RESUMO
Protein phosphorylation cascades play a central role in the regulation of cell growth and protein kinases PKA, Sch9 and Ypk1 take center stage in regulating this process in S. cerevisiae To understand how these kinases co-ordinately regulate cellular functions we compared the phospho-proteome of exponentially growing cells without and with acute chemical inhibition of PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the previously reported RxRxxS/T-, but also in an RRxS/T-motif. Validation experiments revealed that neutral trehalase Nth1, a known PKA target, is additionally phosphorylated and activated downstream of Ypk1. Signaling through Ypk1 is therefore more closely related to PKA- and Sch9-signaling than previously appreciated and may perform functions previously only attributed to the latter kinases.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimologia , Saccharomyces cerevisiae/genética , Motivos de Aminoácidos , Sequência de Aminoácidos , Sequência Consenso , Proteínas Quinases Dependentes de AMP Cíclico/química , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/química , Proteoma/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Trealase/metabolismoRESUMO
TORC1 is a conserved multisubunit kinase complex that regulates many aspects of eukaryotic growth including the biosynthesis of ribosomes. The TOR protein kinase resident in TORC1 is responsive to environmental cues and is potently inhibited by the natural product rapamycin. Recent characterization of the rapamycin-sensitive phosphoproteome in yeast has yielded insights into how TORC1 regulates growth. Here, we show that Sch9, an AGC family kinase and direct substrate of TORC1, promotes ribosome biogenesis (Ribi) and ribosomal protein (RP) gene expression via direct inhibitory phosphorylation of the transcriptional repressors Stb3, Dot6 and Tod6. Deletion of STB3, DOT6 and TOD6 partially bypasses the growth and cell size defects of an sch9 strain and reveals interdependent regulation of both Ribi and RP gene expression, and other aspects of Ribi. Dephosphorylation of Stb3, Dot6 and Tod6 enables recruitment of the RPD3L histone deacetylase complex to repress Ribi/RP gene promoters. Taken together with previous studies, these results suggest that Sch9 is a master regulator of ribosome biogenesis through the control of Ribi, RP, ribosomal RNA and tRNA gene transcription.
Assuntos
Regulação Fúngica da Expressão Gênica , RNA Ribossômico/biossíntese , Proteínas Ribossômicas/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais , RNA de Transferência/biossíntese , Transcrição GênicaRESUMO
The wing somatic mutation and recombination test (SMART) using transheterozygotes for the Drosophila melanogaster third chromosome markers mwh (multiple wing hairs) and flr(3) (flare-3) has proved to be efficient in genotoxicity screens. The genetic background of the D. melanogaster strains affects the frequency of identification of somatic mutant spots. The genetic background appears to be also of importance in hybrid dysgenesis (HD) revealed in crosses between flies from genetically distant D. melanogaster strains. In this study, we analyzed mwh and flr3/Ser D. melanogaster strains for the presence of the P and hobo transposable elements that induce genetic instability in P-M and H-E dysgenic crosses, respectively. According to PCR these strains lack the P-element. The mwh strain does not contain the hobo-element too, while hobo is present as a full-length variant and its numerous defective derivatives in the flr(3)/Ser genome. Based on the crosses the reference E- and H-strains to strains used in SMART, the flr/Ser were assigned to the H-type and the mwh to the E-type. Fluorescence in situ hybridization reveals over 50 hobo hybridization sites scattered throughout the flr(3)/Ser genome. Thus, there is a basis for H-E HD in the strains we studied. However the mwh/flr(3), regardless of cross-direction showed higher fertility than the mwh females. Cross-direction had also no affect on hybrid fertility. This meant that they did not exhibit the major HD symptoms. In our view, the significance of the TE activity as a cause of HD has been overestimated, in the case of H-E HD at least. The majority of the mutant spots in SMART result not so much from mutations or TE transposition as from recombination events, even in the case of balancer individuals.