The Target of Rapamycin (TOR) protein is a Ser/Thr kinase that functions in two distinct multiprotein complexes: TORC1 and TORC2. These conserved complexes regulate many different aspects of cell growth in response to intracellular and extracellular cues. Here we report that the AGC kinase Sch9 is a substrate of yeast TORC1. Six amino acids in the C terminus of Sch9 are directly phosphorylated by TORC1. Phosphorylation of these residues is lost upon rapamycin treatment as well as carbon or nitrogen starvation and transiently reduced following application of osmotic, oxidative, or thermal stress. TORC1-dependent phosphorylation is required for Sch9 activity, and replacement of residues phosphorylated by TORC1 with Asp/Glu renders Sch9 activity TORC1 independent. Sch9 is required for TORC1 to properly regulate ribosome biogenesis, translation initiation, and entry into G0 phase, but not expression of Gln3-dependent genes. Our results suggest that Sch9 functions analogously to the mammalian TORC1 substrate S6K1 rather than the mTORC2 substrate PKB/Akt.

The irreversible G 1 arrest in senescent human diploid fibroblasts is probably caused by inactivation of the G 1 cyclin-cyclin-dependent kinase (Cdk) complexes responsible for phosphorylation of the retinoblastoma protein (pRb). We show that the Cdk inhibitor p21 Sdi1,Cip1,Waf1, which accumulates progressively in aging cells, binds to and inactivates all cyclin E-Cdk2 complexes in senescent cells, whereas in young cells only p21-free Cdk2 complexes are active. Furthermore, the senescent-cell-cycle arrest occurs prior to the accumulation of the Cdk4-Cdk6 inhibitor p16Ink4a , suggesting that p21 may be sufficient for this event. Accordingly, cyclin D1-associated phosphorylation of pRb at Ser-780 is lacking even in newly senescent fibroblasts that have a low amount of p16. Instead, the cyclin D1-Cdk4 and cyclin D1-Cdk6 complexes in these cells are associated with an increased amount of p21, suggesting that p21 may be responsible for inactivation of both cyclin E-and cyclin D1-associated kinase activity at the early stage of senescence. Moreover, even in the late stage of senescence when p16 is high, cyclin D1-Cdk4 complexes are persistent, albeit reduced by <50% compared to young cells. We also provide new evidence that p21 may play a role in inactivation of the DNA replication factor proliferating cell nuclear antigen during early senescence. Finally, because p16 accumulates in parallel with the increases in senescence-associated ␤-Gal activity and cell volume that characterize the senescent phenotype, we suggest that p16 upregulation may be part of a differentiation program that is turned on in senescent cells. Since p21 decreases after senescence is achieved, this upregulation of p16 may be essential for maintenance of the senescent-cell-cycle arrest.Human diploid fibroblasts (HDF) have a finite proliferative lifespan, at the end of which they are unable to enter S phase in response to mitogenic stimuli. Senescent HDF are also enlarged and flattened and synthesize an altered repertoire of cell-type-specific proteins, suggesting that they have differentiated as well as aged (5). Serum-stimulated senescent HDF fail to phosphorylate the retinoblastoma protein (pRb) (51), an event that is necessary for the release of E2F transcription factors that promote the expression of late G 1 genes whose products are required for S-phase initiation and progression (39, 55). The inhibition of DNA synthesis in senescent nuclei can be overcome by factors or treatments that block or inactivate the inhibitory activity of pRb and its family of related proteins. For example, transfection or microinjection of simian virus 40 (SV40) T antigen into senescent HDF induces DNA synthesis, but this effect is lost when SV40 T antigen deficient in pRb binding is used (22). These data suggest that failure to phosphorylate pRb is a key mechanism for the cell cycle arrest of senescent cells.Phosphorylation of pRb during G 1 phase is carried out by cyclin D-Cdk4 and cyclin D-Cdk6 (cyclin D-Cdk4/6) and cyclin E-Cdk2 complexes (44,50,55). In quiesce...

The regulation of ribosome biogenesis in response to environmental conditions is a key aspect of cell growth control. Ribosomal protein (RP) genes are regulated by the nutrient-sensitive, conserved target of rapamycin (TOR) signaling pathway. TOR controls the subcellular localization of protein kinase A (PKA) and the PKA-regulated kinase YAK1. However, the target transcription factor(s) of the TOR-PKA pathway are unknown. We show that regulation of RP gene transcription via TOR and PKA in yeast involves the Forkhead-like transcription factor FHL1 and the two cofactors IFH1 (a coactivator) and CRF1 (a corepressor). TOR, via PKA, negatively regulates YAK1 and maintains CRF1 in the cytoplasm. Upon TOR inactivation, activated YAK1 phosphorylates and activates CRF1. Phosphorylated CRF1 accumulates in the nucleus and competes with IFH1 for binding to FHL1 at RP gene promoters, and thereby inhibits transcription of RP genes. Thus, we describe a signaling mechanism linking an environmental sensor to ribosome biogenesis.

In yeast TOR and PKA pathways both control cell growth but how TORC1 and PKA signaling are linked is unknown. Here we show that TORC1 inhibition prevents the phosphorylation of some but not all PKA targets. We further demonstrate that TORC1 controls PKA by inhibiting the phosphorylation of the PKA regulatory subunit BCY1 by the MAP kinase MPK1.

Yeast Las17 protein is homologous to the Wiskott-Aldrich Syndrome protein, which is implicated in severe immunodeficiency. Las17p/Bee1p has been shown to be important for actin patch assembly and actin polymerization. Here we show that Las17p interacts with the Arp2/3 complex. LAS17 is an allele-specific multicopy suppressor of ARP2 and ARP3 mutations; overexpression restores both actin patch organization and endocytosis defects in ARP2 temperature-sensitive (ts) cells. Six of seven ARP2 ts mutants and at least one ARP3 ts mutant are synthetically lethal with las17Delta ts confirming functional interaction with the Arp2/3 complex. Further characterization of las17Delta cells showed that receptor-mediated internalization of alpha factor by the Ste2 receptor is severely defective. The polarity of normal bipolar bud site selection is lost. Las17-gfp remains localized in cortical patches in vivo independently of polymerized actin and is required for the polarized localization of Arp2/3 as well as actin. Coimmunoprecipitation of Arp2p with Las17p indicates that Las17p interacts directly with the complex. Two hybrid results also suggest that Las17p interacts with actin, verprolin, Rvs167p and several other proteins including Src homology 3 (SH3) domain proteins, suggesting that Las17p may integrate signals from different regulatory cascades destined for the Arp2/3p complex and the actin cytoskeleton.

The Target of Rapamycin (TOR), a protein kinase, is the central node of a highly conserved signaling network that regulates cell growth in response to nutrients, hormones, and stresses. TOR is found in two functionally distinct complexes, TORC1 and TORC2. In this review we address the most recent advances in TOR signaling in invertebrate model organisms, including yeasts, plants, worms, and insects.

In Saccharomyces cerevisiae, the WASP (Wiskott-Aldrich syndrome protein) homologue Las17p (also called Bee1p) is an important component of cortical actin patches. Las17p is part of a high-molecular-weight protein complex that regulates Arp2/3 complex-dependent actin polymerization at the cell cortex and that includes the type I myosins Myo3p and Myo5p and verprolin (Vrp1p). To identify other factors implicated with this complex in actin regulation, we isolated proteins that bind to Las17p by two-hybrid screening and affinity chromatography. Here, we report the characterization of Lsb7/Bzz1p (for Las seventeen binding protein 7), an Src homology 3 (SH3) domain protein that interacts directly with Las17p via a polyproline-SH3 interaction. Bzz1p coimmunoprecipitates in a complex with Las17p, Vrp1p, Myo3/5p, Bbc1p, Hsp70p, and actin. It colocalizes with cortical actin patches and with Las17p. This localization is dependent on Las17p, but not on F-actin. Bzz1p interacts physically and genetically with type I myosins. While deletion of BZZ1 shows no obvious phenotype, simultaneous deletion of the BZZ1, MYO3, and MYO5 genes is lethal. Overexpression of Bzz1p inhibits cell growth, and a bzz1⌬ myo5⌬ double mutant is unable to restore actin polarity after NaCl stress. Finally, Bzz1p in vitro is able to recruit a functional actin polymerization machinery through its SH3 domains. Its interactions with Las17p, Vrp1p, and the type I myosins are essential for this process. This suggests that Bzz1p could be implicated in the regulation of actin polymerization.

The pathogenic yeast Candida albicans is both a powerful commensal and a pathogen of humans that can infect wide range of organs and body sites. Metabolic flexibility promotes infection and commensal colonization by this opportunistic pathogen. Yeast cell survival depends upon assimilation of fermentable and non-fermentable locally available carbon sources. Physiologically relevant sugars like glucose and fructose are present at low levels in host niches. However, because glucose is the preferred substrate for energy and biosynthesis of structural components, its efficient detection and metabolism are fundamental for the metabolic adaptation of the pathogen. We explored and characterized the C. albicans hexose kinase system composed of one hexokinase ( Ca Hxk2) and two glucokinases ( Ca Glk1 and Ca Glk4). Using a set of mutant strains, we found that hexose phosphorylation is mostly performed by Ca Hxk2, which sustains growth on hexoses. Our data on hexokinase and glucokinase expression point out an absence of cross regulation mechanisms at the transcription level and different regulatory pathways. In the presence of glucose, Ca Hxk2 migrates in the nucleus and contributes to the glucose repression signaling pathway. In addition, Ca Hxk2 participates in oxidative, osmotic and cell wall stress responses, while glucokinases are overexpressed under hypoxia. Hexose phosphorylation is a key step necessary for filamentation that is affected in the hexokinase mutant. Virulence of this mutant is clearly impacted in the Galleria mellonella and macrophage models. Filamentation, glucose phosphorylation and stress response defects of the hexokinase mutant prevent host killing by C. albicans . By contributing to metabolic flexibility, stress response and morphogenesis, hexose kinase enzymes play an essential role in the virulence of C. albicans .