The Tor and PKA signaling pathways independently target the Atg1/Atg13 protein kinase complex to control autophagy. Proc Natl Acad Sci USA

Department of Molecular Genetics, The Ohio State University, Columbus, OH 43210, USA.
Proceedings of the National Academy of Sciences (Impact Factor: 9.67). 10/2009; 106(40):17049-54. DOI: 10.1073/pnas.0903316106
Source: PubMed


Macroautophagy (or autophagy) is a conserved degradative pathway that has been implicated in a number of biological processes, including organismal aging, innate immunity, and the progression of human cancers. This pathway was initially identified as a cellular response to nutrient deprivation and is essential for cell survival during these periods of starvation. Autophagy is highly regulated and is under the control of a number of signaling pathways, including the Tor pathway, that coordinate cell growth with nutrient availability. These pathways appear to target a complex of proteins that contains the Atg1 protein kinase. The data here show that autophagy in Saccharomyces cerevisiae is also controlled by the cAMP-dependent protein kinase (PKA) pathway. Elevated levels of PKA activity inhibited autophagy and inactivation of the PKA pathway was sufficient to induce a robust autophagy response. We show that in addition to Atg1, PKA directly phosphorylates Atg13, a conserved regulator of Atg1 kinase activity. This phosphorylation regulates Atg13 localization to the preautophagosomal structure, the nucleation site from which autophagy pathway transport intermediates are formed. Atg13 is also phosphorylated in a Tor-dependent manner, but these modifications appear to occur at positions distinct from the PKA phosphorylation sites identified here. In all, our data indicate that the PKA and Tor pathways function independently to control autophagy in S. cerevisiae, and that the Atg1/Atg13 kinase complex is a key site of signal integration within this degradative pathway.

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Available from: Joe Stephan, Nov 18, 2015
    • "Moreover, it has been shown that Rsp5 can be phosphorylated in vitro by Tpk1 and several other kinases (Ptacek et al., 2005). PKAdependent signaling is important for the regulation of growth in response to glucose, in the regulation of mitochondrial functions and autophagy (Graef and Nunnari, 2011; Kim et al., 2013; Leadsham et al., 2009; Stephan et al., 2009). PKA is also implicated in stress response, since the transcription factor Skn7, which has a unique role in oxidative stress response, is negatively regulated by PKA (Charizanis et al., 1999). "
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    ABSTRACT: Rsp5 ubiquitin ligase belongs to the Nedd4 family of proteins, which affect a wide variety of processes in the cell. Here we document that Rsp5 shows several phosphorylated variants of different mobility and the migration of the phosphorylated forms of Rsp5 was faster for the tpk1Δ tpk3Δ mutant devoid of two alternative catalytic subunits of protein kinase A (PKA), indicating that PKA possibly phosphorylates Rsp5 in vivo. We demonstrated by immunoprecipitation and Western blot analysis of GFP-HA-Rsp5 protein using the anti-phospho PKA substrate antibody that Rsp5 is phosphorylated in PKA sites. Rsp5 contains the sequence 758-RRFTIE-763 with consensus RRXS/T in the catalytic HECT domain and four other sites with consensus RXXS/T, which might be phosphorylated by PKA. The strain bearing the T761D substitution in Rsp5 which mimics phosphorylation grew more slowly at 28°C and did not grow at 37°C, and showed defects in pre-tRNA processing and protein sorting. The rsp5-T761D strain also demonstrated a reduced ability to form colonies, an increase in the level of reactive oxygen species (ROS) and hypersensitivity to ROS-generating agents. These results indicate that PKA may downregulate many functions of Rsp5, possibly affecting its activity. Rsp5 is found in the cytoplasm, nucleus, multivesicular body and cortical patches. The rsp5-T761D mutation led to a strongly increased cortical localization while rsp5-T761A caused mutant Rsp5 to locate more efficiently in internal spots. Rsp5-T761A protein was phosphorylated less efficiently in PKA sites under specific growth conditions. Our data suggests that Rsp5 may be phosphorylated by PKA at position T761 and that this regulation is important for its localization and function.
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    • "Consequently, in addition to p62 and the ubiquitination machinery, cAMP signalling could be misregulated in these cases, leading to hyperphosphorylation of p62 at Ser24, suppression of homopolymerisation and hence a reduction in autophagy . Interestingly, recent data from the yeast S. cerevisiae shows that increased cAMP/PKA activity blocks autophagy [39], and that inactivation of the cAMP pathway induces autophagy [40]. However, yeast do not express p62 or homologues thereof, and the autophagyregulating protein Atg13 was identified as the responsible PKA substrate in this case. "
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    ABSTRACT: p62, also known as SQSTM1, is a multi-domain signalling scaffold protein involved in numerous critical cellular functions such as autophagy, apoptosis and inflammation. Crucial interactions relevant to these functions are mediated by the N-terminal Phox and Bem1p (PB1) domain, which is divided into two interaction surfaces, one of predominantly acidic and one of basic character. Most known interaction partners, including atypical protein kinase C (aPKC), bind to the basic surface, and acidic-basic interactions at this interface also allow for p62 homopolymerisation. We identify here that the coupling of p62 to the cAMP signalling system is conferred by both the direct binding of cAMP degrading phosphodiesterase-4 (PDE4) to the acidic surface of the p62 PB1 domain and the phosphorylation of the basic surface of this domain by cAMP-dependent protein kinase (PKA). Such phosphorylation is a previously unknown means of regulating PB1 domain interaction partnerships by disrupting the interaction of p62 with basic surface binding partners, such as aPKCs, as well as p62 homopolymerisation. Thus, we uncover a new regulatory mechanism that connects cAMP signalling with the p62 multi-domain signalling scaffold and autophagy cargo receptor protein.
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    • "The inhibition of MTOR signaling by glucagon was later shown to proceed through a mechanism dependent on protein kinase A ( Kimball et al . 2004 ) , which stimulates autophagy ( Mavrakis et al . 2006 ) . It must be pointed out that , in contrast to mammalian cells , activation of protein kinase A in yeast cells inhibits autophagy ( Stephan et al . 2009 ) . In this context , it is important to stress that the function of cAMP in yeast , in contrast to that in mamma - lian cells , is to stimulate cell growth ( Thevelein and de Winde 1999 ) ."
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    ABSTRACT: Amino acids not only participate in intermediary metabolism but also stimulate insulin-mechanistic target of rapamycin (MTOR)-mediated signal transduction which controls the major metabolic pathways. Among these is the pathway of autophagy which takes care of the degradation of long-lived proteins and of the elimination of damaged or functionally redundant organelles. Proper functioning of this process is essential for cell survival. Dysregulation of autophagy has been implicated in the etiology of several pathologies. The history of the studies on the interrelationship between amino acids, MTOR signaling and autophagy is the subject of this review. The mechanisms responsible for the stimulation of MTOR-mediated signaling, and the inhibition of autophagy, by amino acids have been studied intensively in the past but are still not completely clarified. Recent developments in this field are discussed.
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