X. F. Steven Zheng

Nutrient regulates Tor1 nuclear localization and association with rDNA promoter

TOR is the target of the immunosuppressant rapamycin and a key regulator of cell growth. It modulates diverse cellular processes in the cytoplasm and nucleus, including the expression of amino acid transporters, ribosomal RNAs and ribosomal proteins. Despite considerable recent progress, little is known about the spatial and temporal regulation of TOR signalling, particularly that leading into the nucleus. Here we show that Tor1 is dynamically distributed in the cytoplasm and nucleus in yeast. Tor1 nuclear localization is nutrient dependent and rapamycin sensitive: starvation or treatment with rapamycin causes Tor1 to exit from the nucleus. Tor1 nuclear localization is critical for 35S rRNA synthesis, but not for the expression of amino acid transporters and ribosomal protein genes. We show further that Tor1 is associated with 35S ribosomal DNA (rDNA) promoter chromatin in a rapamycin- and starvation-sensitive manner; this association is necessary for 35S rRNA synthesis and cell growth. These results indicate that the spatial regulation of TOR complex 1 (TORC1) might be involved in differential control of its target genes. TOR is known as a classic cytoplasmic kinase that mediates the cytoplasm-to-nucleus signalling by controlling the localization of transcription factors. Our data indicate that TOR might be more intimately involved in gene regulation than previously thought.

Tripartite Regulation of Gln3p by TOR, Ure2p, and Phosphatases

Gln3p is a GATA-type transcription factor responsive to different nitrogen nutrients and starvation in yeastSaccharomyces cerevisiae. Recent evidence has linked TOR signaling to Gln3p. Rapamycin causes dephosphorylation and nuclear translocation of Gln3p, thereby activating nitrogen catabolite repressible-sensitive genes. However, a detailed mechanistic understanding of this process is lacking. In this study, we show that Tor1p physically interacts with Gln3p. An intact TOR kinase domain is essential for the phosphorylation of Gln3p, inhibition of Gln3p nuclear entry and repression of Gln3p-dependent transcription. In contrast, at least two distinct protein phosphatases, Pph3p and the Tap42p-dependent phosphatases, are involved in the activation of Gln3p. The yeast pro-prion protein Ure2p binds to both hyper- and hypo-phosphorylated Gln3p. In contrast to the free Gln3p, the Ure2p-bound Gln3p is signifcantly resistant to dephosphorylation. Taken together, these results reveal a tripartite regulatory mechanism by which the phosphorylation of Gln3p is regulated.

FKBP12-Rapamycin-associated Protein or Mammalian Target of Rapamycin (FRAP/mTOR) Localization in the Endoplasmic Reticulum and the Golgi Apparatus

FKBP12-rapamycin-associated protein (FRAP) or mammalian target of rapamycin (mTOR) and its effector proteins form a critical signaling pathway that regulates eukaryotic cell growth and proliferation. Although the protein components in this pathway have begun to be identified, little is known about their subcellular localization or the physiological significance of their localization. By immunofluorescence, we find that both endogenous and recombinant FRAP/mTOR proteins show localization predominantly in the endoplasmic reticulum (ER) and the Golgi apparatus. Consistent with this finding, FRAP/mTOR is cofractionated with calnexin, an ER marker protein. Biochemical characterization suggests that FRAP/mTOR is a peripheral ER/Golgi protein with tight membrane association. Finally, we have identified domains of FRAP/mTOR which may mediate its association with the ER and the Golgi apparatus.

Chromatin-mediated regulation of nucleolar structure and RNA Pol I localization by TOR

The target of rapamycin (TOR) protein is a conserved regulator of ribosome biogenesis, an important process for cell growth and proliferation. However, how TOR is involved remains poorly understood. In this study, we find that rapamycin and nutrient starvation, conditions inhibiting TOR, lead to significant nucleolar size reduction in both yeast and mammalian cells. In yeast, this morphological change is accompanied by release of RNA polymerase I (Pol I) from the nucleolus and inhibition of ribosomal DNA (rDNA) transcription. We also present evidence that TOR regulates association of Rpd3–Sin3 histone deacetylase (HDAC) with rDNA chromatin, leading to site‐specific deacetylation of histone H4. Moreover, histone H4 hypoacetylation mutations cause nucleolar size reduction and Pol I delocalization, while rpd3Ü and histone H4 hyperacetylation mutations block the nucleolar changes as a result of TOR inhibition. Taken together, our results suggest a chromatin‐mediated mechanism by which TOR modulates nucleolar structure, RNA Pol I localization and rRNA gene expression in response to nutrient availability.

Targeting the mTOR kinase domain: the second generation of mTOR inhibitors

The mTOR signaling pathway is dysregulated in ∼50% of all human malignancies and is a major cancer drug target. Although rapamycin analogs (rapalogs) have shown clinical efficacy in a subset of cancers, they do not fully exploit the antitumor potential of mTOR targeting. Because the mTOR kinase domain is important for rapamycin-sensitive and -insensitive functions, mTOR catalytic inhibitors have been developed recently as the second generation of anti-mTOR agents. Importantly, they have shown marked improvement of antitumor activity in vivo and in vitro. This review will detail the potential therapeutic value and issues of these novel antineoplastic agents, with emphasis placed on those that have already entered clinical trials.

Regulation of Subtelomeric Silencing during Stress Response

Sir proteins play a critical role in silent chromatin domains. While mutations can cause derepression of heterochromatin, it remains unclear whether silencing is actively involved in transcriptional control under changing environmental conditions. We find that TOR inhibits Sir3 phosphorylation. Rapamycin or stress induced by chlorpromazine leads to activation of MAP kinase Mpk1/Slt2, which phosphorylates Sir3. Sir3 hyperphosphorylation is correlated with reduced subtelomeric silencing, increased subtelomeric cell wall gene expression, and stress resistance to chlorpromazine, but does not affect the silent HML and rDNA loci. Based on these observations, we propose that regulation of silencing may be used to control gene expression at specific silent chromatin domains in response to stress and possibly other environmental changes.

Convergence of TOR-Nitrogen and Snf1-Glucose Signaling Pathways onto Gln3

ABSTRACT Carbon and nitrogen are two basic nutrient sources for cellular organisms. They supply precursors for energy metabolism and metabolic biosynthesis. In the yeast Saccharomyces cerevisiae, distinct sensing and signaling pathways have been described that regulate gene expression in response to the quality of carbon and nitrogen sources, respectively. Gln3 is a GATA-type transcription factor of nitrogen catabolite-repressible (NCR) genes. Previous observations indicate that the quality of nitrogen sources controls the phosphorylation and cytoplasmic retention of Gln3 via the target of rapamycin (TOR) protein. In this study, we show that glucose also regulates Gln3 phosphorylation and subcellular localization, which is mediated by Snf1, the yeast homolog of AMP-dependent protein kinase and a cytoplasmic glucose sensor. Our data show that glucose and nitrogen signaling pathways converge onto Gln3, which may be critical for both nutrient sensing and starvation responses.

Mechanisms of regulation of RNA polymerase III‐dependent transcription by TORC1

We have found earlier that Tor1 binds to 5S rDNA chromatin but the functional significance has not been established. Here, we show that association with 5S rDNA chromatin is necessary for TOR complex 1 (TORC1) to regulate the synthesis of 5S ribosomal RNA and transfer RNAs (tRNAs) by RNA polymerase (Pol) III, as well as the phosphorylation and binding to Pol III‐transcribed genes of the Pol III repressor Maf1. Interestingly, TORC1 does not bind to tRNA genes, suggesting that TORC1 modulates tRNA synthesis indirectly through Maf1 phosphorylation at the rDNA loci. We also find that Maf1 cytoplasmic localization is dependent on the SSD1‐v allele. In W303 cells that carry the SSD1‐d allele, Maf1 is constitutively nuclear but its nucleolar localization is inhibited by TORC1, indicating that TORC1 regulates nucleoplasm‐to‐nucleolus transport of Maf1. Finally, we show that TORC1 interacts with Maf1 in vivo and phosphorylates Maf1 in vitro, and regulates Maf1 nucleoplasm‐to‐nucleolus translocation. Together, these observations provide new insights into the chromatin‐dependent mechanism by which TORC1 controls transcription by Pol III.

The 14-3-3 proteins positively regulate rapamycin-sensitive signaling

BACKGROUND The kinase Tor is the target of the immunosuppressive drug rapamycin and is a member of the phosphatidylinositol kinase (PIK)-related kinase family. It plays an essential role in progression through the G1 phase of the cell cycle. The molecular details of Tor signaling remain obscure, however. RESULTS We isolated two Saccharomyces cerevisiae genes, BMH1 and BMH2, as multicopy suppressors of the growth-inhibitory phenotype caused by rapamycin in budding yeast. BMH1 and BMH2 encode homologs of the 14-3-3 signal transduction proteins. Deletion of one or both BMH genes caused hypersensitivity to rapamycin in a manner that was dependent on gene dosage. In addition, alterations in the phosphopeptide-binding pocket of the 14-3-3 proteins had dramatically different effects on their ability to relieve the growth-arresting rapamycin phenotype. Mutations that prevented 14-3-3 from binding to a phosphoserine motif abolished its ability to confer rapamycin resistance. In contrast, substitution of two residues in 14-3-3 that surround these phosphoserine-binding sites conferred a dominant rapamycin-resistant phenotype. CONCLUSIONS Our studies reveal 14-3-3 as an important component in rapamycin-sensitive signaling and provide significant new insights into the structure and function of 14-3-3 proteins.

The FKBP12‐rapamycin‐associated protein (FRAP) is a CLIP‐170 kinase

CLIP‐170/Restin belongs to a family of conserved microtubule (MT)‐associated proteins, which are important for MT organization and functions. CLIP‐170 is a phosphoprotein and phosphorylation is thought to regulate the binding of CLIP‐170 to MTs. However, little is known about the kinase(s) involved. In this study, we show that FKBP12‐rapamycin‐associated protein (FRAP, also called mTOR/RAFT) interacts with CLIP‐170. CLIP‐170 is phosphorylated in vivo at multiple sites, including rapamycin‐sensitive and ‐insensitive sites, and is phosphorylated by FRAP in vitro at the rapamycin‐sensitive sites. In addition, rapamycin inhibited the ability of CLIP‐170 to bind to MTs. Our observations suggest that multiple CLIP‐170 kinases are involved in positive and negative control of CLIP‐170, and FRAP is a CLIP‐170 kinase positively regulating the MT‐binding behavior of CLIP‐170.