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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.


Related in: MedlinePlus

Tor1, Gad8, and Psk1 associate to translating ribosomes, and regulate Pck2 levels during stress independently of Rps6 phosphorylation.(A) Absorbance profile at 254 nm of a representative polysome sucrose gradient. Soluble (S), monosomal (M), and polysomal (P) fractions are indicated. (B) Ribosomes from cycloheximide-treated cultures of strains BA192 (nmt1-HA-Tor1), JW960 (Gad8-HA), AN0179 (Psk1-13myc), and AN071 (Cpc2-GFP; 40 S ribosomal protein), were purified by sedimentation through sucrose gradients. Identical volumes of representative monosomal and polysomal fractions were analyzed by Western blot with either anti-HA, anti-Myc, or anti-GFP antibodies. Anti-tubulin was used as a non-ribosomal negative control. (C) Cell extracts from strains described in (B) were incubated with 0.5 mg/ml RNAse A for 20 min before sucrose sedimentation, and monosomal and polysomal fractions analyzed as above. (D) Western blot analysis of monosomal and polysomal ribosomal fractions from cycloheximide-treated cultures of strains BV397 (tor1Δ Gad8-HA), and BV84 (tor1Δ Psk1-13myc). (E) Growing cultures of strains BV8 (Pck2-HA; control) and BV78 (rps601Δ rps602-AA Pck2-HA) were treated with 1 μg/ml Caspofungin (left panel), or shifted to the same medium without glucose (right panel). Pck2 levels were detected after incubation with anti-HA antibodies. Anti-Cdc2 was used as loading control. *P < 0.05 in mutant strain as compared to the control.
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f3: Tor1, Gad8, and Psk1 associate to translating ribosomes, and regulate Pck2 levels during stress independently of Rps6 phosphorylation.(A) Absorbance profile at 254 nm of a representative polysome sucrose gradient. Soluble (S), monosomal (M), and polysomal (P) fractions are indicated. (B) Ribosomes from cycloheximide-treated cultures of strains BA192 (nmt1-HA-Tor1), JW960 (Gad8-HA), AN0179 (Psk1-13myc), and AN071 (Cpc2-GFP; 40 S ribosomal protein), were purified by sedimentation through sucrose gradients. Identical volumes of representative monosomal and polysomal fractions were analyzed by Western blot with either anti-HA, anti-Myc, or anti-GFP antibodies. Anti-tubulin was used as a non-ribosomal negative control. (C) Cell extracts from strains described in (B) were incubated with 0.5 mg/ml RNAse A for 20 min before sucrose sedimentation, and monosomal and polysomal fractions analyzed as above. (D) Western blot analysis of monosomal and polysomal ribosomal fractions from cycloheximide-treated cultures of strains BV397 (tor1Δ Gad8-HA), and BV84 (tor1Δ Psk1-13myc). (E) Growing cultures of strains BV8 (Pck2-HA; control) and BV78 (rps601Δ rps602-AA Pck2-HA) were treated with 1 μg/ml Caspofungin (left panel), or shifted to the same medium without glucose (right panel). Pck2 levels were detected after incubation with anti-HA antibodies. Anti-Cdc2 was used as loading control. *P < 0.05 in mutant strain as compared to the control.

Mentions: In mammalian cells mTORC2 association with ribosomes is required for its activation and downstream signaling32. We performed sucrose gradient fractionation to investigate the putative in vivo interaction of Tor1, Gad8, and/or Psk1 with ribosomes. As previously described, a GFP-fused version of Cpc2, the fission yeast ortholog of mammalian RACK1 and a structural component of the 40 S ribosomal subunit33, distributed throughout the gradient within 40 S, monosome, and polysome fractions. On the contrary, tubulin (non-ribosomal protein; negative control), was detected exclusively in the soluble fraction (Fig. 3A,B). Importantly, HA-Tor1, Gad8-HA, and Psk1-13myc fusions also co-sedimented in monosomal and polysomal fractions similar to Cpc2 (Fig. 3B). In these assays a noticeable amount of Tor1 appeared to be constitutively associated to monosomes and/or polysomes, while the ribosome-bound pool of Gad8 and Psk1 was clearly lower than in the soluble fraction (Fig. 3B). When cell extracts were treated before sedimentation with RNAse A, which dissociates polyribosomes to free ribosomes, Cpc2, Tor1, Gad8, and Psk1 were mostly found in the monosome fractions (Fig. 3C), thus confirming its association to this specific organelle. Notably, the association of Gad8 and Psk1 with ribosomes was still maintained in tor1∆ cells (Fig. 3D). Therefore, in fission yeast TORC2 and its target Gad8 associate with both translating and non-translating ribosomes, and Gad8-ribosome interaction is independent of the presence of TORC2.


Multiple crosstalk between TOR and the cell integrity MAPK signaling pathway in fission yeast
Tor1, Gad8, and Psk1 associate to translating ribosomes, and regulate Pck2 levels during stress independently of Rps6 phosphorylation.(A) Absorbance profile at 254 nm of a representative polysome sucrose gradient. Soluble (S), monosomal (M), and polysomal (P) fractions are indicated. (B) Ribosomes from cycloheximide-treated cultures of strains BA192 (nmt1-HA-Tor1), JW960 (Gad8-HA), AN0179 (Psk1-13myc), and AN071 (Cpc2-GFP; 40 S ribosomal protein), were purified by sedimentation through sucrose gradients. Identical volumes of representative monosomal and polysomal fractions were analyzed by Western blot with either anti-HA, anti-Myc, or anti-GFP antibodies. Anti-tubulin was used as a non-ribosomal negative control. (C) Cell extracts from strains described in (B) were incubated with 0.5 mg/ml RNAse A for 20 min before sucrose sedimentation, and monosomal and polysomal fractions analyzed as above. (D) Western blot analysis of monosomal and polysomal ribosomal fractions from cycloheximide-treated cultures of strains BV397 (tor1Δ Gad8-HA), and BV84 (tor1Δ Psk1-13myc). (E) Growing cultures of strains BV8 (Pck2-HA; control) and BV78 (rps601Δ rps602-AA Pck2-HA) were treated with 1 μg/ml Caspofungin (left panel), or shifted to the same medium without glucose (right panel). Pck2 levels were detected after incubation with anti-HA antibodies. Anti-Cdc2 was used as loading control. *P < 0.05 in mutant strain as compared to the control.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5120329&req=5

f3: Tor1, Gad8, and Psk1 associate to translating ribosomes, and regulate Pck2 levels during stress independently of Rps6 phosphorylation.(A) Absorbance profile at 254 nm of a representative polysome sucrose gradient. Soluble (S), monosomal (M), and polysomal (P) fractions are indicated. (B) Ribosomes from cycloheximide-treated cultures of strains BA192 (nmt1-HA-Tor1), JW960 (Gad8-HA), AN0179 (Psk1-13myc), and AN071 (Cpc2-GFP; 40 S ribosomal protein), were purified by sedimentation through sucrose gradients. Identical volumes of representative monosomal and polysomal fractions were analyzed by Western blot with either anti-HA, anti-Myc, or anti-GFP antibodies. Anti-tubulin was used as a non-ribosomal negative control. (C) Cell extracts from strains described in (B) were incubated with 0.5 mg/ml RNAse A for 20 min before sucrose sedimentation, and monosomal and polysomal fractions analyzed as above. (D) Western blot analysis of monosomal and polysomal ribosomal fractions from cycloheximide-treated cultures of strains BV397 (tor1Δ Gad8-HA), and BV84 (tor1Δ Psk1-13myc). (E) Growing cultures of strains BV8 (Pck2-HA; control) and BV78 (rps601Δ rps602-AA Pck2-HA) were treated with 1 μg/ml Caspofungin (left panel), or shifted to the same medium without glucose (right panel). Pck2 levels were detected after incubation with anti-HA antibodies. Anti-Cdc2 was used as loading control. *P < 0.05 in mutant strain as compared to the control.
Mentions: In mammalian cells mTORC2 association with ribosomes is required for its activation and downstream signaling32. We performed sucrose gradient fractionation to investigate the putative in vivo interaction of Tor1, Gad8, and/or Psk1 with ribosomes. As previously described, a GFP-fused version of Cpc2, the fission yeast ortholog of mammalian RACK1 and a structural component of the 40 S ribosomal subunit33, distributed throughout the gradient within 40 S, monosome, and polysome fractions. On the contrary, tubulin (non-ribosomal protein; negative control), was detected exclusively in the soluble fraction (Fig. 3A,B). Importantly, HA-Tor1, Gad8-HA, and Psk1-13myc fusions also co-sedimented in monosomal and polysomal fractions similar to Cpc2 (Fig. 3B). In these assays a noticeable amount of Tor1 appeared to be constitutively associated to monosomes and/or polysomes, while the ribosome-bound pool of Gad8 and Psk1 was clearly lower than in the soluble fraction (Fig. 3B). When cell extracts were treated before sedimentation with RNAse A, which dissociates polyribosomes to free ribosomes, Cpc2, Tor1, Gad8, and Psk1 were mostly found in the monosome fractions (Fig. 3C), thus confirming its association to this specific organelle. Notably, the association of Gad8 and Psk1 with ribosomes was still maintained in tor1∆ cells (Fig. 3D). Therefore, in fission yeast TORC2 and its target Gad8 associate with both translating and non-translating ribosomes, and Gad8-ribosome interaction is independent of the presence of TORC2.

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