The double-stranded RNA sensor kinase PKR is one of four integrated stress response (ISR) sensor kinases that phosphorylate the α subunit of eukaryotic initiation factor 2 (eIF2α) in response to stress. The current model of PKR activation considers the formation of back-to-back PKR dimers as a prerequisite for signal propagation. Here we show that PKR signaling involves the assembly of dynamic PKR clusters. PKR clustering is driven by ligand binding to PKR’s sensor domain and by front-to-front interfaces between PKR’s kinase domains. PKR clusters are discrete, heterogeneous, autonomous coalescences that share some protein components with processing bodies. Strikingly, eIF2α is not recruited to PKR clusters, and PKR cluster disruption enhances eIF2α phosphorylation. Together, these results support a model in which PKR clustering may limit encounters between PKR and eIF2α to buffer downstream signaling and prevent the ISR from misfiring.

Rapamycin-insensitive TORC2 signaling promotes amino acid starvation–induced autophagy through inhibition of the calcium-dependent phosphatase calcineurin. Vlahakis et al. show that this regulation requires the ER calcium channel regulatory protein Mid1, which couples mitochondrial ROS accumulation in TORC2 mutants to calcineurin activation and autophagy repression.

Macroautophagy/autophagy is a starvation and stress-induced catabolic process critical for cellular homeostasis and adaptation. Several Atg proteins are involved in the formation of the autophagosome and subsequent degradation of cytoplasmic components, a process termed autophagy flux. Additionally, the expression of several Atg proteins, in particular Atg8, is modulated transcriptionally, yet the regulatory mechanisms involved remain poorly understood. Here we demonstrate that the AGC kinase Ypk1, target of the rapamycin-insensitive TORC2 signaling pathway, controls ATG8 expression by repressing the heterodimeric Zinc-finger transcription factors Msn2 and Msn4. We find that Msn2 and Msn4 promote ATG8 expression downstream of the histone deacetylase complex (HDAC) subunit Ume6, a previously identified negative regulator of ATG8 expression. Moreover, we demonstrate that TORC2-Ypk1 signaling is functionally linked to distinct mitochondrial respiratory complexes. Surprisingly, we find that autophagy flux during amino acid starvation is also dependent upon Msn2-Msn4 activity, revealing a broad role for these transcription factors in the autophagy response.

The target of rapamycin (TOR) kinase is a conserved regulator of cell growth and functions within 2 different protein complexes, TORC1 and TORC2, where TORC2 positively controls macroautophagy/autophagy during amino acid starvation. Under these conditions, TORC2 signaling inhibits the activity of the calcium-regulated phosphatase calcineurin and promotes the general amino acid control (GAAC) response and autophagy. Here we demonstrate that TORC2 regulates calcineurin by controlling the respiratory activity of mitochondria. In particular, we find that mitochondrial oxidative stress affects the calcium channel regulatory protein Mid1, which we show is an essential upstream activator of calcineurin. Thus, these findings describe a novel regulation for autophagy that involves TORC2 signaling, mitochondrial respiration, and calcium homeostasis.

The double-stranded RNA sensor kinase PKR is one of four integrated stress response (ISR) sensor kinases that phosphorylate the alpha subunit of the eukaryotic initiation factor 2 (eIF2α) in response to stress. The current model of PKR activation considers the formation of back-to-back PKR dimers as a prerequisite for signal propagation. Here we show that PKR signaling involves the assembly of dynamic PKR clusters. PKR clustering is driven by ligand binding to PKR’s sensor domain and by front-to-front interfaces between PKR’s kinase domains. PKR clusters are discrete, heterogeneous, autonomous coalescences that share some protein components with processing bodies. Strikingly, eIF2α is not recruited to PKR clusters, and PKR cluster disruption enhances eIF2α phosphorylation. Together, these results support a model in which PKR clustering buffers downstream signaling, which may enable proofreading the ISR.