Activation of protein kinase C-mitogen-activated protein kinase signaling in response to inositol starvation triggers Sir2p-dependent telomeric silencing in yeast

Abstract

Depriving wild type yeast of inositol, a soluble precursor for phospholipid, phosphoinositide, and complex sphingolipid synthesis, activates the protein kinase C (PKC)-MAPK signaling pathway, which plays a key role in the activation of NAD(+)-dependent telomeric silencing. We now report that triggering PKC-MAPK signaling by inositol deprivation or by blocking inositol-containing sphingolipid synthesis with aureobasidin A results in increased telomeric silencing regulated by the MAPK, Slt2p, and the NAD(+)-dependent deacetylase, Sir2p. Consistent with the dependence on NAD(+) in Sir2p-regulated silencing, we found that inositol depletion induces the expression of BNA2, which is required for the de novo synthesis of NAD(+). Moreover, telomeric silencing is greatly reduced in bna2Δ and npt1Δ mutants, which are defective in de novo and salvage pathways for NAD(+) synthesis, respectively. Surprisingly, however, omitting nicotinic acid from the growth medium, which reduces cellular NAD(+) levels, leads to increased telomeric silencing in the absence of inositol and/or at high temperature. This increase in telomeric silencing in response to inositol starvation is correlated to chronological life span extension but is Sir2p-independent. We conclude that activation of the PKC-MAPK signaling by interruption of inositol sphingolipid synthesis leads to increased Sir2p-dependent silencing and is dependent upon the de novo and salvage pathways for NAD(+) synthesis but is not correlated with cellular NAD(+) levels.

Keywords: Inositol Phospholipid; NAD Biosynthesis; Protein Kinase C (PKC); Sirtuins; Sphingolipid.

FIGURE 1.

NAD⁺ in S. cerevisiae is synthesized from NA via the salvage pathway and from tryptophan through the de novo pathway. NA and tryptophan imported from the growth medium serve as precursors of NAD⁺. A gray box indicates the activity of sirtuins (Sir2p and Hst1p-4p), NAD⁺-dependent protein deacetylases, which hydrolyze NAD⁺ into nicotinamide and ADP-ribose, in a reaction coupled with protein/histone deacetylation. The enzymes involved in the salvage pathway for NAD⁺ synthesis are primarily localized in the nucleus (52, 62), whereas the enzymes in the de novo pathway are distributed in the nucleus and cytoplasm (52). The de novo pathway converts tryptophan to nicotinic acid mononucleotide in a series of reactions catalyzed by Bna1p–6p. Nicotinic acid mononucleotide is the point of convergence for the de novo and salvage pathways for NAD⁺ biosynthesis. Abbreviations used are as follows: NAD⁺, nicotinamide adenine dinucleotide; NA, nicotinic acid; NAM, nicotinamide; NaMN, nicotinic acid mononucleotide; NaAD, deamido-NAD.

FIGURE 2.

Increased silencing in the absence of inositol and/or at 37 °C is dependent on Sir2p and Slt2p. Wild type, sir2Δ, and slt2Δ strains carrying the TELV_R::URA3 construct were grown on I⁺ medium until mid-logarithmic phase, diluted back to A_{600 nm} = 0.2 in I⁺ and I⁻ media at 30 or 37 °C, and allowed to grow until an A_{600 nm} of 1.0. Gene expression levels were examined by RT-PCR and calculated using the comparative C_T method (ΔΔC_T) as described under “Experimental Procedures.” The changes in URA3 expression levels are log₂ ratios of the level of expression measured by RT-PCR in a specific strain, under the given growth condition, relative to URA3 expression in the control culture. The asterisk refers to the mRNA derived from the wild type control grown in I⁺NA⁺ medium at 30 °C and served as a reference (unit = 1) for measuring relative URA3 expression levels. A indicates the strains grown at 30 °C. B indicates the strains grown at 37 °C. Data represent the mean ± S.E. of three independent experiments, n = 3.

FIGURE 3.

Inhibiting complex sphingolipid synthesis results in increased silencing. Wild type and slt2Δ strains carrying the TELV_R::URA3 construct were grown on I⁺ medium until mid-logarithmic phase, diluted back to A_{600 nm} = 0.2 in I⁺ and I⁻ media at 30 °C, and allowed to grow until an A_{600 nm} of 1.0. An aliquot of the cultures growing in I⁺ medium were treated with AbA to final concentration of 2 μg/ml, incubated at 30 °C for 120 min, and harvested. Gene expression levels were examined by RT-PCR and calculated using the comparative C_T method (ΔΔC_T) as described under “Experimental Procedures.” The changes in URA3 expression levels are log₂ ratios of the level of expression measured by RT-PCR in a specific strain, under the given growth condition, relative to URA3 expression in the control culture. The asterisk refers to the mRNA derived from the wild type control grown in I⁺NA⁺ medium at 30 °C and served as a reference (unit = 1) for measuring relative URA3 expression levels. Data represent the mean ± S.E.) of three independent experiments, n = 3.

FIGURE 4.

Influence of the de novo and salvage pathways for NAD⁺ biosynthesis on Sir2-dependent telomeric silencing. Wild type, bna2Δ, and npt1Δ strains carrying the TELV_R::URA3 construct were grown on I⁺ medium until mid-logarithmic phase, diluted back to A_{600 nm} = 0.2 in I⁺ and I⁻ media at 30 or 37 °C, and allowed to grow until an A_{600 nm} of 1.0. As a control experiment, wild type cells were grown under the same conditions with the exception that NA was omitted from the medium. Gene expression levels were examined by RT-PCR and calculated using the comparative C_T method (ΔΔC_T) as described under “Experimental Procedures.” The changes in URA3 expression levels are log₂ ratios of the level of expression measured by RT-PCR in a specific strain, under the given growth condition, relative to URA3 expression in the control culture. The asterisk refers to the mRNA derived from the wild type control grown in I⁺NA⁺ medium at 30 °C and served as a reference (unit = 1) for measuring relative URA3 expression levels. White bars indicate the strains grown at 30 °C, and black bars indicate the strains grown at 37 °C. A indicates the strains grown in the presence of NA. B indicates the strain grown in the absence of NA. Data represent the mean ± S.E. of three independent experiments, n = 3.

FIGURE 5.

Inositol availability affects expression of the BNA2 and THI4 genes in wild type cells. Gene expression levels were determined by quantitative RT-PCR and calculated using the comparative C_T method (ΔΔC_T) as described under “Experimental Procedures.” Cells were grown in medium containing inositol (I⁺NA⁺ and I⁺NA⁻) to mid-logarithmic phase. The cultures were divided in half. One-half was shifted to inositol-free medium (I⁻NA⁺ and I⁻NA⁻), while maintaining constant temperature at 30 or 37 °C. The remaining culture was shifted to medium containing inositol (I⁺NA⁺ and I⁺NA⁻) and was also incubated at 30 or 37 °C. RNA samples were collected at 3 h following the media shift. The ACT1 gene served as an endogenous control. Data represent the mean ± S.E. of three independent experiments, n = 3. A, BNA2 expression levels in cells grown in NA⁺. B, BNA2 expression levels in cells grown in NA⁻. C, THI4 expression levels in cells grown in NA⁺. The growth conditions indicated are: 30 °C (white bars) and 37 °C (black bars).

FIGURE 6.

Exogenous inositol influences NAD⁺ levels in wild type cells grown in the presence of NA. NAD⁺ levels were measured as described under “Experimental Procedures” using the alcohol dehydrogenase assay. A, wild type, bna2Δ, and npt1Δ strains were grown continuously up to A_{600 nm} = 1 in I⁺NA⁺ and I⁻NA⁺ medium at 30 and 37 °C. B, wild type cells were grown in I⁺NA⁻ and I⁻NA⁻ at 30 and 37 °C and were harvested at A_{600 nm} = 1. Data represent the mean ± S.E. of at least three independent experiments, n = 3 or 4. The growth conditions indicated are: 30 °C (white bars) and 37 °C (black bars).

FIGURE 7.

Absence of inositol causes an increase in yeast chronological life span. Cells were inoculated into I⁺ or I⁻ medium and allowed to grow into late stationary phase. Aliquots from the cultures were diluted in water and plated into YEPD plates such that single colonies could be counted (CFU). Two dilutions per culture were used to reduce the fluctuations due to manual error. CFU were counted after 2 days. The number of CFUs at day 2 was considered to be the initial survival (100% survival) and was used to determine the age-dependent percent survival.