Limits...
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.


Related in: MedlinePlus

Ryh1 promotes proper plasma membrane localization and/or processing of upstream activators of the cell integrity pathway.(A) Total extracts from growing cultures of control, ryh1Δ, tor1Δ, and gad8Δ strains expressing Ksg1-HA, Rgf1-GFP, GFP-Rho1, Rho2-HA, Pck2-HA, and Pmk1-HA fusions were resolved by SDS-PAGE and the levels of the respective fusions detected by incubation with anti-HA or anti-GFP antibodies. Anti-Cdc2 was used as a loading control. (B) Quantification of Western blot experiments shown in (A). *P < 0.05; **P < 0.005; ***P < 0.001. (C) Images by fluorescence microscopy of growing control, ryh1Δ, and tor1Δ cells expressing either Rgf1-GFP, nmt1:Ksg1-GFP, GFP-Rho1, GFP-Rho2, or GFP-CRB fusions. (D) The percentage at the plasma membrane/cell tips of the GFP fusions described in (C) with respect to total cell fluorescence was determined. N > 15 cells; **P < 0.005; ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC5120329&req=5

f5: Ryh1 promotes proper plasma membrane localization and/or processing of upstream activators of the cell integrity pathway.(A) Total extracts from growing cultures of control, ryh1Δ, tor1Δ, and gad8Δ strains expressing Ksg1-HA, Rgf1-GFP, GFP-Rho1, Rho2-HA, Pck2-HA, and Pmk1-HA fusions were resolved by SDS-PAGE and the levels of the respective fusions detected by incubation with anti-HA or anti-GFP antibodies. Anti-Cdc2 was used as a loading control. (B) Quantification of Western blot experiments shown in (A). *P < 0.05; **P < 0.005; ***P < 0.001. (C) Images by fluorescence microscopy of growing control, ryh1Δ, and tor1Δ cells expressing either Rgf1-GFP, nmt1:Ksg1-GFP, GFP-Rho1, GFP-Rho2, or GFP-CRB fusions. (D) The percentage at the plasma membrane/cell tips of the GFP fusions described in (C) with respect to total cell fluorescence was determined. N > 15 cells; **P < 0.005; ***P < 0.001.

Mentions: Ryh1 and orthologs like mammalian Rab6 or budding yeast Ypt6 are major regulators of Golgi membrane trafficking in eukaryotic cells23. The main upstream activators of the CIP, PDK ortholog Ksg1, Rho1 GEF Rgf1, and GTPases Rho1 and Rho2, activate Pmk1 via Pck2 during growth and in response to stress273637. Importantly, they are mostly targeted to the plasma membrane and/or cell poles3637. We therefore hypothesized that Ryh1 might regulate the activity of the CIP independently of TORC2 signaling by promoting an adequate processing and/or trafficking of one or several upstream regulators of this signaling cascade. Indeed, protein levels of Ksg1were very low in ryh1∆ cells as compared to control cells (Fig. 5A,B). tor1∆ and gad8∆ cells also showed some decrease in Ksg1 levels, but this defect was not as evident as in the ryh1∆ mutant (Fig. 5A,B). Protein levels of Rgf1were also strongly down-regulated in ryh1∆ cells, but remained unchanged in tor1∆ and gad8∆ cells (Fig. 5A,B).


Multiple crosstalk between TOR and the cell integrity MAPK signaling pathway in fission yeast
Ryh1 promotes proper plasma membrane localization and/or processing of upstream activators of the cell integrity pathway.(A) Total extracts from growing cultures of control, ryh1Δ, tor1Δ, and gad8Δ strains expressing Ksg1-HA, Rgf1-GFP, GFP-Rho1, Rho2-HA, Pck2-HA, and Pmk1-HA fusions were resolved by SDS-PAGE and the levels of the respective fusions detected by incubation with anti-HA or anti-GFP antibodies. Anti-Cdc2 was used as a loading control. (B) Quantification of Western blot experiments shown in (A). *P < 0.05; **P < 0.005; ***P < 0.001. (C) Images by fluorescence microscopy of growing control, ryh1Δ, and tor1Δ cells expressing either Rgf1-GFP, nmt1:Ksg1-GFP, GFP-Rho1, GFP-Rho2, or GFP-CRB fusions. (D) The percentage at the plasma membrane/cell tips of the GFP fusions described in (C) with respect to total cell fluorescence was determined. N > 15 cells; **P < 0.005; ***P < 0.001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f5: Ryh1 promotes proper plasma membrane localization and/or processing of upstream activators of the cell integrity pathway.(A) Total extracts from growing cultures of control, ryh1Δ, tor1Δ, and gad8Δ strains expressing Ksg1-HA, Rgf1-GFP, GFP-Rho1, Rho2-HA, Pck2-HA, and Pmk1-HA fusions were resolved by SDS-PAGE and the levels of the respective fusions detected by incubation with anti-HA or anti-GFP antibodies. Anti-Cdc2 was used as a loading control. (B) Quantification of Western blot experiments shown in (A). *P < 0.05; **P < 0.005; ***P < 0.001. (C) Images by fluorescence microscopy of growing control, ryh1Δ, and tor1Δ cells expressing either Rgf1-GFP, nmt1:Ksg1-GFP, GFP-Rho1, GFP-Rho2, or GFP-CRB fusions. (D) The percentage at the plasma membrane/cell tips of the GFP fusions described in (C) with respect to total cell fluorescence was determined. N > 15 cells; **P < 0.005; ***P < 0.001.
Mentions: Ryh1 and orthologs like mammalian Rab6 or budding yeast Ypt6 are major regulators of Golgi membrane trafficking in eukaryotic cells23. The main upstream activators of the CIP, PDK ortholog Ksg1, Rho1 GEF Rgf1, and GTPases Rho1 and Rho2, activate Pmk1 via Pck2 during growth and in response to stress273637. Importantly, they are mostly targeted to the plasma membrane and/or cell poles3637. We therefore hypothesized that Ryh1 might regulate the activity of the CIP independently of TORC2 signaling by promoting an adequate processing and/or trafficking of one or several upstream regulators of this signaling cascade. Indeed, protein levels of Ksg1were very low in ryh1∆ cells as compared to control cells (Fig. 5A,B). tor1∆ and gad8∆ cells also showed some decrease in Ksg1 levels, but this defect was not as evident as in the ryh1∆ mutant (Fig. 5A,B). Protein levels of Rgf1were also strongly down-regulated in ryh1∆ cells, but remained unchanged in tor1∆ and gad8∆ cells (Fig. 5A,B).

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