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Multiple crosstalk between TOR and the cell integrity MAPK signaling pathway in fission yeast

View Article: PubMed Central - PubMed

ABSTRACT

In eukaryotic cells, the highly conserved Target of Rapamycin (TOR) and the Mitogen Activated Protein Kinase (MAPK) signaling pathways elicit adaptive responses to extra- and intracellular conditions by regulating essential cellular functions. However, the nature of the functional relationships between both pathways is not fully understood. In the fission yeast Schizosaccharomyces pombe the cell integrity MAPK pathway (CIP) regulates morphogenesis, cell wall structure and ionic homeostasis. We show that the Rab GTPase Ryh1, a TORC2 complex activator, cross-activates the CIP and its core member, the MAPK Pmk1, by two distinct mechanisms. The first one involves TORC2 and its downstream effector, Akt ortholog Gad8, which together with TORC1 target Psk1 increase protein levels of the PKC ortholog Pck2 during cell wall stress or glucose starvation. Also, Ryh1 activates Pmk1 in a TORC2-independent fashion by prompting plasma membrane trafficking and stabilization of upstream activators of the MAPK cascade, including PDK ortholog Ksg1 or Rho1 GEF Rgf1. Besides, stress-activated Pmk1 cross-inhibits Ryh1 signaling by decreasing the GTPase activation cycle, and this ensures cell growth during alterations in phosphoinositide metabolism. Our results reveal a highly intricate cross-regulatory relationship between both pathways that warrants adequate cell adaptation and survival in response to environmental changes.

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Rab-family GTPase Ryh1 regulates the cell integrity pathway in a TORC2-dependent and -independent fashion.(A) Serial dilutions of suspensions of strains MM913 (Pck2-HA; control), BV38 (ryh1Δ Pck2-HA), BV56 (Ryh1-QL Pck2-HA), MM1205 (tor1Δ Pck2-HA), BV11 (gad8Δ Pck2-HA), and MM1200 (pmk1Δ Pck2-HA), were spotted on YES plates supplemented with different concentrations of CaCl2, KCl, Caspofungin, or glucose, and incubated for 3 or 5 days at 28 °C. (B) Upper panels. Growing cultures of strains BV8 (Pck2-HA; control), and BV38 (ryh1Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 1 μg/ml Caspofungin (left panel), starved for glucose (middle panel), or treated with 0.6 M KCl (right panel). Cell extracts were resolved by SDS-PAGE and Pck2 levels detected after incubation with anti-HA antibodies. Anti-Cdc2 was used as a loading control. Lower panels. Purification and detection of activated/total Pmk1 was performed as described above. *P < 0.05; **P < 0.005; ***P < 0.001. (C) Growing cultures of strains BV8 (Pck2-HA; control), BV38 (ryh1Δ Pck2-HA) and MM1205 (tor1Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 0.6 M KCl for the indicated times. Activated/total Pmk1 were detected with anti-phospho-p44/42 and anti-HA antibodies, respectively. **P < 0.005 (D) Growing cultures of strains BV8 (Pck2-HA; control) and BV14 (psk1Δ gad8Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 0.6 M KCl. Purification and detection of activated/total Pmk1 was performed as described above.
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f4: Rab-family GTPase Ryh1 regulates the cell integrity pathway in a TORC2-dependent and -independent fashion.(A) Serial dilutions of suspensions of strains MM913 (Pck2-HA; control), BV38 (ryh1Δ Pck2-HA), BV56 (Ryh1-QL Pck2-HA), MM1205 (tor1Δ Pck2-HA), BV11 (gad8Δ Pck2-HA), and MM1200 (pmk1Δ Pck2-HA), were spotted on YES plates supplemented with different concentrations of CaCl2, KCl, Caspofungin, or glucose, and incubated for 3 or 5 days at 28 °C. (B) Upper panels. Growing cultures of strains BV8 (Pck2-HA; control), and BV38 (ryh1Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 1 μg/ml Caspofungin (left panel), starved for glucose (middle panel), or treated with 0.6 M KCl (right panel). Cell extracts were resolved by SDS-PAGE and Pck2 levels detected after incubation with anti-HA antibodies. Anti-Cdc2 was used as a loading control. Lower panels. Purification and detection of activated/total Pmk1 was performed as described above. *P < 0.05; **P < 0.005; ***P < 0.001. (C) Growing cultures of strains BV8 (Pck2-HA; control), BV38 (ryh1Δ Pck2-HA) and MM1205 (tor1Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 0.6 M KCl for the indicated times. Activated/total Pmk1 were detected with anti-phospho-p44/42 and anti-HA antibodies, respectively. **P < 0.005 (D) Growing cultures of strains BV8 (Pck2-HA; control) and BV14 (psk1Δ gad8Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 0.6 M KCl. Purification and detection of activated/total Pmk1 was performed as described above.

Mentions: In its GTP-bound form, Rab-family GTPase Ryh1 associates in vivo and activates TORC2 to promote phosphorylation and activation of Gad821. As compared to control cells, ryh1∆ cells displayed strong growth sensitivity to CaCl2, KCl, Caspofungin, or low glucose concentrations (Fig. 4A). Many of these phenotypes are shared by tor1∆ or gad8∆ mutants, but the overall sensitivity to stress was more pronounced in ryh1∆ cells (Fig. 4A), suggesting that Ryh1 signaling in response to environmental changes might be elicited through additional mechanisms different to those mediated by TORC2-Gad8. Similar to tor1∆ cells, Pck2 levels were strongly abrogated in ryh1∆ cells during growth and in response to cell wall stress or upon glucose removal (Fig. 4B), and the drop in Pmk1 activation was even stronger than in tor1∆ cells (Fig. 1). Remarkably, Pmk1 activation was also very low in ryh1∆ cells treated with KCl (Fig. 4B). This finding was unexpected, since Pmk1 became strongly activated by this stimulus in tor1∆ cells (Fig. 4C). Moreover, Pmk1 activation in salt stressed gad8∆ psk1∆ cells was similar to that of control cells (Fig. 4D). Thus, Ryh1 positively regulates the cell integrity pathway through additional, TORC2-independent mechanisms. Such control seems specific for the cell integrity pathway, since both the magnitude and kinetics of activation of Sty1, the core member of the stress activated MAPK pathway (SAPK) in fission yeast11, was identical in control and ryh1∆ cells under salt stress (Suppl. Figure S2). Human Rab6 GTPase can stimulate TORC2-Gad8 signaling S. pombe21. Indeed, ryh1∆ cells expressing a cDNA encoding human Rab6 under the control of the ryh1 + 5´UTR effectively suppressed the growth sensitivity of the parental mutant strain in the presence of CaCl2, KCl, Caspofungin, or low glucose concentrations (Suppl. Figure S3). This was accompanied by a moderate increase in the Pck2 levels during glucose starvation, and in Pmk1 activation by saline stress (Suppl. Figure S3). Hence, this novel regulatory role for Ryh1 might be evolutionary conserved.


Multiple crosstalk between TOR and the cell integrity MAPK signaling pathway in fission yeast
Rab-family GTPase Ryh1 regulates the cell integrity pathway in a TORC2-dependent and -independent fashion.(A) Serial dilutions of suspensions of strains MM913 (Pck2-HA; control), BV38 (ryh1Δ Pck2-HA), BV56 (Ryh1-QL Pck2-HA), MM1205 (tor1Δ Pck2-HA), BV11 (gad8Δ Pck2-HA), and MM1200 (pmk1Δ Pck2-HA), were spotted on YES plates supplemented with different concentrations of CaCl2, KCl, Caspofungin, or glucose, and incubated for 3 or 5 days at 28 °C. (B) Upper panels. Growing cultures of strains BV8 (Pck2-HA; control), and BV38 (ryh1Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 1 μg/ml Caspofungin (left panel), starved for glucose (middle panel), or treated with 0.6 M KCl (right panel). Cell extracts were resolved by SDS-PAGE and Pck2 levels detected after incubation with anti-HA antibodies. Anti-Cdc2 was used as a loading control. Lower panels. Purification and detection of activated/total Pmk1 was performed as described above. *P < 0.05; **P < 0.005; ***P < 0.001. (C) Growing cultures of strains BV8 (Pck2-HA; control), BV38 (ryh1Δ Pck2-HA) and MM1205 (tor1Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 0.6 M KCl for the indicated times. Activated/total Pmk1 were detected with anti-phospho-p44/42 and anti-HA antibodies, respectively. **P < 0.005 (D) Growing cultures of strains BV8 (Pck2-HA; control) and BV14 (psk1Δ gad8Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 0.6 M KCl. Purification and detection of activated/total Pmk1 was performed as described above.
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f4: Rab-family GTPase Ryh1 regulates the cell integrity pathway in a TORC2-dependent and -independent fashion.(A) Serial dilutions of suspensions of strains MM913 (Pck2-HA; control), BV38 (ryh1Δ Pck2-HA), BV56 (Ryh1-QL Pck2-HA), MM1205 (tor1Δ Pck2-HA), BV11 (gad8Δ Pck2-HA), and MM1200 (pmk1Δ Pck2-HA), were spotted on YES plates supplemented with different concentrations of CaCl2, KCl, Caspofungin, or glucose, and incubated for 3 or 5 days at 28 °C. (B) Upper panels. Growing cultures of strains BV8 (Pck2-HA; control), and BV38 (ryh1Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 1 μg/ml Caspofungin (left panel), starved for glucose (middle panel), or treated with 0.6 M KCl (right panel). Cell extracts were resolved by SDS-PAGE and Pck2 levels detected after incubation with anti-HA antibodies. Anti-Cdc2 was used as a loading control. Lower panels. Purification and detection of activated/total Pmk1 was performed as described above. *P < 0.05; **P < 0.005; ***P < 0.001. (C) Growing cultures of strains BV8 (Pck2-HA; control), BV38 (ryh1Δ Pck2-HA) and MM1205 (tor1Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 0.6 M KCl for the indicated times. Activated/total Pmk1 were detected with anti-phospho-p44/42 and anti-HA antibodies, respectively. **P < 0.005 (D) Growing cultures of strains BV8 (Pck2-HA; control) and BV14 (psk1Δ gad8Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 0.6 M KCl. Purification and detection of activated/total Pmk1 was performed as described above.
Mentions: In its GTP-bound form, Rab-family GTPase Ryh1 associates in vivo and activates TORC2 to promote phosphorylation and activation of Gad821. As compared to control cells, ryh1∆ cells displayed strong growth sensitivity to CaCl2, KCl, Caspofungin, or low glucose concentrations (Fig. 4A). Many of these phenotypes are shared by tor1∆ or gad8∆ mutants, but the overall sensitivity to stress was more pronounced in ryh1∆ cells (Fig. 4A), suggesting that Ryh1 signaling in response to environmental changes might be elicited through additional mechanisms different to those mediated by TORC2-Gad8. Similar to tor1∆ cells, Pck2 levels were strongly abrogated in ryh1∆ cells during growth and in response to cell wall stress or upon glucose removal (Fig. 4B), and the drop in Pmk1 activation was even stronger than in tor1∆ cells (Fig. 1). Remarkably, Pmk1 activation was also very low in ryh1∆ cells treated with KCl (Fig. 4B). This finding was unexpected, since Pmk1 became strongly activated by this stimulus in tor1∆ cells (Fig. 4C). Moreover, Pmk1 activation in salt stressed gad8∆ psk1∆ cells was similar to that of control cells (Fig. 4D). Thus, Ryh1 positively regulates the cell integrity pathway through additional, TORC2-independent mechanisms. Such control seems specific for the cell integrity pathway, since both the magnitude and kinetics of activation of Sty1, the core member of the stress activated MAPK pathway (SAPK) in fission yeast11, was identical in control and ryh1∆ cells under salt stress (Suppl. Figure S2). Human Rab6 GTPase can stimulate TORC2-Gad8 signaling S. pombe21. Indeed, ryh1∆ cells expressing a cDNA encoding human Rab6 under the control of the ryh1 + 5´UTR effectively suppressed the growth sensitivity of the parental mutant strain in the presence of CaCl2, KCl, Caspofungin, or low glucose concentrations (Suppl. Figure S3). This was accompanied by a moderate increase in the Pck2 levels during glucose starvation, and in Pmk1 activation by saline stress (Suppl. Figure S3). Hence, this novel regulatory role for Ryh1 might be evolutionary conserved.

View Article: PubMed Central - PubMed

ABSTRACT

In eukaryotic cells, the highly conserved Target of Rapamycin (TOR) and the Mitogen Activated Protein Kinase (MAPK) signaling pathways elicit adaptive responses to extra- and intracellular conditions by regulating essential cellular functions. However, the nature of the functional relationships between both pathways is not fully understood. In the fission yeast Schizosaccharomyces pombe the cell integrity MAPK pathway (CIP) regulates morphogenesis, cell wall structure and ionic homeostasis. We show that the Rab GTPase Ryh1, a TORC2 complex activator, cross-activates the CIP and its core member, the MAPK Pmk1, by two distinct mechanisms. The first one involves TORC2 and its downstream effector, Akt ortholog Gad8, which together with TORC1 target Psk1 increase protein levels of the PKC ortholog Pck2 during cell wall stress or glucose starvation. Also, Ryh1 activates Pmk1 in a TORC2-independent fashion by prompting plasma membrane trafficking and stabilization of upstream activators of the MAPK cascade, including PDK ortholog Ksg1 or Rho1 GEF Rgf1. Besides, stress-activated Pmk1 cross-inhibits Ryh1 signaling by decreasing the GTPase activation cycle, and this ensures cell growth during alterations in phosphoinositide metabolism. Our results reveal a highly intricate cross-regulatory relationship between both pathways that warrants adequate cell adaptation and survival in response to environmental changes.

No MeSH data available.


Related in: MedlinePlus