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. 2007 Oct;18(10):4180-9.
doi: 10.1091/mbc.e07-05-0485. Epub 2007 Aug 15.

Protein kinase A and Sch9 cooperatively regulate induction of autophagy in Saccharomyces cerevisiae

Tomohiro Yorimitsu  1 , Shadia Zaman, James R Broach, Daniel J Klionsky
Affiliations

Protein kinase A and Sch9 cooperatively regulate induction of autophagy in Saccharomyces cerevisiae

Tomohiro Yorimitsu et al. Mol Biol Cell. 2007 Oct.

Abstract

Autophagy is a highly conserved, degradative process in eukaryotic cells. The rapamycin-sensitive Tor kinase complex 1 (TORC1) has a major role in regulating induction of autophagy; however, the regulatory mechanisms are not fully understood. Here, we find that the protein kinase A (PKA) and Sch9 signaling pathways regulate autophagy cooperatively in yeast. Autophagy is induced in cells when PKA and Sch9 are simultaneously inactivated. Mutant alleles of these kinases bearing a mutation that confers sensitivity to the ATP-analogue inhibitor C3-1'-naphthyl-methyl PP1 revealed that autophagy was induced independently of effects on Tor kinase. The PKA-Sch9-mediated autophagy depends on the autophagy-related 1 kinase complex, which is also essential for TORC1-regulated autophagy, the transcription factors Msn2/4, and the Rim15 kinase. The present results suggest that autophagy is controlled by the signals from at least three partly separate nutrient-sensing pathways that include PKA, Sch9, and TORC1.

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Figures

Figure 1.
Figure 1.
Inactivation of PKA and Sch9 induces autophagy. (A) PKA-Sch9 inactivation stimulates Atg8 expression and lipidation. Wild-type (W303-1B), pka (Y3527), sch9 (Y3507), and pka sch9 (a-9; Y3528) cells were grown for 6 h in SMD with or without rapamycin or 1NM-PP1. Proteins were precipitated with TCA and resolved by SDS-PAGE followed by immunoblotting with anti-Ape1 and anti-Pgk1 antiserum (as a loading control). Atg8 and Atg8 conjugated with phosphatidylethanolamine (Atg8–PE) were separated by 12% SDS-PAGE in the presence of 6 M urea followed by immunoblotting with anti-Atg8 or anti-Pgk1 antiserum. (B) GFP-Atg8 processing is enhanced by inactivation of PKA and Sch9. Protein extracts from wild-type, pka, sch9, pka sch9 (a-9), atg1Δ, and atg1Δ pka sch9 (TYY167) cells expressing GFP-Atg8 were analyzed as described in A by using anti-GFP antibodies. (C) Kinetics of GFP-Atg8 processing. Wild-type and pka sch9 cells expressing GFP-Atg8 grown to early log-phase were incubated in SMD containing rapamycin or 1NM-PP1. At the indicated times, TCA-precipitated proteins were subjected to immunoblotting, as described in B.
Figure 2.
Figure 2.
Nonspecific and specific autophagy are induced with inactivation of PKA and Sch9. (A) Wild-type (TYY172), pka (TYY173), sch9 (TYY187), pka sch9 (TYY174) atg1Δ (TYY181), and atg1Δ pka sch9 (TYY182) cells expressing Pho8Δ60, a marker for nonspecific autophagy, were grown for 6 h in SMD with or without rapamycin or 1NM-PP1. The Pho8Δ60 activity was measured as described in Materials and Methods, and it was normalized to the activity of the wild-type cells with rapamycin treatment, which was set to 100%. Error bars indicate the SD of at least three independent experiments. (B) Protein extracts from vac8Δ (TYY175), vac8Δ pka (TYY176), vac8Δ sch9 (TYY177), vac8Δ pka sch9 (a-9; TYY178) vac8Δ atg1Δ (TYY179), and vac8Δ atg1Δ pka sch9 (TYY180) cells were analyzed by immunoblotting, as described in Figure 1, by using antiserum to Ape1.
Figure 3.
Figure 3.
Atg1 kinase activity is required for PKA-Sch9 regulation of autophagy. (A) Inactivation of PKA and Sch9 does not induce autophagy without the Atg1–Atg13–Atg17 kinase complex. Protein extracts from wild-type and pka pkb strains, and these strains harboring deletions in ATG1, ATG13, or ATG17 and expressing GFP-Atg8 were subjected to immunoblotting, as described in Figure 1. An Atg17 mutant defective in association with Atg13 (B) and a kinase-defective Atg1 mutant (C) block autophagy when PKA and Sch9 are inactivated. Protein extracts from the atg17Δ pka sch9 (TYY191) (B) or atg1Δ pka sch9 (TYY167) (C) cells harboring the empty vector, and a plasmid expressing wild-type Atg17 or the Atg17C24R mutant (B) or wild-type Atg1 or the Atg1K54A mutant (C) were subjected to immunoblotting, as described in A. (D) atg1Δ sch9 (TYY166) and atg1Δ pka sch9 cells harboring the empty vector, or a plasmid expressing wild-type Atg1 or the Atg1S508,515A mutant (AA) were grown and TCA-precipitated proteins were subjected to immunoblotting, as described in A.
Figure 4.
Figure 4.
Tor kinase acts in parallel with PKA and Sch9. (A) Gln3 is phosphorylated when PKA and Sch9 are inactivated. Wild-type (TYY201), and pka sch9 (a-9; TYY202) cells expressing Gln3-myc were grown for 2 h in SMD with or without rapamycin or 1NM-PP1. TCA-precipitated proteins were subjected to immunoblotting with anti-myc antibody, as described in Figure 1. (B) Inactivation of PKA and Sch9 does not induce dephosphorylation of Atg13. Wild-type (W303-1B) and pka sch9 (a-9; Y3528) cells harboring a plasmid containing ATG13 (YEp351[APG13]) were grown for 2 h in SMD with or without rapamycin or 1NM-PP1. TCA-precipitated proteins were subjected to immunoblotting with anti-Atg13 antiserum, as described in A. (C) Inactivation of PKA and Sch9 partially affects dephosphorylation of Atg13 that occurs in −N conditions. Wild-type and pka sch9 (a-9) cells harboring the Atg13 plasmid were grown for 2 h in SMD with or without 1NM-PP1, shifted to SD-N for 30 min, and then YNB, amino acids, and vitamins were added to cultures to the same concentrations as those in SMD. At each time point, TCA-precipitated proteins were subjected to immunoblotting, as described in B. (D) Nonspecific autophagy is elevated with inactivation of both the Tor and PKA-Sch9 signaling pathways. Wild-type (TYY172) and pka sch9 (TYY174) cells expressing Pho8Δ60 were grown for 4 h in SMD with or without either or both rapamycin and 1NM-PP1 as indicated. The Pho8Δ60 activity was measured as described in Materials and Methods, and it was normalized to the activity of the cells treated with rapamycin, which was set at 100%. Error bars indicate the SD of at least three independent experiments. (E) pka sch9 (Y3528) cells expressing GFP-Atg8 grown at early log phase were incubated in SMD containing rapamycin and/or 1NM-PP1. At the indicated times, TCA-precipitated proteins were subjected to immunoblotting, as described in Figure 1.
Figure 5.
Figure 5.
Msn2/4 and Rim15 are involved in autophagic flux, but not induction, resulting from inactivation of PKA and Sch9, but not from inactivation of the Tor signaling pathway. (A) Autophagic flux assessed by GFP-Atg8 processing was defective in the absence of Msn2/4 and/or Rim15. Protein extracts from pka sch9 (Y3528), msn2/4Δ pka sch9 (TYY193), rim15Δ pka sch9 (TYY197), and msn2/4Δ rim15Δ pka sch9 (TYY199) cells expressing GFP-Atg8 were subjected to immunoblotting, as in described in Figure 1. (B) Depletion of Msn2/4 and Rim15 does not affect enhancement of Atg8 expression or lipidation resulting from inactivation of PKA and Sch9. The cells were grown, and TCA-precipitated proteins were subjected to immunoblotting, as described in A.
Figure 6.
Figure 6.
Constitutively active PKA and Sch9 suppress autophagy. (A) Constitutively active PKA suppresses autophagy. Wild-type (W303-1B), atg1Δ (TYY164), and bcy1Δ (TYY220) cells expressing GFP-Atg8 were grown for 4 h in SMD with or without rapamycin, or in SD-N. TCA-precipitated proteins were subjected to immunoblotting, as described in Figure 1. (B) A hyperactive Sch9 mutant delays processing of GFP-Atg8 by rapamycin treatment, but not under starvation conditions. Wild-type cells expressing GFP-Atg8 with a plasmid carrying wild type (WT) or hyperactive mutant Sch9 (DE) were grown in SMD containing rapamycin, or in SD-N. At the indicated times, TCA-precipitated proteins were subjected to immunoblotting, as described in A. (C) Band intensities of GFP-Atg8 and free GFP were quantified and normalized to those of wild-type cells treated with rapamycin or under starvation conditions for 6 h. Error bars indicate the SD of at least three independent experiments. DE, hyperactive Sch9. (D) The hyperactive Sch9 mutant blocks bulk autophagy by rapamycin treatment, but not under starvation conditions. Wild-type (TYY172) cells with a plasmid expressing WT or DE, or atg1Δ (TYY181) cells with the empty vector were grown for 4 h in SMD containing rapamycin, or in SD-N. The Pho8Δ60 activity was measured as described in Materials and Methods and normalized to the activity of the wild-type cells, which was set at 100%. Error bars indicate the SD of at least three independent experiments. (E) Hyperactive Sch9 does not affect dephosphorylation of Atg13 in response to rapamycin or nitrogen starvation. Wild-type (W303-1B) cells harboring the Atg13 plasmid along with a plasmid carrying WT or DE Sch9 were grown for 30 min in SMD with or without rapamycin, or in SD-N. TCA-precipitated proteins were subjected to immunoblotting, as described in Figure 4.
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