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, rapamycininsensitive 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.Cell growth (increased cell mass and size) is a prerequisite for proliferation (increased cell number), since a cell will divide only after it has reached a critical mass (38,49,55,58). Thus, factors that govern cell cycle progression must also regulate growth in an interrelated fashion. Cell growth is not however, unconditionally dependent on cell cycle progression, as mutations in the budding yeast Saccharomyces cerevisiae and the fruit fly Drosophila melanogaster that block or disrupt cell division do not necessarily arrest cell growth (34,44). Recent studies have demonstrated that cell growth and cell cycle progression in proliferating mammalian cells, like lower organisms, are also separable processes (8, 50, 63). Thus, detailed knowledge of the biochemical and molecular mechanisms governing cell size will be essential to understanding how the cell division cycle is coupled to growth and how this process is uncoupled during differentiation or is perturbed during diseases associated with deregulated growth. Our knowledge of cell cycle regulatory mechanisms has advanced considerably over the past decade. In contrast, information on the mechanisms of regulating cell growth in mammalian cells is limited.Increased protein synthesis is one of the major anabolic events required for the growth response (28). Recent studies suggest that one of the key nodal points upon which signaling pathwa...

The protein encoded by the retinoblastoma susceptibility gene (Rb) functions as a tumour suppressor and negative growth regulator. As actively growing cells require the ongoing synthesis of ribosomal RNA, we considered that Rb might interact with the ribosomal DNA transcription apparatus. Here we report that (1) there is an accumulation of Rb protein in the nucleoli of differentiated U937 cells which correlates with inhibition of rDNA transcription; (2) addition of Rb to an in vitro transcription system inhibits transcription by RNA polymerase I; (3) this inhibition requires a functional Rb pocket; and (4) Rb specifically inhibits the activity of the RNA polymerase I transcription factor UBF (upstream binding factor) in vitro. This last observation was confirmed by affinity chromatography and immunoprecipitation, which demonstrated an interaction between Rb and UBF. These results indicate that there is an additional mechanism by which Rb suppresses cell growth, namely that Rb directly represses transcription of the rRNA genes.

Cycloheximide inhibits ribosomal DNA (rDNA) transcription in vivo. The mouse homologue of yeast Rrn3, a polymerase-associated transcription initiation factor, can complement extracts from cycloheximide-treated mammalian cells. Cycloheximide inhibits the phosphorylation of Rrn3 and causes its dissociation from RNA polymerase I. Rrn3 interacts with the rpa43 subunit of RNA polymerase I, and treatment with cycloheximide inhibits the formation of a Rrn3⅐rpa43 complex in vivo. Rrn3 produced in Sf9 cells but not in bacteria interacts with rpa43 in vitro, and such interaction is dependent upon the phosphorylation state of Rrn3. Significantly, neither dephosphorylated Rrn3 nor Rrn3 produced in Escherichia coli can restore transcription by extracts from cycloheximide-treated cells. These results suggest that the phosphorylation state of Rrn3 regulates rDNA transcription by determining the steady-state concentration of the Rrn3⅐RNA polymerase I complex within the nucleolus.In the early 1970s Feigelson and colleagues (1-3) reported that cycloheximide caused a rapid cessation of nucleolar RNA synthesis (ribosomal DNA transcription) and concluded that a rapidly turning over protein was required for RNA polymerase I (pol I) 1 activity in vivo. Subsequent studies have demonstrated that transcription by RNA polymerase I is subject to regulation at many levels (4, 5). At least three, and possibly more, polymerase-associated proteins, TIF-IA, Factor C*, and TFIC (6 -8), have been demonstrated to contribute to the regulation of rDNA transcription. TIF-IA and Factor C* were identified as factors that were required for the complementation of extracts of quiescent or cycloheximide-treated cells. TFIC was identified as that activity required to reconstitute transcription by extracts of glucocorticoid-treated P1798 cells.This lymphosarcoma cell line exits the cell cycle in response to the synthetic glucocorticoid dexamethasone (DEX) (6). Interestingly, TIF-IA, Factor C*, and TFIC shared several properties, including a tight association with the core polymerase (8 -10). TIF-IA and TFIC were purified and consisted of different polypeptides (10, 11). However, the lack of immunological and molecular tools precluded a definitive statement that TIF-IA and TFIC were the same or different proteins (reviewed in Refs. 4 and 5).The formation of the stable preinitiation complex in yeast requires an interaction between the upstream activating factor bound to the upstream promoter element and core factor, bound to the core promoter element. This complex then recruits transcriptionally competent RNA polymerase I to the transcription initiation site (Ref. 12 and references therein). Mechanistically, Rrn3 appears to "bridge" the polymerase and transcription initiation complexes (13-15). Thus, only pol I molecules in complex with Rrn3 are able to recognize the preinitiation complex and initiate transcription.Studies comparing the state of RNA polymerase I in growing and stationary yeast cells demonstrated that ϳ2% of the pol I in whole cell extracts was...

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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.