Negative Regulation of TSC1-TSC2 by Mammalian D-Type Cyclins

Zacharek, Sima J.; Shumway, Stuart D.

doi:10.1158/0008-5472.can-05-2236

Cited by 66 publications

(60 citation statements)

References 63 publications

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“…Although MEK itself was required for hyperexpression of cyclin D1, MTOR had additional but not major roles in this phenomenon. Noteworthy, it has been reported that cyclin D1 can, in turn, further activate MTOR, 69 and vice versa inhibition of MTOR can decrease cyclin D1 levels, 60,67 establishing positive feedback between them.…”

Section: Discussionmentioning

confidence: 99%

MEK drives cyclin D1 hyperelevation during geroconversion

2013

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When the cell cycle becomes arrested, MTOR (mechanistic Target of Rapamycin) converts reversible arrest into senescence (geroconversion). Hyperexpression of cyclin D1 is a universal marker of senescence along with hypertrophy, beta-Gal staining and loss of replicative/regenerative potential (RP), namely, the ability to restart proliferation when the cell cycle is released. Inhibition of MTOR decelerates geroconversion, although only partially decreases cyclin D1. Here we show that in p21-and p16-induced senescence, inhibitors of mitogen-activated/extracellular signal-regulated kinase (MEK) (U0126, PD184352 and siRNA) completely prevented cyclin D1 accumulation, making it undetectable. We also used MEL10 cells in which MEK inhibitors do not inhibit MTOR. In such cells, U0126 by itself induced senescence that was remarkably cyclin D1 negative. In contrast, inhibition of cyclin-dependent kinase (CDK) 4/6 by PD0332991 caused cyclin D1-positive senescence in MEL10 cells. Both types of senescence were suppressed by rapamycin, converting it into reversible arrest. We confirmed that the inhibitor of CDK4/6 caused cyclin D1 positive senescence in normal RPE cells, whereas U0126 prevented cyclin D1 expression. Elimination of cyclin D1 by siRNA did not prevent other markers of senescence that are consistent with the lack of its effect on MTOR. Our data confirmed that a mere inhibition of the cell cycle was sufficient to cause senescence, providing MTOR was active, and inhibition of MEK partially inhibited MTOR in a cell-type-dependent manner. Second, hallmarks of senescence may be dissociated, and hyperelevated cyclin D1, a marker of hyperactivation of senescent cells, did not necessarily determine other markers of senescence. Third, inhibition of MEK was sufficient to eliminate cyclin D1, regardless of MTOR. In proliferating cells, MTOR (mechanistic Target of Rapamycin) stimulates cellular mass growth, whereas cyclins D and E initiate S-phase transition.1-4 Withdrawal of growth factors causes reversible cell-cycle arrest (quiescence) with low levels of cyclins and MTOR activity, reversible by re-addition of growth factors. In contrast, cell-cycle arrest in the presence of growth stimuli, which activate MTOR, leads to cellular senescence. 5,6 In other words, when the cell cycle is blocked, while growth-promoting pathways such as MTOR remain active, cells continue to grow in size and undergo senescence. A process of conversion from normal reversible arrest into senescence was named as gerogenic conversion (geroconversion).7 Cellular senescence is characterized by large flat morphology (hypertrophy), beta-Gal staining, very high levels of cyclins D1 and E, senescence-associated secretory phenotype and loss of replicative/regenerative potential (RP).7-12 Loss of RP means that cells cannot proliferate even when cell-cycle block is removed and cells re-enter the cell cycle.13,14 Rapamycin decreases hypertrophy and loss of RP, in a dose-dependent manner proportional to S6 dephosphorylation (a marker of MTORC1 activity). 15Other ...

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Section: Discussionmentioning

confidence: 99%

MEK drives cyclin D1 hyperelevation during geroconversion

2013

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“…Several molecules critical for cell cycle progression have recently been shown to interact with TSC1 or TSC2 in mammalian cells, including: D-type cyclins (Zacharek et al, 2005), polo-like kinase 1 (Astrinidis et al, 2006) and the cyclin-dependent kinase inhibitor p27 , These findings are not surprising; well before the mammalian homolog of TOR was discovered, it was surmised that whatever molecule rapamycin targeted must have a significant effect on cell cycle progression, given the profound effects of rapamycin treatment in T lymphocytes and in yeast (Sabers et al, 1995). Other growth regulatory proteins that have been shown to affect mTOR via TSC1/2 include the SMAD (mothers against decapentaplegic) proteins (Birchenall-Roberts et al, 2004), the focal adhesion kinase interacting protein FIP200 (Gan et al, 2005), the estrogen receptor (Finlay et al, 2004) and many more than space will allow us to mention in detail.…”

Section: Negative and Positive Feedbackmentioning

confidence: 99%

Upstream of the mammalian target of rapamycin: do all roads pass through mTOR?

2006

Oncogene

348

309

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The mammalian target of rapamycin (mTOR) is a serine/ threonine kinase that controls many aspects of cellular physiology, including transcription, translation, cell size, cytoskeletal organization and autophagy. Recent advances in the mTOR signaling field have found that mTOR exists in two heteromeric complexes, mTORC1 and mTORC2. The activity of mTORC1 is regulated by the integration of many signals, including growth factors, insulin, nutrients, energy availability and cellular stressors such as hypoxia, osmotic stress, reactive oxygen species and viral infection. In this review we highlight recent advances in the mTOR signaling field that relate to how the two mTOR complexes are regulated, and we discuss stress conditions linked to the mTOR signaling network that have not been extensively covered in other reviews. Given the diversity of signals that have been shown to impinge on mTOR, we also speculate on other signaltransduction pathways that may be linked to mTOR in the future.

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“…Antibodies to CUL4A, DDB1, ROC1, CAND1, TSC1, and TSC2 were described previously (Shumway et al 2003;Hu et al 2004;Zacharek et al 2005). Polyclonal antibody to Gigas was raised against the C-terminal epitope of Gigas protein (DMDDQRGDFIKYT).…”

Section: Antibodies Proteins and Immunological Proceduresmentioning

confidence: 99%

WD40 protein FBW5 promotes ubiquitination of tumor suppressor TSC2 by DDB1–CUL4–ROC1 ligase

Hu¹,

Zacharek²,

He³

et al. 2008

Genes Dev.

Self Cite

145

129

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Tuberous sclerosis (TSC) is an autosomal dominant disease characterized by hamartoma formation in various organs and is caused by mutations targeting either theThe tuberous sclerosis 1 (TSC1) locus encodes a 130-kDa protein termed "hamartin," and the TSC2 locus encodes a 180-kDa protein termed "tuberin" that contains a coiled-coil domain and a GAP (GTPase-activating protein) domain. Genetic studies in Drosophila melanogaster and biochemical analyses in mammalian cells have established the TSC1-TSC2 heterodimeric complex as a critical regulator in coupling various cellular conditions to cell growth through stimulation of GTP hydrolysis of RheB to antagonize the mTOR signaling pathway (Inoki et al. 2005). These studies have identified both TSC subunits as substrates of various kinases that mediate different cellular conditions such as nutrient availability, energy, hormones, and growth factor stimulation. TSC2 is a short-lived protein, and it is actively ubiquitinated (Chong-Kopera et al. 2006). Many disease-associated mutations in TSC1 and TSC2 result in a substantial decrease in the level of hamartin and tuberin, respectively (Inoki et al. 2002;Nellist et al. 2005), suggesting that protein turnover plays a critical role in TSC regulation. Identification of the ubiquitin ligase for TSC2 would shed light on the understanding of the regulation of TSC and cell growth pathways. Results and Discussion TSC2 binds to WD40 protein FBW5, DDB1, and CUL4We used a yeast two-hybrid screen and coupled immunoprecipitation and immunoblotting (IP-Western) analyses to search for TSC2-interacting protein with potential ubiquitin ligase activity and identified two F-box proteins-FBW5 (Fig. 1A) and FBL6 (data not shown)-that bind with TSC2. Three additional control F-box proteins-SKP2, ␤-TrCP, and FBX5-did not bind with TSC2 in the same assay (data not shown), indicating specificity of the FBL6-TSC2 and FBW5-TSC2 interactions and also suggesting that the F-box motif is not sufficient for binding to TSC2. Consistent with TSC1-TSC2 dimerization, FBW5 also associates with TSC1 (Fig. 1A). Our subsequent genetic study in Drosophila (see below) led us to focus on determining the function and mechanism of the FBW5-TSC interaction. The functional significance of the FBL6-TSC interaction remains to be determined.Human FBW5 is a 60-kDa (566 residues) protein containing two recognizable domains; an N-terminally located F-box motif and seven WD40 repeats occupying most of the rest of the sequence. The F-box motif functions to bridge a substrate protein to the CUL1-ROC1 E3 ligase via the SKP1 linker (Bai et al. 1996;Feldman et al. 1997;Skowyra et al. 1997). Recently, we and others found that DDB1 binds to a subset of WD40 proteins referred to as DWD proteins (also known as DCAF, for Ddb1-and Cul4-associated factors; or CDW, for CUL4-and DDB1-associated WD40 repeat proteins), and bridges them to CUL4-ROC1 to constitute a potentially large and distinct family of DWD-DDB1-CUL4-ROC1 E3 ubiquitin ligases (Angers et al. 2006;He et al. 2006;Higa et al. ...

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Negative Regulation of TSC1-TSC2 by Mammalian D-Type Cyclins

Cited by 66 publications

References 63 publications

MEK drives cyclin D1 hyperelevation during geroconversion

MEK drives cyclin D1 hyperelevation during geroconversion

Upstream of the mammalian target of rapamycin: do all roads pass through mTOR?

WD40 protein FBW5 promotes ubiquitination of tumor suppressor TSC2 by DDB1–CUL4–ROC1 ligase

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