TOR Complex 2- independent mutations in the regulatory PIF pocket of Gad8AKT1/SGK1 define separate branches of the stress response mechanisms in fission yeast

Abstract

The Target of rapamycin (TOR) protein kinase forms part of TOR complex 1 (TORC1) and TOR complex 2 (TORC2), two multi-subunit protein complexes that regulate growth, proliferation, survival and developmental processes by phosphorylation and activation of AGC-family kinases. In the fission yeast, Schizosaccharomyces pombe, TORC2 and its target, the AGC kinase Gad8 (an orthologue of human AKT or SGK1) are required for viability under stress conditions and for developmental processes in response to starvation cues. In this study, we describe the isolation of gad8 mutant alleles that bypass the requirement for TORC2 and reveal a separation of function of TORC2 and Gad8 under stress conditions. In particular, osmotic and nutritional stress responses appear to form a separate branch from genotoxic stress responses downstream of TORC2-Gad8. Interestingly, TORC2-independent mutations map into the regulatory PIF pocket of Gad8, a highly conserved motif in AGC kinases that regulates substrate binding in PDK1 (phosphoinositide dependent kinase-1) and kinase activity in several AGC kinases. Gad8 activation is thought to require a two-step mechanism, in which phosphorylation by TORC2 allows further phosphorylation and activation by Ksg1 (an orthologue of PDK1). We focus on the Gad8-K263C mutation and demonstrate that it renders the Gad8 kinase activity independent of TORC2 in vitro and independent of the phosphorylation sites of TORC2 in vivo. Molecular dynamics simulations of Gad8-K263C revealed abnormal high flexibility at T387, the phosphorylation site for Ksg1, suggesting a mechanism for the TORC2-independent Gad8 activity. Significantly, the K263 residue is highly conserved in the family of AGC-kinases, which may suggest a general way of keeping their activity in check when acting downstream of TOR complexes.

Fig 1. Isolation of tor1-independent alleles of gad8.

A, Serial dilutions of wild-type (WT) and Δtor1 cells transformed with the indicated plasmids. pREP1, in grey, allows strong overexpression, while pREP81, in black, allows weak overexpression of the cloned gene. Cells were spotted onto minimal medium with, or without 0.8M KCl and were incubated for 4 days at 30°C (no stress) or at high temperature (37°C). B, Schematic representation of the screen carried out to isolate tor1-independent gad8 alleles. The gad8⁺ open reading frame was amplified under PCR mutagenesis conditions using primers that contained sequences homologous to pREP81 sequences (in red) and sequences homologous to gad8⁺ (in blue). The mutagenized gad8 fragments were co-transformed together with linearized pREP81 plasmids into the tor1-L2045D strain and transformants were selected on minimal plates at 37°C. C, Serial dilutions of wild-type (WT) and Δtor1 cells as described in A. D, Schematic representation of Gad8. Indicated in red are the tor1-independent gad8 mutations, T260C and K263C, and in black the Ksg1-dependent phosphorylation site, T387, and the Tor1-dependent phosphorylation sites, S527 and S546. E, Alignment of the amino acid sequences surrounding the T260C and K263C point mutations in several members of the AGC kinases. The tor1-independent gad8 mutations are in red. The K263, but not T260, residue is conserved in all the examined AGC kinases.

Fig 2. gad8-K263C or gad8-T260C suppress high temperature, osmotic stress, low-glucose and sterility in TORC2 mutant cells.

A-B, Suppression by gad8-K263C and gad8-T260C when expressed from multicopy plasmids. Serial dilutions of wild-type (WT), and Δtor1 cells transformed with the indicated plasmids. Cells were spotted onto minimal medium at 30°C (no stress), 37°C, or at 30°C with 1M KCl or low glucose levels (A). Mating efficiencies of WT and Δtor1 strains transformed with the indicated plasmids. The results are the mean values of three independent experiments (B). C-E, Chromosomal expression of the gad8-K263C mutation suppresses growth and cellular defects in Δtor1 cells. Serial dilutions of cells plated onto rich medium (YES) medium at 30°C (no stress), 37°C, or at 30°C with 1M KCl (C) or low glucose levels (D). Mating efficiencies of the indicated strains. The results are the mean values of three separate experiments (E).

Fig 3. Gad8-K263C activity is independent of Tor1 in vitro and in vivo.

A, Western blot analysis of protein extracts isolated from strains expressing no-tag Gad8 (lane 1), Gad8-HA or Gad8-K263C-HA (lanes 2–11). Cells were grown to mid-log phase and left untreated in YES medium or shifted for 1 h to EMM containing no carbon source (EMM-Glu) or to YES containing 1M KCl or 1 M NaCl. Gad8-HA and Gad8-K263C-HA were immunoprecipitated and assayed in vitro for their kinase activity using the Fkh2-GST peptide as a substrate. Phosphorylation of Fkh2-GST was detected with anti-PAS antibodies recognizing the phosphorylated form of the consensus sequence of AGC kinases (Fkh2-P). Phosphorylation of Gad8 at S456 was detected with anti-Gad8-S546-P phsophospecific antibodies (S546-P). B, Serial dilutions of the indicated strains spotted onto YES (no stress) or YES medium containing KCl and incubated at 30⁰ C or 37⁰ for 3 days. All mutations are chromosomal. C, Western blot analysis of protein extracts isolated from wild-type (tor1⁺) or Δtor1 cells grown in YES medium to mid-log phase or shifted for 1 hour into EMM containing no carbon source (-Glu). The status of phosphorylation of Gad8 at T387, the target site for Ksg1 (PDK1) was detected with anti-Gad8-T387-P phosphospecific antibodies. D, Serial dilutions of the indicated strains as described in B.

Fig 4. The gad8-K263C mutation is located at the PIF pocket and induces higher flexibility at the PIF pocket and activation loop A, 3D model of S. pombe Gad8 structure.

The protein is shown as a ribbon diagram (grey), the ATP is shown as sticks, and the Mg ion is shown as a green sphere (ATP is colored according to atom types). The activation loop (A-loop) is shown in red. The highly conserved structures in AGC kinases, the DFG-motif and the Gly-rich loop are colored in blue and magenta, respectively. The C-terminal extension in cyan. The residues K263, T387, S527, and S546 are shown in balls and sticks. B, RMSF plot of the backbone atoms for non-phosphorylated Gad8 (blue), Gad8-pS527/pS546 (yellow, p denotes phosphorylation), and non-phosphorylated K263C-Gad8 (orange).

Fig 5. gad8-Q298L partially suppresses the mating type defect, but not osmotic stress, high temperature or low-glucose stress sensitivities in Δtor1 cells A, Mating efficiencies of the indicated strains.

The results are the mean values of three separate experiments. B, Serial dilutions of cells plated onto minimal medium containing no stress, 1M KCl, or reduced glucose levels. Plates were incubated at 30°C, unless otherwise indicated.

Fig 6. gad8-K263C or gad8-T260 confer sensitivity to DNA damaging conditions.

A, Plasmids containing gad8-K263C or gad8-T260 confer genotoxic stress in wild-type cells and are unable to suppress genotoxic sensitivity in Δtor1 mutant cells. WT or Δtor1 cells transformed with empty vector (pREP81), or with vector carrying tor1⁺, gad8⁺, gad8-T260C or gad8-K263C were serially diluted and spotted onto minimal medium with no stress or containing 5mM HU or 5μM CPT. Plates were incubated at 30⁰ C for 3 days. B, The chromosomal K263C mutation confers sensitivity to genotoxic stress. WT, Δtor1, Δgad8, gad8-K263C or Δtor1gad8-K263C cells were serially diluted and spotted onto minimal medium with no stress or containing 5mM HU or 5μM CPT and incubated at 30⁰ C for 3 days. Cells spotted on 0.8M KCl are used as control. C, gad8-K263C confers dominant-negative effect under genotoxic stress conditions. Serial dilutions of diploid cells were spotted onto the indicated plates. Plates were incubated at 30⁰ for 3 days. D, The gad8-Q298L partially suppresses genotoxic stress sensitivity in Δtor1 cells. Serial dilutions of WT or Δtor1 cells harboring the indicated plasmids were spotted onto minimal medium or minimal medium containing 5mM HU or 5μM CPT and incubated at the indicated temperatures for 4 days.

Fig 7. gad8-K263C cells show similar defects to Δtor1 cells with respect to DNA damage responses A, Expression levels of cdt2⁺, cdc18⁺ and cdc22⁺ in WT, Δtor1, gad8-K263C and Δtor1gad8-K263C cells were determined by qRT-PCR.

Total RNA was prepared from untreated cells or cells treated with 12mM HU for 3 h. The level of act1⁺ mRNA was used as a reference. Error bars (SD) were calculated from biological triplets and significant differences were determined by the Student's t-test (*** P < 0.001). B, Chk1 phosphorylation is delayed in gad8-K263C in response to CPT. Wild-type, Δtor1 or gad8-K263C cells containing an HA-tagged Chk1 were grown to mid-log phase and treated with 30 μm CPT. Protein extracts from each of the indicated time points were analyzed by Western blotting. Membranes were probed with anti-HA to visualize Chk1 and with anti-Actin as a loading control. The upper band observed with anti-HA antibodies represent the phosphorylated and activated band of Chk1. C, De-phosphorylation of Chk1 is delayed in gad8-K263C mutant. Cells were arrested for 3 h in 30μM CPT before being released into fresh medium. Protein samples from the indicated time points were analyzed by Western blot as describe above.