F-box and WD Repeat Domain-containing-7 (Fbxw7) Protein Targets Endoplasmic Reticulum-anchored Osteogenic and Chondrogenic Transcriptional Factors for Degradation

Yumimoto, Kanae; Matsumoto, Masaki; Onoyama, Ichiro; Imaizumi, Kazunori; Nakayama, Keiichi I.

doi:10.1074/jbc.m113.465179

Cited by 39 publications

(41 citation statements)

References 52 publications

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“…Recently, SCF FBW7 was shown to inhibit osteogenesis by regulating OASIS expression through ubiquitin-mediated proteasome degradation (23). In osteoblasts, Runx2 levels are regulated coordinately with the cell cycle machinery, apparently by E3 ubiquitin ligases (14,24).…”

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confidence: 99%

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation

Kumar

Kapoor

Khan

et al. 2015

Journal of Biological Chemistry

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Runx2, a master regulator of osteoblast differentiation, is tightly regulated at both transcriptional and post-translational levels. Post-translational modifications such as phosphorylation and ubiquitination have differential effects on Runx2 functions. Here, we show that the reduced expression and functions of Runx2 upon its phosphorylation by GSK3␤ are mediated by its ubiquitin-mediated degradation through E3 ubiquitin ligase Fbw7␣. Fbw7␣ through its WD domain interacts with Runx2 both in a heterologous (HEK293T cells) system as well as in osteoblasts. GSK3␤ was also present in the same complex as determined by co-immunoprecipitation. Furthermore, overexpression of either Fbw7␣ or GSK3␤ was sufficient to down-regulate endogenous Runx2 expression and function; however, both failed to inhibit endogenous Runx2 when either of them was depleted in osteoblasts. Fbw7␣-mediated inhibition of Runx2 expression also led to reduced Runx2 transactivation and osteoblast differentiation. In contrast, inhibition of Fbw7␣ restored Runx2 levels and promoted osteoblast differentiation. We also observed reciprocal expression levels of Runx2 and Fbw7␣ in models of bone loss such as lactating (physiological bone loss condition) and ovariectomized (induction of surgical menopause) animals that show reduced Runx2 and enhanced Fbw7␣, whereas this was reversed in the estrogen-treated ovariectomized animals. In addition, methylprednisolone (a synthetic glucocorticoid) treatment to neonatal rats showed a temporal decrease in Runx2 with a reciprocal increase in Fbw7 in their calvarium. Taken together, these data demonstrate that Fbw7␣ negatively regulates osteogenesis by targeting Runx2 for ubiquitin-mediated degradation in a GSK3␤-dependent manner and thus provides a plausible explanation for GSK3␤-mediated bone loss as described before.Runt domain-containing protein Runx2 is a critical regulator of the osteogenic lineage (1). Null mutation of Runx2 gene results in unmineralized skeleton in mice, and its heterozygotic loss results in human cleidocranial dysplasia (CCD) 2 phenotype (2, 3), which demonstrates the importance of Runx2 in bone biology. Runx2 induces transcription of osteopontin (OP) and osteocalcin (OC) by binding to osteoblast-specific cis-acting OSE2 elements in the promoter regions of these genes (4, 5). Runx2 expression is regulated by vitamin D 3 , estrogen, TGF-␤/ BMP2, and Wnt signaling pathways (6 -10).In comparison with transcriptional regulation, the posttranslational regulation of Runx2 in osteoblasts is relatively unexplored (11). Recent studies indicate that phosphorylation, acetylation, SUMOylation, and ubiquitination regulate Runx2 functions differently (11). The effects of phosphorylation by different kinases on the Runx2 protein stability and its subsequent function have been reported earlier (12-15); however, the identification of E3 ubiquitin ligases in such processes has remained elusive.F-box protein Fbw7 (also known as Fbwx7 and cdc4) is a component of the SCF (Skp-Cullin-F box) ubiquitin ligase complex...

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confidence: 99%

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation

Kumar

Kapoor

Khan

et al. 2015

Journal of Biological Chemistry

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“…Recombinant SCF FBXO21 and HA‐EID1 were purified and subjected to an in vitro ubiquitylation assay as described previously (Yumimoto et al . ).…”

Section: Methodsmentioning

confidence: 97%

FBXO21 mediates the ubiquitylation and proteasomal degradation of EID1

2015

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Although identification of substrates for ubiquitin ligase (E3) is important for understanding its biological functions, detection of the interaction between an E3 and its substrates has remained challenging. We recently developed a new approach, termed differential proteomics-based identification of ubiquitylation substrates (DiPIUS), for the discovery of substrates of a given E3 ligase. We have now applied this approach to an uncharacterized human F-box protein, FBXO21, which serves as the substrate-recognition subunit of a SKP1-CUL1-F-box protein (SCF)-type E3, thereby identifying EID1 (EP300-interacting inhibitor of differentiation 1) as a candidate substrate. The central and COOH-terminal portion of FBXO21 was found to interact with the COOH-terminal region of EID1 in transfected cells. Over-expression of FBXO21 resulted in the down-regulation of EID1, whereas disruption of the FBXO21 gene with the CRISPR/Cas9 system stabilized EID1 and led to its accumulation in both the cytoplasm and nucleus. An in vitro ubiquitylation assay showed that EID1 is a direct substrate of SCF FBXO21 . Collectively, our results suggest that EID1 is a bona fide substrate of FBXO21 and that the control of EID1 abundance by SCF FBXO21 might affect the transcriptional repression activity of EID1.

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“…F‐box proteins constitute a large family of >70 eukaryotic proteins (Jin et al, ), but the substrates of each F‐box protein have remained largely unknown. To identify substrates for a given F‐box protein, we previously developed a method termed differential proteomics‐based identification of ubiquitylation substrates (DiPIUS; Yumimoto, Matsumoto, Oyamada, Moroishi, & Nakayama, ), and we have applied this method to several systems (Nakajima et al, ; Nakayama, Yumimoto, Kawamura, & Nakayama, ; Nishiyama, Nita, Yumimoto, & Nakayama, ; Watanabe, Yumimoto, & Nakayama, ; Yamauchi, Nishiyama, Moroishi, Kawamura, & Nakayama, ; Yumimoto, Matsumoto, Onoyama, Imaizumi, & Nakayama, ).…”

Section: Introductionmentioning

confidence: 99%

“…It was subsequently shown to play a pivotal role in the control of cell proliferation and differentiation through ubiquitylation of cyclin E (Koepp et al, ; Moberg, Bell, Wahrer, Haber, & Hariharan, ; Strohmaier et al, ), c‐Myc (Welcker et al, ; Yada et al, ), Notch (Gupta‐Rossi et al, ; Hubbard et al, ; Oberg et al, ), c‐Jun (Nateri, Riera‐Sans, Da Costa, & Behrens, ; Wei, Jin, Schlisio, Harper, & Kaelin, ) and Krüppel‐like factor (KLF) 5 (Liu et al, ; Zhao, Zheng, Zhou, & Chen, ). In addition, Fbxw7 was found to contribute to cell differentiation and development in many tissues through ubiquitylation of sterol response element‐binding proteins (SREBPs; Onoyama et al, ; Sundqvist et al, ), OASIS (Yumimoto et al, ), BBF2H7 (Yumimoto et al, ) and MyRF (Nakayama et al, ).…”

Section: Introductionmentioning

confidence: 99%

SCF^Fbxw7 ubiquitylates KLF7 for degradation in a manner dependent on GSK‐3‐mediated phosphorylation

et al. 2019

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The biological relation between ubiquitin ligases and their substrates has been largely unclear. We previously developed a method—differential proteomics‐based identification of ubiquitylation substrates (DiPIUS)—for the comprehensive identification of substrates for a given ubiquitin ligase. We have now applied DiPIUS to the F‐box protein Fbxw7 in three cell lines (mHepa, Neuro2A and C2C12) and thereby identified Krüppel‐like factor 7 (KLF7) as a candidate substrate of the SCFFbxw7 ubiquitin ligase complex. KLF7 was shown to interact with Fbxw7 and to undergo Fbxw7‐mediated polyubiquitylation. The stability of KLF7 was increased by depletion of Fbxw7, mutation of a putative Cdc4 phosphodegron (CPD) of KLF7 or exposure to inhibitors of glycogen synthase kinase‐3 (GSK‐3). Over‐expression of Fbxw7 in Neuro2A cells down‐regulated expression of the p21Cip1 gene, which is a transcriptional target of KLF7 in neuronal differentiation and maintenance. Despite the presence of an almost identical CPD sequence in KLF6, the closest paralog of KLF7, mutation of this sequence affected neither the interaction of KLF6 with Fbxw7 nor its half‐life. Our results suggest that KLF7, but not KLF6, is a bona fide substrate of SCFFbxw7, and that control of KLF7 abundance by SCFFbxw7 might contribute to the regulation of neuronal differentiation and maintenance.

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F-box and WD Repeat Domain-containing-7 (Fbxw7) Protein Targets Endoplasmic Reticulum-anchored Osteogenic and Chondrogenic Transcriptional Factors for Degradation

Cited by 39 publications

References 52 publications

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation

FBXO21 mediates the ubiquitylation and proteasomal degradation of EID1

SCF^Fbxw7 ubiquitylates KLF7 for degradation in a manner dependent on GSK‐3‐mediated phosphorylation

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F-box and WD Repeat Domain-containing-7 (Fbxw7) Protein Targets Endoplasmic Reticulum-anchored Osteogenic and Chondrogenic Transcriptional Factors for Degradation

Cited by 39 publications

References 52 publications

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation

E3 Ubiquitin Ligase Fbw7 Negatively Regulates Osteoblast Differentiation by Targeting Runx2 for Degradation

FBXO21 mediates the ubiquitylation and proteasomal degradation of EID1

SCFFbxw7 ubiquitylates KLF7 for degradation in a manner dependent on GSK‐3‐mediated phosphorylation

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SCF^Fbxw7 ubiquitylates KLF7 for degradation in a manner dependent on GSK‐3‐mediated phosphorylation