Control of PHAS-I phosphorylation in 3T3-L1 adipocytes: effects of inhibiting protein phosphatases and the p70 S6K signalling pathway

Lin, Teng Nan; Lawrence, John C.

doi:10.1007/s001250051391

Cited by 21 publications

(22 citation statements)

References 36 publications

(59 reference statements)

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“…This is in agreement with others who have shown that calyculin A can stimulate S6K activity and can separately increase PHAS-1 phosphorylation (44,45). Because calyculin A is a broad specificity phosphatase that inhibits both PP2A and PP1 equipotently, we also tested the effects of okadaic acid, which is more specific for PP2A, and tautomycin, which is more specific for PP1, on osmotic stress inhibition of S6K (40).…”

Section: Figsupporting

confidence: 90%

Osmotic Stress Inhibits p70/85 S6 Kinase through Activation of a Protein Phosphatase

Parrott

Templeton

1999

Journal of Biological Chemistry

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While studying the stress regulation of p70/85 S6 kinase (S6K), we observed that anisomycin and UV light stimulated S6K activity, but that sorbitol inactivated S6K. Pretreatment with hyperosmotic stress also prevented the activation of S6K by both 12-O-tetradecanoylphorbol-13-acetate and anisomycin. Comparison of sorbitol and rapamycin revealed that both agents inactivated S6K and caused dephosphorylation of Ser/ThrPro sites in the COOH terminus of S6K, including Thr 412 , a residue essential to S6K regulation, as determined by phospho-specific antibodies. Rapamycin-resistant S6K truncation mutants were similarly resistant to deactivation by sorbitol. Additionally, the PHAS-1 mobility shift, which is sensitive to rapamycin, was also found to be sensitive to osmotic stress. Experiments using the p38 inhibitor SB203580 and dominant negative mutants involving both stress-activated protein kinase/c-Jun NH 2 -terminal kinase and p38 stress pathways indicated that these pathways are probably not involved in osmotic stress inhibition of S6K. Examining the potential involvement of a phosphatase, we found that sodium pyrophosphate, sodium vanadate, cyclosporin A, tautomycin, and okadaic acid had no effect on osmotic stress inhibition of S6K. However, calyculin A prevented both rapamycin-and sorbitol-mediated deactivation of S6K. Our results suggest that osmotic stress and rapamycin act through a calyculin A-sensitive phosphatase to cause dephosphorylation and deactivation of S6K.The p70/85 ribosomal S6 kinase (S6K) 1 is stimulated by a variety of mitogens, including insulin, TPA, and growth factors (1, 2). The activation of S6K involves phosphorylation on multiple sites (3-5). Ser/Thr kinases such as MAP kinase family members and cdc2/cyclin B can phosphorylate Ser/Thr-Pro residues in the COOH terminus of S6K (6, 7). RAFT1/FRAP/ mTOR can phosphorylate Thr 412 , whereas a kinase termed PDK can phosphorylate Thr 252 (8 -11). These and other phosphorylation events contribute to the multistep activation of S6K (3). Such findings suggest that S6K may be an integrator of signals from various kinase cascades.S6K function is also controlled via inhibitory domains at both the NH 2 terminus and COOH terminus of the kinase (12, 13). Deletion of these domains confers relative resistance of these truncated kinases to the inhibitory effects of rapamycin. A model that explains these findings is that phosphorylation of S6K negates the inhibitory effect of these domains. Rapamycin might thus promote dephosphorylation events within these domains, a principle first suggested by Ferrari et al. (5).Upon activation, S6K phosphorylates the S6 protein of the 40S ribosomal subunit (14). Phosphorylated S6 directs the translational machinery toward increased translation of transcripts containing an oligopyrimidine tract (5Ј TOP), such as those encoding ribosomal proteins and elongation factors (15-17). Thus, the most well understood function of S6K involves a contribution to cell growth by increasing the production of translational machinery. It i...

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Section: Figsupporting

confidence: 90%

Osmotic Stress Inhibits p70/85 S6 Kinase through Activation of a Protein Phosphatase

Parrott

Templeton

1999

Journal of Biological Chemistry

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“…Given the observation that MG132 caused a slight shift in TSC2 mobility indicative of phosphorylation changes and the previous involvement of PP2A in mTORC1 signaling (18)(19)(20)(21)(22), we tested whether PP2A was involved in disrupting mTORC1 formation. Treatment of cells with the PP2A inhibitor okadaic acid was able to restore mTOR/raptor complex formation and signaling, suggesting that PP2A regulates mTORC1 formation (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…Under conditions where TOR is inactivated (e.g., nutrient starvation or rapamycin treatment), the Tip41 protein binds Tap42, allowing for SIT4 dissociation and restoration of phosphatase activity (33,36). Similarly, in mammalian cells, small molecule inhibition of PP2A activity blocks rapamycin-induced dephosphorylation of the mTORC1 substrates 4E-BP1 and S6K1 (18)(19)(20)(21)(22), suggesting that PP2A directly dephosphorylates mTORC1 substrates when mTORC1 activity is inhibited by rapamycin, or that mTORC1 constrains PP2A activity. However, with the latter scenario, it is unclear whether the analogous regulatory paradigm with the Tip41L/α4/PP2A axis in mammals is conserved from yeast (37)(38)(39).…”

Section: Discussionmentioning

confidence: 99%

Control of mTORC1 signaling by the Opitz syndrome protein MID1

Liu

Knutzen

Krauß

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Mutations in the MID1 gene are causally linked to X-linked Opitz BBB/G syndrome (OS), a congenital disorder that primarily affects the formation of diverse ventral midline structures. The MID1 protein has been shown to function as an E3 ligase targeting the catalytic subunit of protein phosphatase 2A (PP2A-C) for ubiquitinmediated degradation. However, the molecular pathways downstream of the MID1/PP2A axis that are dysregulated in OS and that translate dysfunctional MID1 and elevated levels of PP2A-C into the OS phenotype are poorly understood. Here, we show that perturbations in MID1/PP2A affect mTORC1 signaling. Increased PP2A levels, resulting from proteasome inhibition or depletion of MID1, lead to disruption of the mTOR/Raptor complex and downregulated mTORC1 signaling. Congruously, cells derived from OS patients that carry MID1 mutations exhibit decreased mTORC1 formation, S6K1 phosphorylation, cell size, and cap-dependent translation, all of which is rescued by expression of wild-type MID1 or an activated mTOR allele. Our findings define mTORC1 signaling as a downstream pathway regulated by the MID1/PP2A axis, suggesting that mTORC1 plays a key role in OS pathogenesis.ubiquitin ligase | proteasome-mediated degradation M utations in the MID1 gene are causally linked to X-linked Opitz BBB/G syndrome (OS), a congenital disorder that primarily affects the formation of ventral midline structures. The clinical manifestations of Opitz syndrome are characterized by a diverse spectrum of symptoms, including hypertelorism and hypospadias, cleft lip/palate, dysphagia, heart defects, and mental retardation (1).The MID1 protein is a member of the RBCC/TRIM family, which contains a conserved module of the RING domain, followed by B-boxes and a coiled-coil domain (2). MID1 also contains fibronectin type III and B30.2/SPRY domains. The RING domain has been well characterized in ubiquitin-mediated protein degradation, whereas the other domains mediate proteinprotein interactions (3-6). Opitz syndrome-derived mutations in MID1 have been identified throughout the protein and show several functional consequences such as compromised association with microtubules and/or transport along microtubules (4, 7-9). In addition to its microtubule-binding function, MID1 also functions as an E3 ligase that targets the microtubule-associated pool of the catalytic subunit of protein phosphatase 2A (PP2A-C) for ubiquitin-mediated degradation through an interaction with the protein α4 (3). Perturbation of the E3 ligase function of MID1 in OS cells leads to the accumulation of PP2A-C and the dramatic dephosphorylation of microtubule-associated proteins, which is postulated to contribute to OS pathogenesis (3).Signaling from the mammalian target of rapamycin (mTOR) controls diverse cellular processes such as growth, autophagy, stress responses, cytoskeletal reorganization, cell motility, metabolism, and aging (10-12). mTORC1 consists of mTOR and the interacting proteins, Raptor, and mLST8. In particular, Raptor plays an essential scaffolding...

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“…In addition, the assay was conducted with recombinant GST-4E-BP1 as an in vitro substrate; hence, any modifications on 4E-BP1 in vivo would not be accounted for. Conversely, it is unlikely that inhibition of a phosphatase specific for 4E-BP1 is involved, because calyculin A and okadaic acid-induced 4E-BP1 phosphorylation was sensitive to rapamycin (26). Furthermore, the ability of wortmannin and PI-103, which, like rapamycin, inhibits mTORC1, to attenuate this reemerging phosphorylation suggests that phosphatase inhibition would be insufficient (Fig.…”

Section: Discussionmentioning

confidence: 99%

Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation

Choo

Yoon²,

Kim³

et al. 2008

Proc. Natl. Acad. Sci. U.S.A.

726

595

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The mammalian translational initiation machinery is a tightly controlled system that is composed of eukaryotic initiation factors, and which controls the recruitment of ribosomes to mediate cap-dependent translation. Accordingly, the mTORC1 complex functionally controls this cap-dependent translation machinery through the phosphorylation of its downstream substrates 4E-BPs and S6Ks. It is generally accepted that rapamycin, a specific inhibitor of mTORC1, is a potent translational repressor. Here we report the unexpected discovery that rapamycin's ability to regulate cap-dependent translation varies significantly among cell types. We show that this effect is mechanistically caused by rapamycin's differential effect on 4E-BP1 versus S6Ks. While rapamycin potently inhibits S6K activity throughout the duration of treatment, 4E-BP1 recovers in phosphorylation within 6 h despite initial inhibition (1-3 h). This reemerged 4E-BP1 phosphorylation is rapamycinresistant but still requires mTOR, Raptor, and mTORC1's activity. Therefore, these results explain how cap-dependent translation can be maintained in the presence of rapamycin. In addition, we have also defined the condition by which rapamycin can control cap-dependent translation in various cell types. Finally, we show that mTOR catalytic inhibitors are effective inhibitors of the rapamycin-resistant phenotype.cap-dependent translation ͉ mTORC1 ͉ rapamycin resistance T he mammalian translational initiation machinery governs the recruitment of ribosomes to mRNA to commence the production of protein synthesis. This machinery consists of various eukaryotic initiation factors (eIFs) that tightly regulate protein synthesis based on environmental cues. Importantly, initiation is an important step for cellular control because it is the ratelimiting step of translation (1).Two predominant pathways translate mammalian mRNA through cap-dependent and independent mechanisms. The capping of the 5Ј end of mRNA by m 7 GTP allows the recruitment of the eIF4F complex, eIF3, and the 40S ribosomal subunit to the 5Ј mRNA cap. Capindependent translation is mediated by an internal RNA structure called internal ribosome entry site (IRES), which recruits the ribosome independent of both the cap and the entire eIF4F complex (2).The initiation of cap-dependent translation is tightly regulated by extracellular conditions including glucose, nutrient, and growth factor levels. These factors control cap-dependent translation by regulating the evolutionarily conserved mTORC1 (mTOR, Raptor, mLST8) pathway (3). Activation of mTORC1 positively stimulates mRNA translation via its downstream substrates S6Ks and 4E-BP1/ eIF4E (4-7). Phosphorylation of 4E-BP1 by mTORC1 results in its dissociation from eIF4E, promoting assembly of the eIF4F complex. It is thought that S6K1 can phosphorylate translational regulators such as eIF4B and PDCD4 to enhance the translational efficiency of mRNAs with highly structured 5Ј UTRs (8-10).Therefore, growth factors positively regulate cap-dependent translation via mTORC...

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Control of PHAS-I phosphorylation in 3T3-L1 adipocytes: effects of inhibiting protein phosphatases and the p70 S6K signalling pathway

Cited by 21 publications

References 36 publications

Osmotic Stress Inhibits p70/85 S6 Kinase through Activation of a Protein Phosphatase

Osmotic Stress Inhibits p70/85 S6 Kinase through Activation of a Protein Phosphatase

Control of mTORC1 signaling by the Opitz syndrome protein MID1

Rapamycin differentially inhibits S6Ks and 4E-BP1 to mediate cell-type-specific repression of mRNA translation

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