In Saccharomyces cerevisiae, the Tap42-phosphatase complexes are major targets of the Tor kinases in the rapamycinsensitive signaling pathway. The immunosuppressive agent, rapamycin, induces a prompt activation of the Tap42-associated phosphatases, which is vitally important in Tor-mediated transcriptional regulation. However, the mechanism for the rapid phosphatase activation is poorly understood. In this study, we show that the Tap42-phosphatase complexes exist mainly on membrane structures through their association with Tor complex 1 (TORC1). Rapamycin abrogates this association and releases the Tap42-phosphatase complexes into the cytosol. Disassembly of the Tap42-phosphatase complexes occurs subsequently, following the release but at a much slower rate, presumably caused by Tap42 dephosphorylation. Release of the Tap42-phosphatase complexes from membrane structures also occurs when cells are deprived of nutrient. These findings suggest that the association of the Tap42-phosphatase complexes with TORC1 represents an important mechanism by which nutrient controls Tor signaling activity. In addition, our data support a model in which rapamycin acts not by inhibiting the kinase activity of Tor but by disrupting its interaction with downstream targets.

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Folliculin (FLCN) is the tumor suppressor associated withBirt-Hogg-Dubé (BHD) syndrome that predisposes patients to incident of hamartomas and cysts in multiple organs. Its inactivation causes deregulation in the mammalian target of rapamycin complex 1 (mTORC1) signaling pathway. However, the underlying mechanism is poorly defined. In this study, we show that FLCN is a ciliary protein that functions through primary cilia to regulate mTORC1. In response to flow stress, FLCN associates with LKB1 and recruits the kinase to primary cilia for activation of AMPK resided at basal bodies, which causes mTORC1 down-regulation. In cells depleted of FLCN, LKB1 fails to accumulate in primary cilia and AMPK at the basal bodies remains inactive, thus nullifying the inhibitory effect of flow stress on mTORC1 activity. Our results demonstrate that FLCN is part of a flow sensory mechanism that regulates mTORC1 through primary cilia. Birt-Hogg-Dubé (BHD)4 syndrome is a rare autosomal dominant genetic disorder characterized by development of hamartomas and cysts in multiple organs, including skin, lung, colon, and kidney (1, 2). The syndrome is caused by germ-line mutations in the BHD gene, which encodes the folliculin protein (FLCN), a 64-kDa polypeptide that shares little sequence similarity with any other known proteins (3). FLCN is found to complex with AMPK and FNIP1, although the significance of the complex for FLCN function remains unclear (4). In mouse models FLCN deficiency leads to development of polycystic kidneys and renal cell carcinoma that are characteristically similar to those found in BHD patients (5, 6). Inactivation of FNIP1, together with its homolog FNIP2, in mice also produces similar phenotypes as does by FLCN deficiency (7), suggesting that FNIP1 may be required for FLCN function. Analyses of tumors derived from BHD patients and FLCN deficient animals have revealed deregulation in mammalian target of rapamycin complex 1 (mTORC1) signaling, a key event in tumorigenesis (5, 8 -12). This abnormality in mTORC1 signaling is believed to be a major contributor to the pathological conditions in BHD, as inhibition of mTORC1 with rapamycin has been found to reduce the BHD tumor growth in animal models (5, 6). However, the mechanism by which FLCN regulates mTORC1 remains poorly understood.At non-cycling resting state, most eukaryotic cells possess a microtubule-based membranous protrusion from cell surface termed as primary cilium (13). This unique structure plays a critical role in maintaining tissue homeostasis by functioning as a sensor for extracellular fluidic shear stress and chemicals (14, 15). Many signaling pathways involved in cell growth and proliferation are regulated by this environmental sensor, among which is the mTORC1 pathway (16 -18). Several upstream regulators of mTORC1 have been found to localize to primary cilia, including the tuberous sclerosis complex proteins, LKB1 and AMPK (19 -21). A recent study has shown that primary cilia are able to act through a LKB1-and AMPK-dependent mechanism to dow...

In eukaryotic cells, two conserved protein kinases, Gcn2 and TOR complex 1 (TORC1), couple amino acid conditions to protein translation. Gcn2 functions as an amino acid sensor and is activated by uncharged tRNAs that accumulate when intracellular amino acids are limited. Activated Gcn2 phosphorylates and inhibits eukaryotic initiation factor-2α (eIF2α), resulting in repression of general protein synthesis. Like Gcn2, TORC1 is also involved in sensing amino acid conditions. However, the underlying mechanism remains unclear. In the present study, we show that TORC1 is a direct target of Gcn2 kinase in the yeast In response to amino acid starvation, Gcn2 binds to TORC1 and phosphorylates Kog1, the unique regulatory subunit of TORC1, resulting in down-regulation of TORC1 kinase activity. In the absence of Gcn2, TORC1 signaling activity increases and becomes unresponsive to amino acid starvation. Our findings demonstrate that TORC1 is an effector of Gcn2 in amino acid signaling, hence defining a novel mechanism by which TORC1 senses amino acid starvation.

In the yeast Saccharomyces cerevisiae the guanosine triphosphatase (GTPase) Rho1 controls actin polarization and cell wall expansion. When cells are exposed to various environmental stresses that perturb the cell wall, Rho1 activates Pkc1, a mammalian Protein Kinase C homologue, and Mpk1, a mitogen activated protein kinase (MAPK), resulting in actin depolarization and cell wall remodeling. In this study, we demonstrate a novel feedback loop in this Rho1-mediated Pkc1-MAPK pathway that involves regulation of Rom2, the guanine nucleotide exchange factor of Rho1, by Mpk1, the end kinase of the pathway. This previously unrecognized Mpk1-depedent feedback is a critical step in regulating Rho1 function. Activation of this feedback mechanism is responsible for redistribution of Rom2 and cell wall synthesis activity from the bud to cell periphery under stress conditions. It is also required for terminating Rho1 activity toward the Pkc1-MAPK pathway and for repolarizing actin cytoskeleton and restoring growth after the stressed cells become adapted.