for rapid inactivation of phosphorylated cyclin E by nucleolar sequestration prior to its multiubiquitylation and degradation.

International audience; Acquiring high proliferation rate is crucial for carcinogenic transformation of cells. We report here proteome profiling of human breast epithelial cells with low (184A1) and high (MCF10A) proliferation rates. We identified 183 proteins in 184A1 and 318 proteins in MCF10A cells. These datasets provide the most comprehensive proteome annotations of 184A1 and MCF10A cells. Proteins were taken for identification from 2-D gels in a systematic and unbiased way. Functional clustering of the identified proteins showed similarities in distribution of proteins to the same functional domains, indicating similarities in proteomes of 184A1 and MCF10A cells. Among observed differences in protein expression, we validated correlation of expression of endogenous cyclin-dependent kinase 4 (CDK4), cyclin D3, cdc25B, and p38γ with cell proliferation. Furthermore, down-regulation of CDK4 and cyclin D3 with specific siRNA inhibited cell proliferation, which emphasized the role of CDK4 and cyclin D3 in enhancement of cell proliferation rate of human breast epithelial cells.

Transforming growth factor-beta (TGFbeta) is a key regulator of cell proliferation, death, migration, and differentiation. To explore mechanisms of TGFbeta action, we performed systemic analysis of functional dependencies between 153 proteins which changed their expression and synthesis upon treatment of human breast epithelial cells with TGFbeta1. We found that TGFbeta1 initiated signaling via a scale-free network of proteins which affect primary cellular metabolism, stress response, signal transduction, transport, transcription, cytoskeleton, and cell death. Multiple inputs into each functional domain were observed, emphasizing robustness of TGFbeta1 signaling. Network analysis demonstrated importance of a Plag1/CNK1/RASSF1A/Src-dependent prozone effect, as a systemic feature which is crucial for TGFbeta1-dependent activation of Erk1/2 and regulation of cell proliferation. We showed that the balance between Plag1, CNK1, RASSF1A and Src defined whether TGFbeta1 will stimulate, inhibit or will have no effect on a long-term activation of Erk1/2 and subsequent TGFbeta1 inhibitory or stimulatory effect on cell proliferation. This is the first demonstration of importance of systemic features for incorporation of Erk1/2 activation into TGFbeta1 signaling.

Smad2 is a crucial component of transforming growth factor-β (TGFβ) signaling, and is involved in the regulation of cell proliferation, death and differentiation. Phosphorylation, ubiquitylation and acetylation of Smad2 have been found to regulate its activity. We used mass spectrometry to search for novel post-translational modifications (PTMs) of Smad2. Peptide mass fingerprinting (PMF) indicated that Smad2 can be acetylated, methylated, citrullinated, phosphorylated and palmitoylated. Sequencing of selected peptides validated methylation at Gly122 and hydroxylation at Trp18 of Smad2. We also observed a novel, so far unidentified modification at Tyr128 and Tyr151. Our observations open for further exploration of biological importance of the detected PTMs.

Iron Sensor Intracellular iron is an essential cofactor for many proteins, but can also damage macromolecules, so its levels are carefully controlled. Cellular iron homeostasis is mediated by iron regulatory proteins that regulate the expression of genes involved in iron uptake and storage. However, it is not clear how cells sense iron bioavailability (see the Perspective by Rouault ). Using different approaches, Salahudeen et al. (p. 722 , published online 17 September) and Vashisht et al. (p. 718 , published online 17 September) have identified the F-box protein FBXL5 as a human iron sensor. FBXL5 is part of an E3 ubiquitin ligase complex that regulates the degradation of iron regulatory proteins and thereby cellular iron levels. It contains a hemerythrin domain that binds iron and acts as an iron-dependent regulatory switch, causing the degradation of FBXL5 under low iron conditions. This alternative pathway for the regulation of iron homeostasis has implications for both normal cellular physiology and disease.