| 1. |
- Akhoondi, Shahab, et al.
(author)
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FBXW7/hCDC4 is a general tumor suppressor in human cancer
- 2007
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In: Cancer Research. - 0008-5472 .- 1538-7445. ; 67:19, s. 9006-9012
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Journal article (peer-reviewed)abstract
- The ubiquitin-proteasome system is a major regulatory pathway of protein degradation and plays an important role in cellular division. Fbxw7 (or hCdc4), a member of the F-box family of proteins, which are substrate recognition components of the multisubunit ubiquitin ligase SCF (Skpl-Cdc53/ Cullin-F-box-protein), has been shown to mediate the ubiquitin-dependent proteolysis of several oncoproteins including cyclin El, c-Myc, c-Jun, and Notch. The oncogenic potential of Fbxw7 substrates, frequent allelic loss in human cancers, and demonstration that mutation of FBXW7 cooperates with p53 in mouse tumorigenesis have suggested that Fbxw7 could function as a tumor suppressor in human cancer. Here, we carry out an extensive genetic screen of primary tumors to evaluate the role of FBXW7 as a tumor suppressor in human tumorigenesis. Our results indicate that FBXW7 is inactivated by mutation in diverse human cancer types with an overall mutation frequency of ∼ 6%. The highest mutation frequencies were found in tumors of the bile duct (cholangio-carcinomas, 35%), blood (T-cell acute lymphocytic leukemia, 31%), endometrium (9%), colon (9%), and stomach (6%). Approximately 43% of all mutations occur at two mutational "hotspots," which alter Arg residues (Arg465 and Arg479) that are critical for substrate recognition. Furthermore, we show that Fbxw7Arg465 hotspot mutant can abrogate wild-type Fbxw7 function through a dominant negative mechanism. Our study is the first comprehensive screen of FBXW7 mutations in various human malignancies and shows that FBXW7 is a general tumor suppressor in human cancer.
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| 2. |
- Cepeda, Diana
(author)
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F‐box proteins as regulators of oncogenic pathways by ubiquitylation
- 2011
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Doctoral thesis (other academic/artistic)abstract
- F-box proteins are the substrate-recognition components of the SCF E3 ubiquitin ligases that catalyze the ubiquitylation of many key cell cycle regulators. Functional studies indicate that the ubiquitin-proteasome system participates in the control of nearly all cellular processes through the timely degradation of short-lived regulatory proteins. Accordingly, altered protein degradation due to defective E3 ligases has been shown to underlie many human diseases, such as cancer. The studies in this thesis have focused on the functional characterization of two F-box proteins, FBXW7/hCDC4 and FBXO28, in ubiquitylation and degradation of the cell cycle regulatory proteins, cyclin E and Myc, and their potential deregulation in cancer. The tumor suppressor protein FBXW7/hCDC4 has been linked to human tumorigenesis through the targeted degradation of several important oncoproteins, including cyclin E. The ubiquitin-dependent turnover of cyclin E1 is regulated by phosphorylation and isomerization of cyclin E1, and executed by the concerted actions of the FBXW7/hCDC4-α and -γ isoforms. Our results demonstrate that this two-isoform dependence is not employed in conditions where cyclin E1 levels are elevated. Under these circumstances, cyclin E1 can be ubiquitylated by FBXW7/hCDC4-α alone, perhaps through an alternative pathway that does not require isomerization. In the second study, we report that cyclin E2 is targeted for ubiquitin-dependent proteolysis by SCFFBXW7/hCDC4. Interestingly, we found that cyclin E1 enhances the ubiquitin-dependent proteolysis of cyclin E2, suggesting a mechanism by which cyclin E1 regulates the abundance of cyclin E2, allowing it to possibly perform non-redundant functions in cell cycle control. In the last two studies we characterized the novel F-box protein, FBXO28, initially identified in an RNAi screen for F-box genes that regulate cell proliferation. We show that SCFFBXO28 targets Myc for ubiquitylation, without altering Myc protein turnover. Instead, FBXO28 was found to be an important regulator of Myc-driven transcription through the ubiquitin-dependent recruitment of a transcriptional cofactor to Myc target gene promoters. In addition, we found that FBXO28 is a nuclear substrate for cyclin-CDK phosphorylation and that phosphorylation of FBXO28 is significantly associated with poor prognosis in patients with primary breast cancer. FBXO28 may thus constitute an important player in cell proliferation and Myc pathways during tumorigenesis. In summary, in this thesis we present different mechanisms by which SCF-mediated ubiquitylation can regulate proliferation, thus linking ubiquitin- mediated processes to proliferative pathways often altered in human cancer.
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| 3. |
- Lerner, Mikael, et al.
(author)
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The RBCC gene RFP2 (leu5) encodes a novel transmembrane E3 ubiquitin ligase involved in ERAD
- 2007
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In: Molecular Biology of the Cell. - 1059-1524 .- 1939-4586. ; 18:5, s. 1670-1682
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Journal article (peer-reviewed)abstract
- RFP2, a gene frequently lost in various malignancies, encodes a protein with RING finger, B-box, and coiled-coil domains that belongs to the RBCC/TRIM family of proteins. Here we demonstrate that Rfp2 is an unstable protein with auto-polyubiquitination activity in vivo and in vitro, implying that Rfp2 acts as a RING E3 ubiquitin ligase. Consequently, Rfp2 ubiquitin ligase activity is dependent on an intact RING domain, as RING deficient mutants fail to drive polyubiquitination in vitro and are stabilized in vivo. Immunopurification and tandem mass spectrometry enabled the identification of several putative Rfp2 interacting proteins localized to the endoplasmic reticulum (ER), including valosin-containing protein (VCP), a protein indispensable for ER-associated degradation (ERAD). Importantly, we also show that Rfp2 regulates the degradation of the known ER proteolytic substrate CD3-delta, but not the N-end rule substrate Ub-R-YFP (yellow fluorescent protein), establishing Rfp2 as a novel E3 ligase involved in ERAD. Finally, we show that Rfp2 contains a C-terminal transmembrane domain indispensable for its localization to the ER and that Rfp2 colocalizes with several ER-resident proteins as analyzed by high-resolution immunostaining. In summary, these data are all consistent with a function for Rfp2 as an ERAD E3 ubiquitin ligase.
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