Alice H. Cavanaugh

mTOR-Dependent Regulation of Ribosomal Gene Transcription Requires S6K1 and Is Mediated by Phosphorylation of the Carboxy-Terminal Activation Domain of the Nucleolar Transcription Factor UBF†

ABSTRACT Mammalian target of rapamycin (mTOR) is a key regulator of cell growth acting via two independent targets, ribosomal protein S6 kinase 1 (S6K1) and 4EBP1. While each is known to regulate translational efficiency, the mechanism by which they control cell growth remains unclear. In addition to increased initiation of translation, the accelerated synthesis and accumulation of ribosomes are fundamental for efficient cell growth and proliferation. Using the mTOR inhibitor rapamycin, we show that mTOR is required for the rapid and sustained serum-induced activation of 45S ribosomal gene transcription (rDNA transcription), a major rate-limiting step in ribosome biogenesis and cellular growth. Expression of a constitutively active, rapamycin-insensitive mutant of S6K1 stimulated rDNA transcription in the absence of serum and rescued rapamycin repression of rDNA transcription. Moreover, overexpression of a dominant-negative S6K1 mutant repressed transcription in exponentially growing NIH 3T3 cells. Rapamycin treatment led to a rapid dephosphorylation of the carboxy-terminal activation domain of the rDNA transcription factor, UBF, which significantly reduced its ability to associate with the basal rDNA transcription factor SL-1. Rapamycin-mediated repression of rDNA transcription was rescued by purified recombinant phosphorylated UBF and endogenous UBF from exponentially growing NIH 3T3 cells but not by hypophosphorylated UBF from cells treated with rapamycin or dephosphorylated recombinant UBF. Thus, mTOR plays a critical role in the regulation of ribosome biogenesis via a mechanism that requires S6K1 activation and phosphorylation of UBF.

RNA polymerase I transcription in confluent cells: Rb downregulates rDNA transcription during confluence-induced cell cycle arrest

When 3T6 cells are confluent, they withdraw from the cell cycle. Concomitant with cell cycle arrest a significant reduction in RNA polymerase I transcription (80% decrease at 100% confluence) is observed. In the present study, we examined mechanism(s) through which transcription of the ribosomal genes is coupled to cell cycle arrest induced by cell density. Interestingly with an increase in cell density (from 3–43% confluence), a significant accumulation in the cellular content of hyperphosphorylated Rb was observed. As cell density increased further, the hypophosphorylated form of Rb became predominant and accumulated in the nucleoli. Co-immunoprecipitation experiments demonstrated there was also a significant rise in the amount of hypophosphorylated Rb associated with the rDNA transcription factor UBF. This increased interaction between Rb and UBF correlated with the reduced rate of rDNA transcription. Furthermore, overexpression of recombinant Rb inhibited UBF-dependent activation of transcription from a cotransfected rDNA reporter in either confluent or exponential cells. The amounts or activities of the rDNA transcription components we examined did not significantly change with cell cycle arrest. Although the content of PAF53, a polymerase associated factor, was altered marginally (decreased 38%), the time course and magnitude of the decrease did not correlate with the reduced rate of rDNA transcription. The results presented support a model wherein regulation of the binding of UBF to Rb and, perhaps the cellular content of PAF53, are components of the mechanism through which cell cycle and rDNA transcription are linked.

Agonist-Driven Maturation and Plasma Membrane Insertion of Calcium-Sensing Receptors Dynamically Control Signal Amplitude

Activation of a G protein–coupled receptor increases its own surface abundance to enhance its signaling. Linking Activity to Abundance Many G protein–coupled receptors decrease their signaling output when activated by ligands. However, the calcium-sensing receptor (CaSR), which maintains the serum concentration of calcium within a narrow range, is constantly exposed to calcium, leading Grant et al. to investigate how CaSR-mediated signaling increases in response to increases in the concentration of extracellular calcium. Activation of CaSRs by calcium or other agonists resulted in mobilization of CaSRs to the plasma membrane from a large intracellular pool, thereby enabling increased and sustained CaSR signaling. Mutations in CASR can result in hypocalcemia or hypercalcemia; thus, understanding its regulation could help in the development of drugs to treat these conditions. In addition, this mechanism might regulate the signaling output of other receptors that are also constantly exposed to ligands. Calcium-sensing receptors (CaSRs) regulate systemic calcium homeostasis in the parathyroid gland, kidney, intestine, and bone and translate fluctuations in serum calcium into peptide hormone secretion, cell signaling, and regulation of gene expression. The CaSR is a G protein (heterotrimeric guanosine triphosphate–binding protein)–coupled receptor that operates in the constant presence of agonist, sensing small changes with high cooperativity and minimal functional desensitization. Here, we used multiwavelength total internal reflection fluorescence microscopy to demonstrate that the signaling properties of the CaSR result from agonist-driven maturation and insertion of CaSRs into the plasma membrane. Plasma membrane CaSRs underwent constitutive endocytosis without substantial recycling, indicating that signaling was determined by the rate of insertion of CaSRs into the plasma membrane. Intracellular CaSRs colocalized with calnexin in the perinuclear endoplasmic reticulum and formed complexes with 14-3-3 proteins. Ongoing CaSR signaling resulted from agonist-driven trafficking of CaSR through the secretory pathway. The intracellular reservoir of CaSRs that were mobilized by agonist was depleted when glycosylation of newly synthesized receptors was blocked, suggesting that receptor biosynthesis was coupled to signaling. The continuous, signaling-dependent insertion of CaSRs into the plasma membrane ensured a rapid response to alterations in the concentrations of extracellular calcium or allosteric agonist despite ongoing desensitization and endocytosis. Regulation of CaSR plasma membrane abundance represents a previously unknown mechanism of regulation that may be relevant to other receptors that operate in the chronic presence of agonist.

Affinity purification of mammalian RNA polymerase I. Identification of an associated kinase.

Overlapping cDNA clones encoding the two largest subunits of rat RNA polymerase I, designated A194 and A127, were isolated from a Reuber hepatoma cDNA library. Analyses of the deduced amino acid sequences revealed that A194 and A127 are the homologues of yeast A190 and A135 and have homology to the β′ and β subunits of Escherichia coli RNA polymerase I. Antibodies raised against the recombinant A194 and A127 proteins recognized single proteins of approximately 190 and 120 kDa on Western blots of total cellular proteins of mammalian origin. N1S1 cell lines expressing recombinant His-tagged A194 and FLAG-tagged A127 proteins were isolated. These proteins were incorporated into functional RNA polymerase I complexes, and active enzyme, containing FLAG-tagged A127, could be immunopurified to approximately 80% homogeneity in a single chromatographic step over an anti-FLAG affinity column. Immunoprecipitation of A194 from 32P metabolically labeled cells with anti-A194 antiserum demonstrated that this subunit is a phosphoprotein. Incubation of the FLAG affinity-purified RNA polymerase I complex with [γ-32P]ATP resulted in autophosphorylation of the A194 subunit of RPI, indicating the presence of associated kinase(s). One of these kinases was demonstrated to be CK2, a serine/threonine protein kinase implicated in the regulation of cell growth and proliferation.

The species-specific RNA polymerase I transcription factor SL-1 binds to upstream binding factor.

Transcription of the 45S rRNA genes is carried out by RNA polymerase I and at least two trans-acting factors, upstream binding factor (UBF) and SL-1. We have examined the hypothesis that SL-1 and UBF interact. Coimmunoprecipitation studies using an antibody to UBF demonstrated that TATA-binding protein, a subunit of SL-1, associates with UBF in the absence of DNA. Inclusion of the detergents sodium dodecyl sulfate and deoxycholate disrupted this interaction. In addition, partially purified UBF from rat cell nuclear extracts and partially purified SL-1 from human cells coimmunoprecipitated with the anti-UBF antibody after mixing, indicating that the UBF-SL-1 complex can re-form. Treatment of UBF-depleted extracts with the anti-UBF antibody depleted the extracts of SL-1 activity only if UBF was added to the extract prior to the immunodepletion reaction. Furthermore, SL-1 activity could be recovered in the immunoprecipitate. Interestingly, these immunoprecipitates did not contain RNA polymerase I, as a monospecific antibody to the 194-kDa subunit of RNA polymerase I failed to detect that subunit in the immunoprecipitates. Treatment of N1S1 cell extracts with the anti-UBF antibody depleted the extracts of SL-1 activity but not TFIIIB activity, suggesting that the binding of UBF to SL-1 is specific and not solely mediated by an interaction between UBF and TATA-binding protein, which is also a component of TFIIIB. These data provide evidence that UBF and SL-1 interact.

Calcium-sensing receptor biosynthesis includes a cotranslational conformational checkpoint and endoplasmic reticulum retention.

Metabolic labeling with [35S]cysteine was used to characterize early events in CaSR biosynthesis. [35S]CaSR is relatively stable (half-life ∼8 h), but maturation to the final glycosylated form is slow and incomplete. Incorporation of [35S]cysteine is linear over 60 min, and the rate of [35S]CaSR biosynthesis is significantly increased by the membrane-permeant allosteric agonist NPS R-568, which acts as a cotranslational pharmacochaperone. The [35S]CaSR biosynthetic rate also varies as a function of conformational bias induced by loss- or gain-of-function mutations. In contrast, [35S]CaSR maturation to the plasma membrane was not significantly altered by exposure to the pharmacochaperone NPS R-568, the allosteric agonist neomycin, or the orthosteric agonist Ca2+ (0.5 or 5 mm), suggesting that CaSR does not control its own release from the endoplasmic reticulum. A CaSR chimera containing the mGluR1α carboxyl terminus matures completely (half-time of ∼8 h) and without a lag period, as does the truncation mutant CaSRΔ868 (half-time of ∼16 h). CaSRΔ898 exhibits maturation comparable with full-length CaSR, suggesting that the CaSR carboxyl terminus between residues Thr868 and Arg898 limits maturation. Overall, these results suggest that CaSR is subject to cotranslational quality control, which includes a pharmacochaperone-sensitive conformational checkpoint. The CaSR carboxyl terminus is the chief determinant of intracellular retention of a significant fraction of total CaSR. Intracellular CaSR may reflect a rapidly mobilizable “storage form” of CaSR and/or may subserve distinct intracellular signaling roles that are sensitive to signaling-dependent changes in endoplasmic reticulum Ca2+ and/or glutathione.

Androgen regulation of ribosomal RNA synthesis in LNCaP cells and rat prostate

Androgen-dependent growth of prostate tissue has been well documented. An additional prerequisite for cellular growth is the accumulation of ribosomes. It is thus reasonable to hypothesize that ribosomal DNA (rDNA) transcription in prostate tissue must be stimulated by androgen either directly or indirectly. This hypothesis was tested using both LNCaP cells, an androgen-dependent tissue culture line and in a rat animal model. Nuclear run-on assays confirmed that the administration of DHT to LNCaP cells resulted in a two- to three-fold increase in the rate of rRNA synthesis when compared to cells maintained in the absence of androgen. Enzymatic analysis and Western blots were carried out to measure the amount (activity and mass) of RNA polymerase I in DHT treated LNCaP cells. These assays demonstrated that neither the catalytic activity of RNA polymerase I nor the amount of the enzyme varied in response to DHT. However, Western blots revealed that the amount of the auxiliary RNA polymerase I transcription factor UBF, was significantly increased (two- to three-fold) in cells grown in the presence of DHT. Similar experiments were carried out with prostatic tissue obtained from orchiectomized rats maintained on either placebo or testosterone pellets. In this model, both the catalytic activity as well as the amount of RNA polymerase I protein decreased. However, in agreement with the tissue culture model, UBF protein decreased in prostates from orchiectomized rats and was maintained in animals supplemented with testosterone. These lines of evidence are consistent with the hypothesis that androgens stimulate rRNA synthesis by increasing the quantities of the components of the rDNA transcription system.

DNA Binding by the Ribosomal DNA Transcription Factor Rrn3 Is Essential for Ribosomal DNA Transcription

Background: Transcription initiation by RNA polymerase I requires protein-protein interactions between Rrn3, polymerase, and core factors. Results: Mutagenesis of a putative DNA binding domain in Rrn3 had no effect on essential protein-protein interactions, but abrogated DNA binding and inactivated Rrn3 function in transcription. Conclusion: DNA binding is essential for Rrn3 to function in transcription. Significance: DNA binding by Rrn3 may provide an additional target to regulate rDNA transcription. The human homologue of yeast Rrn3 is an RNA polymerase I-associated transcription factor that is essential for ribosomal DNA (rDNA) transcription. The generally accepted model is that Rrn3 functions as a bridge between RNA polymerase I and the transcription factors bound to the committed template. In this model Rrn3 would mediate an interaction between the mammalian Rrn3-polymerase I complex and SL1, the rDNA transcription factor that binds to the core promoter element of the rDNA. In the course of studying the role of Rrn3 in recruitment, we found that Rrn3 was in fact a DNA-binding protein. Analysis of the sequence of Rrn3 identified a domain with sequence similarity to the DNA binding domain of heat shock transcription factor 2. Randomization, or deletion, of the amino acids in this region in Rrn3, amino acids 382–400, abrogated its ability to bind DNA, indicating that this domain was an important contributor to DNA binding by Rrn3. Control experiments demonstrated that these mutant Rrn3 constructs were capable of interacting with both rpa43 and SL1, two other activities demonstrated to be essential for Rrn3 function. However, neither of these Rrn3 mutants was capable of functioning in transcription in vitro. Moreover, although wild-type human Rrn3 complemented a yeast rrn3-ts mutant, the DNA-binding site mutant did not. These results demonstrate that DNA binding by Rrn3 is essential for transcription by RNA polymerase I.

Behind the curtain: cellular mechanisms for allosteric modulation of calcium-sensing receptors

Calcium‐sensing receptors (CaSR) are integral to regulation of systemic Ca2+ homeostasis. Altered expression levels or mutations in CaSR cause Ca2+ handling diseases. CaSR is regulated by both endogenous allosteric modulators and allosteric drugs, including the first Food and Drug Administration‐approved allosteric agonist, Cinacalcet HCl (Sensipar®). Recent studies suggest that allosteric modulators not only alter function of plasma membrane‐localized CaSR, but regulate CaSR stability at the endoplasmic reticulum. This brief review summarizes our current understanding of the role of membrane‐permeant allosteric agonists in cotranslational stabilization of CaSR, and highlights additional, indirect, signalling‐dependent role(s) for membrane‐impermeant allosteric drugs. Overall, these studies suggest that allosteric drugs act at multiple cellular organelles to control receptor abundance and hence function, and that drug hydrophobicity can bias the relative contributions of plasma membrane and intracellular organelles to CaSR abundance and signalling.

Factor-assisted DNA binding as a possible general mechanism for steroid receptors. Functional heterogeneity among activated receptor-steroid complexes

We previously reported that activated glucocorticoid receptor-steroid complexes from rat HTC cell cytosol exist as at least two sub-populations, one of which requires a low molecular weight (700-3000 Da) factor(s) for binding to DNA. This factor is removed by Sephadex G-50 chromatography and is found predominantly in extracts of crude HTC cell nuclei. We have now determined that factor is not limited to HTC cells since an apparently identical factor(s) was found in nuclear extracts of rat kidney and liver as well as human HeLa and MCF-7 cells. Furthermore, the DNA binding of a sub-population of human glucocorticoid receptors depends on factor. While these results were obtained with agonist (dexamethasone) bound receptors, a sub-population of HTC cell receptors covalently labeled by the antiglucocorticoid dexamethasone 21-mesylate also displayed factor-dependent DNA binding. This receptor heterogeneity was not an artifact of cell-free activation since the cell-free nuclear binding of dexamethasone mesylate labeled complexes was, as in intact cells, less than that for dexamethasone bound complexes. Earlier results suggested that the increased DNA binding with factor involved a direct interaction of receptor with factor(s). We now find that the factor-induced DNA binding is retained by amino terminal truncated (42 kDa) glucocorticoid receptors from HTC cells. Thus the ability of receptor to interact with factor(s) is encoded by the DNA and/or steroid binding domains. Two dimensional gel electrophoresis analysis of dexamethasone-mesylate labeled 98 kDa receptors revealed multiple charged isoforms for both sub-populations but no differences in the amount of the various isoforms in each sub-population. Finally, activated progesterone and estrogen receptor complexes were also found to be heterogeneous, with a similar, if not identical, small molecular weight factor(s) being required for the DNA binding of one sub-population. The observations that functional heterogeneity of receptors is not unique to glucocorticoid receptors, whether bound by an agonist or antagonist, and that the factor(s) is neither species nor tissue specific suggests that factor-assisted DNA binding may be a general mechanism for all steroid receptors.