Showing papers on "Protein kinase complex published in 2012"

MTORC1 functions as a transcriptional regulator of autophagy by preventing nuclear transport of TFEB

Abstract: The mammalian target of rapamycin (MTOR) protein kinase complex is a key component of a pathway that regulates cell growth and proliferation in response to energy levels, hypoxia, nutrients and insulin. Inhibition of MTORC1 strongly induces autophagy by regulating the activity of the ULK protein kinase complex that is required for the formation of autophagosomes. However, the participation of MTORC1 in the expression of autophagy genes has not been characterized. Here we show that MTORC1 regulates nuclear localization and activity of the transcription factor EB (TFEB), a member of the bHLH leucine-zipper family of transcription factors that drives expression of autophagy and lysosomal genes. Under normal nutrient conditions, TFEB is phosphorylated in Ser211 in an MTORC1-dependent manner. This phosphorylation promotes association of TFEB with members of the YWHA (14-3-3) family of proteins and retention of the transcription factor in the cytosol. Pharmacological or genetic inhibition of MTORC1 causes dissociation of the TFEB/YWHA complex and rapid transport of TFEB to the nucleus where it increases transcription of multiple genes implicated in autophagy and lysosomal function. Active TFEB also associates with late endosomal/lysosomal membranes through interaction with the LAMTOR/RRAG/MTORC1 complex. Our results unveil a novel role for MTORC1 in the maintenance of cellular homeostasis by regulating autophagy at the transcriptional level.

Functional dissection of lysine deacetylases reveals that HDAC1 and p300 regulate AMPK

Abstract: First identified as histone-modifying proteins, lysine acetyltransferases (KATs) and deacetylases (KDACs) antagonize each other through modification of the side chains of lysine residues in histone proteins. Acetylation of many non-histone proteins involved in chromatin, metabolism or cytoskeleton regulation were further identified in eukaryotic organisms, but the corresponding enzymes and substrate-specific functions of the modifications are unclear. Moreover, mechanisms underlying functional specificity of individual KDACs remain enigmatic, and the substrate spectra of each KDAC lack comprehensive definition. Here we dissect the functional specificity of 12 critical human KDACs using a genome-wide synthetic lethality screen in cultured human cells. The genetic interaction profiles revealed enzyme-substrate relationships between individual KDACs and many important substrates governing a wide array of biological processes including metabolism, development and cell cycle progression. We further confirmed that acetylation and deacetylation of the catalytic subunit of the adenosine monophosphate-activated protein kinase (AMPK), a critical cellular energy-sensing protein kinase complex, is controlled by the opposing catalytic activities of HDAC1 and p300. Deacetylation of AMPK enhances physical interaction with the upstream kinase LKB1, leading to AMPK phosphorylation and activation, and resulting in lipid breakdown in human liver cells. These findings provide new insights into previously underappreciated metabolic regulatory roles of HDAC1 in coordinating nutrient availability and cellular responses upstream of AMPK, and demonstrate the importance of high-throughput genetic interaction profiling to elucidate functional specificity and critical substrates of individual human KDACs potentially valuable for therapeutic applications.

Regulation of sphingolipid synthesis through Orm1 and Orm2 in yeast.

Abstract: Sphingolipids are crucial components of membranes, and sphingolipid metabolites serve as signaling molecules. Yeast Orm1 and Orm2 belong to a conserved family of ER membrane proteins that regulate serine palmitoyltransferase, which catalyzes the first and rate-limiting step in sphingolipid synthesis. We now show that sphingolipid synthesis through Orm1 is a target of TOR signaling, which regulates cell growth in response to nutritional signals. Orm1 phosphorylation is dependent on the Tap42–phosphatase complex, which acts downstream of TOR protein kinase complex 1. In temperature-sensitive tap42-11 cells, impaired Orm1 phosphorylation occurs concomitantly with reduced sphingolipid synthesis. A second mechanism for regulating sphingolipid synthesis is through control of Orm2 protein level. The Orm2 protein level responds to ER stress conditions, increasing when cells are treated with tunicamycin or DTT, agents that induce the unfolded protein response (UPR). The sphingolipid intermediates (long chain base and ceramide) are decreased when ORM2 is overexpressed, suggesting that sphingolipid synthesis is repressed under ER stress conditions. Finally, in the absence of the Orms, the UPR is constitutively activated. Lipid dysregulation in the absence of the Orms might signal to the ER from the plasma membrane because UPR activation is dependent on a cell surface sensor and the mitogen-activated protein kinase (MAPK) cell wall integrity pathway. Thus, sphingolipid synthesis and the UPR are coordinately regulated.