Showing papers on "Protein kinase complex published in 2019"

Molecular logic of mTORC1 signalling as a metabolic rheostat

Abstract: The protein kinase complex mechanistic target of rapamycin complex 1 (mTORC1) serves as a key conduit between growth signals and the metabolic processes underlying cell growth. The activation state of mTORC1 is controlled by intracellular nutrients and energy, as well as exogenous hormones and growth factors, thereby integrating local and systemic growth signals. Here we discuss the molecular logic of the mTORC1 signalling network and its importance in coupling growth signals to the control of cellular metabolism. After activation, mTORC1 promotes the conversion of available nutrients and energy into the major macromolecular species contributing to cellular mass, including proteins, nucleic acids and lipids, while suppressing the autophagic recycling of these macromolecules back into their nutrient constituents. Given that uncoupling of mTORC1 from its normal regulatory inputs contributes to many diseases-including cancer, genetic tumour syndromes, metabolic diseases, autoimmune diseases and neurological disorders-understanding the molecular logic of the mTORC1 network and how to modulate it may present therapeutic opportunities for treatment of a broad range of diseases and potentially even for the extension of lifespan.

Members of the autophagy class III phosphatidylinositol 3-kinase complex I interact with GABARAP and GABARAPL1 via LIR motifs.

Abstract: Autophagosome formation depends on a carefully orchestrated interplay between membrane-associated protein complexes. Initiation of macroautophagy/autophagy is mediated by the ULK1 (unc-51 like autophagy activating kinase 1) protein kinase complex and the autophagy-specific class III phosphatidylinositol 3-kinase complex I (PtdIns3K-C1). The latter contains PIK3C3/VPS34, PIK3R4/VPS15, BECN1/Beclin 1 and ATG14 and phosphorylates phosphatidylinositol to generate phosphatidylinositol 3-phosphate (PtdIns3P). Here, we show that PIK3C3, BECN1 and ATG14 contain functional LIR motifs and interact with the Atg8-family proteins with a preference for GABARAP and GABARAPL1. High resolution crystal structures of the functional LIR motifs of these core components of PtdIns3K-C1were obtained. Variation in hydrophobic pocket 2 (HP2) may explain the specificity for the GABARAP family. Mutation of the LIR motif in ATG14 did not prevent formation of the PtdIns3K-C1 complex, but blocked colocalization with MAP1LC3B/LC3B and impaired mitophagy. The ULK-mediated phosphorylation of S29 in ATG14 was strongly dependent on a functional LIR motif in ATG14. GABARAP-preferring LIR motifs in PIK3C3, BECN1 and ATG14 may, via coincidence detection, contribute to scaffolding of PtdIns3K-C1 on membranes for efficient autophagosome formation. Abbreviations: ATG: autophagy-related; BafA1: bafilomycin A1; GABARAP: GABA type A receptor-associated protein; GABARAPL1: GABA type A receptor associated protein like 1; GFP: enhanced green fluorescent protein; KO: knockout; LDS: LIR docking site; LIR: LC3-interacting region; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PIK3R4: phosphoinositide-3-kinase regulatory subunit 4; PtdIns3K: phosphatidylinositol 3-kinase; PtdIns3P: phosphatidylinositol-3-phosphate; SQSTM1/p62: sequestosome 1; VPS: Vacuolar protein sorting; ULK: unc-51 like autophagy activating kinase.

Rab5 GTPases are required for optimal TORC2 function

Abstract: Target of rapamycin complex-2 (TORC2), a conserved protein kinase complex, is an indispensable regulator of plasma membrane homeostasis. In budding yeast (Saccharomyces cerevisiae), the essential downstream effector of TORC2 is protein kinase Ypk1 and its paralog Ypk2. Muk1, a Rab5-specific guanine nucleotide exchange factor (GEF), was identified in our prior global screen for candidate Ypk1 targets. We confirm here that Muk1 is a substrate of Ypk1 and demonstrate that Ypk1-mediated phosphorylation stimulates Muk1 function in vivo. Strikingly, yeast lacking its two Rab5 GEFs (Muk1 and Vps9) or its three Rab5 paralogs (Vps21/Ypt51, Ypt52, and Ypt53) or overexpressing Msb3, a Rab5-directed GTPase-activating protein, all exhibit pronounced reduction in TORC2-mediated phosphorylation and activation of Ypk1. Vps21 coimmunoprecipitates with TORC2, and immuno-enriched TORC2 is less active in vitro in the absence of Rab5 GTPases. Thus, TORC2-dependent and Ypk1-mediated activation of Muk1 provides a control circuit for positive (self-reinforcing) up-regulation to sustain TORC2-Ypk1 signaling.

Modulation of TOR complex 2 signaling by the stress-activated MAPK pathway in fission yeast.

Abstract: Sin1 is a substrate-binding subunit of target of rapamycin complex 2 (TORC2), an evolutionarily conserved protein kinase complex. In fission yeast, Sin1 has also been identified as a protein that interacts with Spc1 (also known as Sty1) in the stress-activated protein kinase (SAPK) pathway. Therefore, this study examined the relationship between TORC2 and Spc1 signaling. We found that the common docking (CD) domain of Spc1 interacts with a cluster of basic amino acid residues in Sin1. Although diminished TORC2 activity in the absence of the functional Spc1 cascade suggests positive regulation of TORC2 by Spc1, such regulation appears to be independent of the Sin1-Spc1 interaction. Hyperosmotic stress transiently inhibits TORC2, and its swift recovery is dependent on Spc1, the transcription factor Atf1, and the glycelrol-3-phosphate dehydrogenase Gpd1, whose expression is induced upon osmostress by the Spc1-Atf1 pathway. Thus, cellular adaptation to osmostress seems important for TORC2 reactivation, though Spc1 and Atf1 contribute to TORC2 activation also in the absence of osmostress. These results indicate coordinated actions of the SAPK and TORC2 pathways, both of which are essential for fission yeast cells to survive environmental stress.

Nutrient Signaling via the TORC1-Greatwall-PP2A B55δ Pathway Is Responsible for the High Initial Rates of Alcoholic Fermentation in Sake Yeast Strains of Saccharomyces cerevisiae.

Abstract: Saccharomyces cerevisiae sake yeast strain Kyokai no. 7 (K7) and its relatives carry a homozygous loss-of-function mutation in the RIM15 gene, which encodes a Greatwall family protein kinase. Disruption of RIM15 in nonsake yeast strains leads to improved alcoholic fermentation, indicating that the defect in Rim15p is associated with the enhanced fermentation performance of sake yeast cells. In order to understand how Rim15p mediates fermentation control, we here focused on target-of-rapamycin protein kinase complex 1 (TORC1) and protein phosphatase 2A with the B55δ regulatory subunit (PP2AB55δ), complexes that are known to act upstream and downstream of Rim15p, respectively. Several lines of evidence, including our previous transcriptomic analysis data, suggested enhanced TORC1 signaling in sake yeast cells during sake fermentation. Fermentation tests of the TORC1-related mutants using a laboratory strain revealed that TORC1 signaling positively regulates the initial fermentation rate in a Rim15p-dependent manner. Deletion of the CDC55 gene, encoding B55δ, abolished the high fermentation performance of Rim15p-deficient laboratory yeast and sake yeast cells, indicating that PP2AB55δ mediates the fermentation control by TORC1 and Rim15p. The TORC1-Greatwall-PP2AB55δ pathway similarly affected the fermentation rate in the fission yeast Schizosaccharomyces pombe, strongly suggesting that the evolutionarily conserved pathway governs alcoholic fermentation in yeasts. It is likely that elevated PP2AB55δ activity accounts for the high fermentation performance of sake yeast cells. Heterozygous loss-of-function mutations in CDC55 found in K7-related sake strains may indicate that the Rim15p-deficient phenotypes are disadvantageous to cell survival.IMPORTANCE The biochemical processes and enzymes responsible for glycolysis and alcoholic fermentation by the yeast S. cerevisiae have long been the subject of scientific research. Nevertheless, the factors determining fermentation performance in vivo are not fully understood. As a result, the industrial breeding of yeast strains has required empirical characterization of fermentation by screening numerous mutants through laborious fermentation tests. To establish a rational and efficient breeding strategy, key regulators of alcoholic fermentation need to be identified. In the present study, we focused on how sake yeast strains of S. cerevisiae have acquired high alcoholic fermentation performance. Our findings provide a rational molecular basis to design yeast strains with optimal fermentation performance for production of alcoholic beverages and bioethanol. In addition, as the evolutionarily conserved TORC1-Greatwall-PP2AB55δ pathway plays a major role in the glycolytic control, our work may contribute to research on carbohydrate metabolism in higher eukaryotes.

Skeletal Muscle mTORC1 Activation Increases Energy Expenditure and Reduces Longevity in Mice

Abstract: The mechanistic target of rapamycin (mTORC1) is a nutrient responsive protein kinase complex that helps co-ordinate anabolic processes across all tissues. There is evidence that signaling through mTORC1 in skeletal muscle may be a determinant of energy expenditure and aging and therefore components downstream of mTORC1 signaling may be potential targets for treating obesity and age-associated metabolic disease. Here, we generated mice with Ckmm-Cre driven ablation of Tsc1, which confers constitutive activation of mTORC1 in skeletal muscle and performed unbiased transcriptional analyses to identify pathways and candidate genes that may explain how skeletal muscle mTORC1 activity regulates energy balance and aging. Activation of skeletal muscle mTORC1 produced a striking resistance to diet- and age-induced obesity without inducing systemic insulin resistance. We found that increases in energy expenditure following a high fat diet were mTORC1-dependent and that elevated energy expenditure caused by ablation of Tsc1 coincided with the upregulation of skeletal muscle-specific thermogenic mechanisms that involve sarcolipin-driven futile cycling of Ca2+ through SERCA2. Additionally, we report that constitutive activation of mTORC1 in skeletal muscle reduces lifespan. These findings support the hypothesis that activation of mTORC1 and its downstream targets, specifically in skeletal muscle, may play a role in nutrient-dependent thermogenesis and aging.