In cycling cells, transcription of ribosomal RNA genes by RNA polymerase I (Pol I) is tightly coordinated with cell growth. Here, we show that the mammalian target of rapamycin (mTOR) regulates Pol I transcription by modulating the activity of TIF-IA, a regulatory factor that senses nutrient and growth-factor availability. Inhibition of mTOR signaling by rapamycin inactivates TIF-IA and impairs transcription-initiation complex formation. Moreover, rapamycin treatment leads to translocation of TIF-IA into the cytoplasm. Rapamycin-mediated inactivation of TIF-IA is caused by hypophosphorylation of Ser 44 (S44) and hyperphosphorylation of Ser 199 (S199). Phosphorylation at these sites affects TIF-IA activity in opposite ways, for example, phosphorylation of S44 activates and S199 inactivates TIF-IA. The results identify a new target for mTOR-signaling pathways and elucidate the molecular mechanism underlying mTOR-dependent regulation of rRNA synthesis.

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The presence of actin and nuclear myosin I (NMI) in the nucleus suggests a role for these motor proteins in nuclear functions. We have investigated the role of actin and nuclear myosin I (NMI) in the transcription of ribosomal RNA genes (rDNA). Both proteins are associated with rDNA and are required for RNA polymerase I (Pol I) transcription. Microinjection of antibodies against actin or NMI, as well as short interfering RNA-mediated depletion of NMI, decreased Pol I transcription in vivo, whereas overexpression of NMI augmented pre-rRNA synthesis. In vitro, recombinant NMI activated Pol I transcription, and antibodies to NMI or actin inhibited Pol I transcription both on naked DNA and pre-assembled chromatin templates. Whereas actin associated with Pol I, NMI bound to Pol I through the transcription-initiation factor TIF-IA. The association with Pol I requires phosphorylation of TIF-IA at Ser 649 by RSK kinase, indicating a role for NMI in the growth-dependent regulation of rRNA synthesis.

The chemokine receptor CXCR4 has recently been shown to be a co-receptor involved in the entry of human immunodeficiency virus type 1 into target cells. This study shows that coexpression of ␤-arrestin with CXCR4 in human embryonic kidney 293 cells attenuated chemokine-stimulated G protein activation and inhibition of cAMP production. Truncation of the C-terminal 34 amino acids of CXCR4 (CXCR4-T) abolished the effects of ␤-arrestin on CXCR4/G protein signaling, indicating the functional interaction of the receptor C terminus with ␤-arrestin. On the other hand, receptor internalization and the subsequent activation of extracellular signal-regulated kinases were significantly promoted by coexpression of ␤-arrestin with CXCR4, whereas the C-terminal truncation of CXCR4 did not affect this regulation of ␤-arrestin, suggesting that ␤-arrestin can functionally interact with CXCR4 with or without the C terminus. Moreover, ␤ 2 V54D, the dominant inhibitory mutant of ␤-arrestin 2, exerted no effects on CXCR4/G protein signaling, but strongly influenced receptor internalization and extracellular signalregulated kinase activation. Further cross-linking experiments demonstrated that ␤-arrestin as well as ␤ 2 V54D could physically contact both CXCR4 and CXCR4-T. Glutathione S-transferase pull-down assay showed that ␤-arrestin was able to bind efficiently in vitro to both the third intracellular loop and the 34-amino acid C terminus of CXCR4. Taken together, our data clearly establish that ␤-arrestin can effectively regulate different functions of CXCR4 and that this is mediated through its distinct interactions with the C terminus and other regions including the third loop of CXCR4.