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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.

No MeSH data available.


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Gad8 regulates Pck2 levels and Pmk1 activation during growth, cell wall stress and glucose deprivation.Upper panels. Growing cultures of strains MM913 (Pck2-HA; control), MM1205 (tor1Δ Pck2-HA), and BV11 (gad8Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 1 μg/ml Caspofungin (left panel), or starved for glucose (right panel). Cell extracts were resolved by SDS-PAGE and Pck2 levels detected with anti-HA antibodies. Anti-Cdc2 was used as loading control. Lower panels. Pmk1-HA6H fusion was purified by affinity chromatography, and activated/total Pmk1 detected with anti-phospho-p44/42 and anti-HA antibodies, respectively. *P < 0.05; **P < 0.005; ***P < 0.001.
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f1: Gad8 regulates Pck2 levels and Pmk1 activation during growth, cell wall stress and glucose deprivation.Upper panels. Growing cultures of strains MM913 (Pck2-HA; control), MM1205 (tor1Δ Pck2-HA), and BV11 (gad8Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 1 μg/ml Caspofungin (left panel), or starved for glucose (right panel). Cell extracts were resolved by SDS-PAGE and Pck2 levels detected with anti-HA antibodies. Anti-Cdc2 was used as loading control. Lower panels. Pmk1-HA6H fusion was purified by affinity chromatography, and activated/total Pmk1 detected with anti-phospho-p44/42 and anti-HA antibodies, respectively. *P < 0.05; **P < 0.005; ***P < 0.001.

Mentions: The fission yeast TORC2 complex does not participate in catalytic activation of Pck2. Nevertheless, this complex contributes to de novo Pck2 synthesis, which is essential to activate the CIP in response to cell wall damage or glucose exhaustion, but not to other stimuli like saline stress29. To identify the main components of this novel regulatory mechanism, we analyzed Pck2 levels and Pmk1 activation in a series of mutants lacking upstream regulators or downstream effectors of the TORC2 signaling cascade. As expected, Pck2 levels were strongly reduced in tor1∆ cells during growth and under cell wall stress (Caspofungin) or glucose deprivation as compared to control cells, resulting in a delayed or lowered Pmk1 activation, respectively (Fig. 1). Notably, similar to tor1∆ cells, Pck2 levels decreased in growing and stressed cells lacking the AGC-kinase family member Gad8, the major target for TORC2 (Fig. 1)20. However, defective Pck2 levels in gad8∆ cells were less severe than in the tor1∆ mutant, although showed a similar defect in Pmk1 activation (Fig. 1). Hence, while both basal and increased Pck2 levels in response to the above stimuli is mostly dependent on the Tor1-Gad8 branch, Gad8 does not appear to be the sole Tor1-target controlling this response.


Multiple crosstalk between TOR and the cell integrity MAPK signaling pathway in fission yeast
Gad8 regulates Pck2 levels and Pmk1 activation during growth, cell wall stress and glucose deprivation.Upper panels. Growing cultures of strains MM913 (Pck2-HA; control), MM1205 (tor1Δ Pck2-HA), and BV11 (gad8Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 1 μg/ml Caspofungin (left panel), or starved for glucose (right panel). Cell extracts were resolved by SDS-PAGE and Pck2 levels detected with anti-HA antibodies. Anti-Cdc2 was used as loading control. Lower panels. Pmk1-HA6H fusion was purified by affinity chromatography, and activated/total Pmk1 detected with anti-phospho-p44/42 and anti-HA antibodies, respectively. *P < 0.05; **P < 0.005; ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5120329&req=5

f1: Gad8 regulates Pck2 levels and Pmk1 activation during growth, cell wall stress and glucose deprivation.Upper panels. Growing cultures of strains MM913 (Pck2-HA; control), MM1205 (tor1Δ Pck2-HA), and BV11 (gad8Δ Pck2-HA) expressing genomic Pmk1-HA6H fusions were treated with 1 μg/ml Caspofungin (left panel), or starved for glucose (right panel). Cell extracts were resolved by SDS-PAGE and Pck2 levels detected with anti-HA antibodies. Anti-Cdc2 was used as loading control. Lower panels. Pmk1-HA6H fusion was purified by affinity chromatography, and activated/total Pmk1 detected with anti-phospho-p44/42 and anti-HA antibodies, respectively. *P < 0.05; **P < 0.005; ***P < 0.001.
Mentions: The fission yeast TORC2 complex does not participate in catalytic activation of Pck2. Nevertheless, this complex contributes to de novo Pck2 synthesis, which is essential to activate the CIP in response to cell wall damage or glucose exhaustion, but not to other stimuli like saline stress29. To identify the main components of this novel regulatory mechanism, we analyzed Pck2 levels and Pmk1 activation in a series of mutants lacking upstream regulators or downstream effectors of the TORC2 signaling cascade. As expected, Pck2 levels were strongly reduced in tor1∆ cells during growth and under cell wall stress (Caspofungin) or glucose deprivation as compared to control cells, resulting in a delayed or lowered Pmk1 activation, respectively (Fig. 1). Notably, similar to tor1∆ cells, Pck2 levels decreased in growing and stressed cells lacking the AGC-kinase family member Gad8, the major target for TORC2 (Fig. 1)20. However, defective Pck2 levels in gad8∆ cells were less severe than in the tor1∆ mutant, although showed a similar defect in Pmk1 activation (Fig. 1). Hence, while both basal and increased Pck2 levels in response to the above stimuli is mostly dependent on the Tor1-Gad8 branch, Gad8 does not appear to be the sole Tor1-target controlling this response.

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