https://archive-ouverte.unige.ch/unige:18229/ATTACHMENT01

TOR signaling in growth and metabolism.

NF-κB (nuclear factor-κB) is a collective name for inducible dimeric transcription factors composed of members of the Rel family of DNA-binding proteins that recognize a common sequence motif. NF-κ...

Phosphorylation meets ubiquitination: the control of NF-[kappa]B activity.

The evolutionarily conserved checkpoint protein kinase, TOR (target of rapamycin), has emerged as a major effector of cell growth and proliferation via the regulation of protein synthesis. Work in the last decade clearly demonstrates that TOR controls protein synthesis through a stunning number of downstream targets. Some of the targets are phosphorylated directly by TOR, but many are phosphorylated indirectly. In this review, we summarize some recent developments in this fast-evolving field. We describe both the upstream components of the signaling pathway(s) that activates mammalian TOR (mTOR) and the downstream targets that affect protein synthesis. We also summarize the roles of mTOR in the control of cell growth and proliferation, as well as its relevance to cancer and synaptic plasticity.

/pdf/upstream-and-downstream-of-mtor-2v5cvgfhmb.pdf

Upstream and downstream of mTOR

In all eukaryotes, the target of rapamycin (TOR) signalling pathway couples energy and nutrient abundance to the execution of cell growth and division, owing to the ability of TOR protein kinase to simultaneously sense energy, nutrients and stress and, in metazoans, growth factors. Mammalian TOR complex 1 (mTORC1) and mTORC2 exert their actions by regulating other important kinases, such as S6 kinase (S6K) and Akt. In the past few years, a significant advance in our understanding of the regulation and functions of mTOR has revealed the crucial involvement of this signalling pathway in the onset and progression of diabetes, cancer and ageing.

/pdf/mtor-from-growth-signal-integration-to-cancer-diabetes-and-503skey4mb.pdf

mTOR: from growth signal integration to cancer, diabetes and ageing

▪ Abstract The conjugation of ubiquitin to other cellular proteins regulates a broad range of eukaryotic cell functions. The high efficiency and exquisite selectivity of ubiquitination reactions reflect the properties of enzymes known as ubiquitin-protein ligases or E3s. An E3 recognizes its substrates based on the presence of a specific ubiquitination signal, and catalyzes the formation of an isopeptide bond between a substrate (or ubiquitin) lysine residue and the C terminus of ubiquitin. Although a great deal is known about the molecular basis of E3 specificity, much less is known about molecular mechanisms of catalysis by E3s. Recent findings reveal that all known E3s utilize one of just two catalytic domains—a HECT domain or a RING finger—and crystal structures have provided the first detailed views of an active site of each type. The new findings shed light on many aspects of E3 structure, function, and mechanism, but also emphasize that key features of E3 catalysis remain to be elucidated.

Mechanisms underlying ubiquitination.

SCFCdc4 (Skp1, Cdc53/cullin, F-box protein) defines a family of modular ubiquitin ligases (E3s) that regulate diverse processes including cell cycle, immune response, and development. Mass spectrometric analysis of proteins copurifying with Cdc53 identified the RING-H2 finger protein Hrt1 as a subunit of SCF. Hrt1 shows striking similarity to the Apc11 subunit of anaphase-promoting complex. Conditional inactivation of hrt1(ts) results in stabilization of the SCFCdc4 substrates Sic1 and Cln2 and cell cycle arrest at G1/S. Hrt1 assembles into recombinant SCF complexes and individually binds Cdc4, Cdc53 and Cdc34, but not Skp1. Hrt1 stimulates the E3 activity of recombinant SCF potently and enables the reconstitution of Cln2 ubiquitination by recombinant SCFGrr1. Surprisingly, SCF and the Cdc53/Hrt1 subcomplex activate autoubiquitination of Cdc34 E2 enzyme by a mechanism that does not appear to require a reactive thiol. The highly conserved human HRT1 complements the lethality of hrt1Delta, and human HRT2 binds CUL-1. We conclude that Cdc53/Hrt1 comprise a highly conserved module that serves as the functional core of a broad variety of heteromeric ubiquitin ligases.

/pdf/cdc53-cullin-and-the-essential-hrt1-ring-h2-subunit-of-scf-2olgvj3d3b.pdf

Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34.

Yeast cells entering into stationary phase decrease rRNA synthesis rate by decreasing both the number of active genes and the transcription rate of individual active genes. Using chromatin immunoprecipitation assays, we found that the association of RNA polymerase I with the promoter and the coding region of rDNA is decreased in stationary phase, but association of transcription factor UAF with the promoter is unchanged. Similar changes were also observed when growing cells were treated with rapamycin, which is known to inhibit the Tor signaling system. Rapamycin treatment also caused a decrease in the amount of Rrn3p-polymerase I complex, similar to stationary phase. Because recruitment of Pol I to the rDNA promoter is Rrn3p-dependent as shown in this work, these data suggest that the decrease in the transcription rate of individual active genes in stationary phase is achieved by the Tor signaling system acting at the Rrn3p-dependent polymerase recruitment step. Miller chromatin spreads of cells treated with rapamycin and cells in post-log phase confirm this conclusion and demonstrate that the Tor system does not participate in alteration of the number of active genes observed for cells entering into stationary phase.

/pdf/tor-pathway-regulates-rrn3p-dependent-recruitment-of-yeast-4ft7dpk8rj.pdf

Tor pathway regulates Rrn3p-dependent recruitment of yeast RNA polymerase I to the promoter but does not participate in alteration of the number of active genes.

We report the results of an analysis of Ty3 transcription and identification of Ty3 regions that mediate pheromone and mating-type regulation to coordinate its expression with the yeast life cycle. A set of strains was constructed which was isogenic except for the number of Ty3 elements, which varied from zero to three. Analysis of Ty3 expression in these strains showed that each of the three elements was transcribed and that each element was regulated. Dissection of the long terminal repeat regulatory region by Northern blot analysis of deletion mutants and reporter gene analysis showed that the upstream junction of Ty3 with flanking chromosomal sequences contained a negative control region. A 19-bp fragment (positions 56-74) containing one consensus copy and one 7 of 8-bp match to the pheromone response element (PRE) consensus was sufficient to mediate pheromone induction in either haploid cell type. Deletion of this region, however, did not abolish expression, indicating that other sequences also activate transcription. A 24-bp block immediately downstream of the PRE region contained a sequence similar to the a1-alpha 2 consensus that conferred mating-type control. A single base pair mutation in the region separating the PRE and a1-alpha 2 sequences blocked pheromone induction, but not mating-type control. Thus, the long terminal repeat of Ty3 is a compact, highly regulated, mobile promoter which is responsive to cell type and mating.

https://www.genetics.org/content/genetics/134/3/685.full.pdf

Positive and negative regulatory elements control expression of the yeast retrotransposon Ty3.

Position-specific integration of the retroviruslike element Ty3 near the transcription initiation sites of tRNA genes requires transcription factors IIIB and IIIC (TFIIIB and TFIIIC). Using a genetic screen, we isolated a mutant with a truncated 95-kDa subunit of TFIIIC (TFIIIC95) that reduced the apparent retrotransposition of Ty3 into a plasmid-borne target site between two divergently transcribed tRNA genes. Although TFIIIC95 is conserved and essential, no defect in growth or transcription of tRNAs was detected in the mutant. Steps of the Ty3 life cycle, such as protein expression, proteolytic processing, viruslike particle formation, and reverse transcription, were not affected by the mutation. However, Ty3 integration into a divergent tDNA target occurred exclusively in one orientation in the mutant strain. Investigation of this orientation bias showed that TFIIIC95 and Ty3 integrase interacted in two-hybrid and glutathione S-transferase pulldown assays and that interaction with the mutant TFIIIC95 protein was attenuated. The orientation bias observed here suggests that even for wild-type Ty3, the protein complexes associated with the long terminal repeats are not equivalent in vivo.

/pdf/a-truncation-mutant-of-the-95-kilodalton-subunit-of-2rs7qa5qlc.pdf

A Truncation Mutant of the 95-Kilodalton Subunit of Transcription Factor IIIC Reveals Asymmetry in Ty3 Integration

Ty3 is a gypsy-type, retrovirus-like element found in the budding yeast Saccharomyces cerevisiae. In cells overexpressing Ty3 under the GAL1 upstream activation sequence, Ty3 RNA, proteins, and DNA are made. Elucidation of the molecular masses and amino-terminal sequences of protease and reverse transcriptase indicated the existence of an additional intervening domain, designated J, in the Ty3 Gag3-Pol3p polyprotein. A region analogous to J can be found in many retrotransposable elements closely related to Ty3; however, J does not correspond to any of the highly conserved retroviral protein domains. Ty3 mutants deleted for the J-coding region showed moderately reduced transposition frequency but greatly reduced levels of Ty3 DNA. These results show that under galactose regulation, the Ty3 J domain is not absolutely essential.

/pdf/ten-kilodalton-domain-in-ty3-gag3-pol3p-between-pr-and-rt-is-1zmnqjf677.pdf

Jonathan A. Claypool

Papers

Cdc53/cullin and the essential Hrt1 RING-H2 subunit of SCF define a ubiquitin ligase module that activates the E2 enzyme Cdc34.

Tor pathway regulates Rrn3p-dependent recruitment of yeast RNA polymerase I to the promoter but does not participate in alteration of the number of active genes.

Positive and negative regulatory elements control expression of the yeast retrotransposon Ty3.

A Truncation Mutant of the 95-Kilodalton Subunit of Transcription Factor IIIC Reveals Asymmetry in Ty3 Integration

Ten-Kilodalton Domain in Ty3 Gag3-Pol3p between PR and RT Is Dispensable for Ty3 Transposition