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. 2003 Jun 16;22(12):3073-83.
doi: 10.1093/emboj/cdg298.

Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases

Tomohiko Matsuo  1 , Yoshiya Kubo, Yoshinori Watanabe, Masayuki Yamamoto
Affiliations

Schizosaccharomyces pombe AGC family kinase Gad8p forms a conserved signaling module with TOR and PDK1-like kinases

Tomohiko Matsuo et al. EMBO J. .

Abstract

The TOR protein is a phosphoinositide kinase-related kinase widely conserved among eukaryotes. Fission yeast tor1 encodes an ortholog of TOR, which is required for sexual development and growth under stressed conditions. We isolated gad8, which encodes a Ser/Thr kinase of the AGC family, as a high-copy suppressor of the sterility of a tor1 mutant. Disruption of gad8 caused phenotypes similar to those of tor1 disruption. Gad8p was less phosphorylated and its kinase activity was undetectable in tor1Delta cells. Three amino acid residues corresponding to conserved phosphorylation sites in the AGC family kinases, namely Thr387 in the activation loop, Ser527 in the turn motif and Ser546 in the hydrophobic motif, were important for the kinase activity of Gad8p. Tor1p was responsible for the phosphorylation of Ser527 and Ser546, whereas Ksg1p, a PDK1-like kinase, appeared to phosphorylate Thr387 directly. Altogether, Tor1p, Ksg1p and Gad8p appear to constitute a signaling module for sexual development and growth under stressed conditions in fission yeast, which resembles the mTOR-PDK1-S6K1 system in mammals and may represent a basic signaling module ubiquitous in eukaryotes.

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Figures

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Fig. 1. The gad2-1 (tor1-g2) mutation does not complement the tor1 disruption. (A) Sporulation assay. Diploid strain JW958 (denoted as gad2/tor1Δ), constructed by crossing a gad2-1 haploid and a tor1Δ haploid, was incubated on MEA medium together with control diploids heterozygous for either gad2-1 (JW956; denoted as gad2/+) or tor1Δ (JW957; denoted as tor1Δ/+). After four days, patches were exposed to iodine vapor, which stains sporulated cells dark brown. (B) Complementation for the growth at high temperature. The same diploid strains as in (A) were streaked on the YE medium and incubated for three days at 37°C.
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Fig. 2. The deduced gad8 gene product, Gad8p, is a Ser/Thr kinase of the AGC family. Gad8p is aligned with some members of the AGC family, including budding yeast Ypk1 and Ypk2/Ykr2 and human PKBα, PKCα and S6K1. The kinase domain is represented by a black box and the C-terminal region conserved in AGC family kinases, by a shaded box. The percentage values indicates the amino acid identity of the respective region compared with the corresponding region in Gad8p. PH, pleckstrin homology domain; C1, diacylglycerol/phorbol ester binding domain; C2, Ca2+/TPA binding domain.
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Fig. 3. Suppression of tor1-g2 and tor1Δ by overexpression of gad8. (A) Suppression of the tor1-g2 mutant. A homothallic haploid tor1-g2 strain (JW948) was transformed with either pREP41-gad8, or pREP41-tor1 or the vector pREP41. Transformants were grown on SSA medium at 30°C for four days. The mating efficiency of each transformant was scored under the microscope (top panel). Each transformant was also incubated on MM medium at 36°C for four days (bottom panel). (B) Suppression of the tor1Δ strain. A homothallic haploid tor1Δ strain (JW951) was examined as in (A), except that the incubation time on MM medium was three days.
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Fig. 4. Phenotypic similarities between gad8Δ and tor1Δ. (A) A comparison of the mating efficiencies of the wild-type (JY476), gad8Δ (JW945), tor1Δ (JW951) and gad8Δ tor1Δ (JW959) strains. Cells were incubated on SSA medium for four days and the mating efficiency was scored under the microscope. (B) Flow-cytometric analysis of the four strains subjected to nitrogen starvation. Cells were grown to logarithmic phase in MM liquid medium at 30°C, harvested, washed once and transferred to MM without a nitrogen source. Their DNA content was measured by FACS analysis at 2 h intervals. (C) Temperature-sensitivity and high-osmolarity sensitivity of each strain. Cells of each strain, grown to logarithmic phase at 30°C, were spotted on YE medium with sequential ten-fold dilution and incubated at either 30°C (left panel) or 36°C (middle panel) for three days. Cells were also spotted similarly on YE medium containing 0.2 M KCl and incubated at 30°C for three days (right panel).
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Fig. 5. Tor1p is essential for the kinase activity of Gad8p and affects its phosphorylation state. (A) Assay of the kinase activity of Gad8p recovered from either the wild-type (JW960) or the tor1Δ (JW965) background. Gad8-6HAp expressed in each strain was immunopurified from the cell extract and assayed for its ability to phosphorylate crosstide, as described in the Materials and methods. (B) Detection of Gad8-6HAp in wild-type and tor1Δ cells. Extracts of JW960 and JW965, expressing Gad8-6HAp, were separated by SDS–PAGE, and the protein was detected by western blotting using anti-HA antibody. Extracts were also treated with CIAP, with or without the addition of the phosphatase inhibitor mixture, to examine possible phosphorylation of Gad8p. The white arrowhead indicates the major band of Gad8p, and the black arrowhead indicates the minor band. (C) Effects of phosphatase treatment on the kinase activity of Gad8p. Gad8-6HAp immunopurified as in (A) was treated with CIAP, either native or boiled, in the presence or absence of the phosphatase inhibitor mixture. The kinase assay was performed as in (A).
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Fig. 6. Effects of mutations in the conserved phosphorylation sites on Gad8p. (A) Alignment of the sequences around the three possible phosphorylation sites on Gad8p, in the activation loop, in the turn motif and in the hydrophobic motif, with those of human PKCα, S6K1, PKBα, PKAα and mouse RSK2. The predicted phosphorylation sites on Gad8p, namely Thr387, Ser527 and Ser546, are indicated by asterisks. (B) Unphosphorylatable forms of Gad8p are not functional. A homothallic haploid gad8Δ strain (JW945) was transformed with pR3C-gad8, pR3C-gad8-T387A, pR3C-gad8-S527A, pR3C-gad8-S546A, pR3C-gad8-S527A S546A or the vector pREP41. Transformants were examined for mating efficiency (left panel), growth at high osmolarity (middle panel) and growth at high temperature (right panel). Their mating efficiency was scored after incubation on SSA medium at 30°C for three days. Growth was examined on MM medium containing 0.6 M KCl at 30°C for six days, or on MM medium at 37°C for six days. (C) The kinase activity of the unphosphorylatable forms of Gad8p. Gad8-6HAp immunopurified from each strain, namely the gad8-T387A (JW962), gad8-S527A (JV107), gad8-S546A (JW963) and gad8-S527A S546A (JV108) mutants was subjected to the in vitro kinase assay, together with the control wild-type (JW960) and the gad8-K259R mutant (JW961) supposed to generate a kinase-dead product. (D) Detection of mutant forms of Gad8-6HAp in SDS–PAGE followed by western blot analysis.
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Fig. 7. Effects of the tor1 disruption were alleviated by gad8-S527D S546D. Homothallic haploid gad8Δ (JW945) or gad8Δ tor1Δ (JW959) strains were transformed with pR3C-gad8, pR3C-gad8-S527D, pR3C-gad8-S546D and pR3C- gad8-S527D S546D. Each transformant was tested for mating efficiency (left panel), growth at high osmolarity (middle panel) and growth at high temperature (right panel). The mating efficiency was scored after incubation on SSA medium at 30°C for five days. Growth was examined on MM medium containing 0.6 M KCl at 30°C for six days, or on MM medium at 37°C for five days.
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Fig. 8. Functional relationship of Gad8p with Ksg1p. (A) Suppression of ksg1-208 by overexpression of gad8. The ksg1-208 mutant (JW237) was transformed with pREP41-gad8, pREP41-gad8-T387A, pREP41-gad8-S546A, pREP41-gad8-S546D, pREP41-ksg1 and the vector pREP41. Each transformant was examined for mating efficiency (left panel) and growth at the restrictive temperature (right panel). Transformants were cultured on SSA at 26.5°C for six days before scoring mating efficiency, and on MM medium at 33°C for three days to estimate temperature sensitivity. (B) The ksg1-208 mutation affects the kinase activity of Gad8p. Cells expressing Gad8-6HAp in either the wild-type (JW960) or the ksg1-208 (JW967) background were grown to logarithmic phase in YE liquid medium at 26.5°C and shifted to 34°C. Cells were harvested before and 4.5 h after the shift, and Gad8-6HAp was immunopurified from each sample. The kinase activity of Gad8-6HAp from each sample was assayed in vitro using crosstide. Immunoprecipitated Gad8-6HAp, detected by western blotting, is also shown. (C) Ksg1p phosphorylates Gad8p in vitro. Wild-type and mutant Gad8p fused to MBP, expressed bacterially and immunopurified, was incubated with GST-tagged Ksg1p in the presence of [γ-32P]ATP. Phosphorylation of MBP–Gad8p was detected by autoradiography. Ksg1-K128Rp, a kinase-dead form of Ksg1, was also examined as a negative control. An autoradiograph is shown in the upper panel and a Coomassie staining pattern in shown in the lower panel. (D) Inhibition of the Ksg1p kinase activity by PIF-tide. Phosphorylation reaction of Gad8p and Gad8-S546Dp was conducted as described in (C), in the presence of PIF-tide at the concentration indicated.
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Fig. 9. A model for the activation of Gad8p by Tor1p and Ksg1p. Gad8p is an essential factor required for sexual development and growth under stressed conditions. Tor1p is responsible for phosphorylation of Ser546 in the hydrophobic motif and Ser527 in the turn motif. Ksg1p phosphorylates Thr387 in the activation loop. Phosphorylation of these three residues is essential for the full activation of Gad8p. Phosphorylation of Ser546 by Tor1p may facilitate the access of Ksg1p to Gad8p and hence enhance the phosphorylation of Thr387.
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