Tsc1+ and tsc2+ Regulate Arginine Uptake and Metabolism in Schizosaccharomyces pombe

Abstract

Mutations in either TSC1 or TSC2 cause tuberous sclerosis complex, an autosomal dominant disorder characterized by seizures, mental retardation, and benign tumors of the skin, brain, heart, and kidneys. Homologs for the TSC1 and TSC2 genes have been identified in mouse, rat, Fugu, Drosophila, and in the yeast Schizosaccharomyces pombe. Here we show that S. pombe lacking tsc1 ؉ or tsc2؉ have similar phenotypes including decreased arginine uptake, decreased expression of three amino acid permeases, and low intracellular levels of four members of the arginine biosynthesis pathway. Recently, the small GTPase Rheb was identified as a target of the GTPase-activating domain of tuberin in mammalian cells and in Drosophila. We show that the defect in arginine uptake in cells lacking tsc2 ؉ is rescued by the expression of a dominant negative form of rhb1 ؉ , the Rheb homolog in S. pombe, but not by expressing wildtype rhb1 ؉ . Expression of the tsc2 ؉ gene with a patientderived mutation within the GAP domain did not rescue the arginine uptake defect in tsc2 ؉ mutant yeast. Taken together, these findings support a model in which arginine uptake is regulated through tsc1 ؉ , tsc2 ؉ , and rhb1 ؉ in S. pombe and also suggest a role for the Tsc1 and Tsc2 proteins in amino acid biosynthesis and sensing.Tuberous sclerosis complex (TSC) 1 is a tumor suppressor syndrome that is characterized by the development of a variety of benign tumors (hamartomas) and severe neurological problems including seizures, mental retardation, and autism. TSC is caused by mutations in either TSC1 (1) or TSC2 (2). Hamartin, the TSC1 gene product, and tuberin, the TSC2 gene product, are known to interact (3, 4) and function in a complex. Tuberin has a highly conserved GTPase-activating protein (GAP) domain with activity for Rheb1 (Ras homolog enriched in brain) (5-11), a small GTPase that may be involved in nutrient signaling and cell cycle regulation (12).Studies in Drosophila and mammalian systems have shown that the hamartin-tuberin complex negatively regulates p70S6 kinase (pS6K) within the PI3K signaling pathway (13,14). The regulation of pS6K is mediated by Rheb and by the target of rapamycin, which are components in pathways that control cell size by integrating mitogenic signals and nutrient availability with protein synthesis (11,13,(15)(16)(17)(18). Both hamartin and tuberin are regulated by phosphorylation. Hamartin is phosphorylated by cyclin-dependent kinase CDK1 (19), and tuberin is a substrate for Akt (protein kinase B) (14,20,21), p38-activated kinase MK2 (22), and the AMP-activated protein kinase (23).Schizosaccharomyces pombe contains genes with significant similarity to TSC1 and TSC2, which were named tsc1 ϩ and tsc2 ϩ (24). The GAP domain of tuberin is particularly highly conserved with 39% identity. In addition to TSC1 and TSC2 homologs, S. pombe also has a Rheb homolog, rhb1 ϩ . Loss of rhb1 ϩ in S. pombe results in growth arrest, similar to that caused by nitrogen starvation (25), and loss of farnesylation of the Rhb1 protein has...

The TSC/Rheb/TOR Signaling Pathway in Fission Yeast and Mammalian Cells: Temperature Sensitive and Constitutive Active Mutants of TOR

“…27,28,33 These tsc -phenotypes could be suppressed by the expression of a dominant negative Rheb, RhebD60K. 27,28 These studies supported the idea that Tsc acts to negatively regulate Rheb in fission yeast. We also found that single amino acid changes in Rheb confer phenotypes similar to those observed with tsc -cells.…”

“…[27][28][29]33 A resistance to the toxic analogues of arginine, lysine and methionine (canavanine, thialysine, and D-ethionine, respectively) highlighted this defect. 27,28,33 These tsc -phenotypes could be suppressed by the expression of a dominant negative Rheb, RhebD60K. 27,28 These studies supported the idea that Tsc acts to negatively regulate Rheb in fission yeast.…”

“…[27][28][29] In contrast to rhb -cells, tsc -mutants exhibit a delay to nitrogen starvation and were shown to exhibit a defect in the uptake of several amino acids. [27][28][29]33 A resistance to the toxic analogues of arginine, lysine and methionine (canavanine, thialysine, and D-ethionine, respectively) highlighted this defect. 27,28,33 These tsc -phenotypes could be suppressed by the expression of a dominant negative Rheb, RhebD60K.…”

“…The tsc1 + and tsc2 + genes analogous to the mammalian TSC genes are present in fission yeast and their gene products form a complex which functions as a downregulator of Rheb GTPase. 8,[27][28][29] Two homologues of mTOR, Tor1 and Tor2, are also present in fission yeast. 8 Tor2 forms a complex with a Raptor homologue, Mip1, as well as with a G b L homologue, Wat1/Pop3.…”

Crystal structure of the yeast TSC1 core domain and implications for tuberous sclerosis pathological mutations

“…Recently, it has been shown that TSC1 has critical roles in neurodevelopment and neurological diseases, immune diseases, diabetes, autophagy and DNA repair [25][26][27][28] . Similar to the signalling mechanism in mammalian cells, the TSC1-TSC2 complex in S. pombe also regulates the TOR kinase activity, and has a role in amino-acid uptake from the environment and amino-acid homeostasis [29][30][31] .…”