XWH - 05 - 1 - 0140 TITLE : Fission Yeast Model Study for Dissection of TSC Pathway

Mutations in the human Tsc1 and Tsc2 genes predispose to tuberous sclerosis complex (TSC), a disorder characterized by the wide spread of benign tumors. Tsc1 and Tsc2 proteins form a complex and serve as a GTPase-activating protein (GAP) for Rheb, a GTPase regulating a downstream kinase, mTOR. The genome of Schizosaccharomyces pombe contains tsc1 and tsc2, homologs of human Tsc1 and Tsc2, respectively. In this study we analyzed the gene expression profile on a genomewide scale and found that deletion of either tsc1 or tsc2 affects gene induction upon nitrogen starvation. Three hours after nitrogen depletion genes encoding permeases and genes required for meiosis are less induced. Under the same condition, retrotransposons, G1-cyclin (pas1), and inv1 are more induced. We also demonstrate that a mutation (cpp1-1) in a gene encoding a b-subunit of a farnesyltransferase can suppress most of the phenotypes associated with deletion of tsc1 or tsc2. When a mutant of rhb1 (homolog of human Rheb), which bypasses the requirement of protein farnesylation, was expressed, the cpp1-1mutation could no longer suppress, indicating that deficient farnesylation of Rhb1 contributes to the suppression. On the basis of these results, we discuss TSC pathology and possible improvement in chemotherapy for TSC. TUBEROUS sclerosis complex (TSC) is an autosomal dominant disorder characterized by the wide spread of benign tumors called hamartomas in different organs including the brain, eyes, heart, kidney, skin, and lungs (Kwiatkowski and Short 1994; Gomez 1995). Seizures and learning and behavioral problems, which are likely due to development of tumors in the brain, are also common in patients with TSC (Kwiatkowski and Short 1994; Gomez et al. 1999). Two human genes, TSC1 and TSC2, are responsible for TSC (European Chromosome 16 Tuberous Sclerosis Consortium 1993; van Slegtenhorst et al. 1997), each of which encodes hamartin and tuberin, respectively. Inactivation of TSC1 and TSC2 causes phenotypes similar each other, suggesting that they might affect the same pathway. TSC1 and TSC2 form a heterodimer and the TSC1–TSC2 interaction appears to be important for the stability of the two proteins (Li et al. 2004b). Therefore, TSC1 and TSC2 are generally considered as a complex (TSC1/2) with a single biological function, and understanding functions of the TSC1/2 complex is clinically important. Genetic studies in mammalian systems (Carbonara et al. 1994; Green et al. 1994a,b; Henske et al. 1996; Kwiatkowski et al. 2002) and Drosophila (Ito and Rubin 1999) have shown that TSC1/2 functions to inhibit cell growth as well as cellular proliferation (Hengstschlager et al. 2001). Appearance of giant cells within hamartomas from TSC patients and the gigas phenotype in the fly mutant highlight the capability of TSC1/2 in controlling cell size (Ito and Rubin 1999). Studies have shown that TSC1/2 controls cell growth/ proliferation by regulating the activity of a small GTPase, RHEB (Zhang et al. 2003). When the environment surrounding the cell is not favorable for growth/proliferation, TSC1 and TSC2, which have a GTPase-activating protein (GAP) domain in their C-terminal region, convert RHEB into an inactive form. A kinase, mTOR is a target of RHEB and promotes protein synthesis when stimulated by RHEB GTPase (Manning and Cantley 2003; Li et al. 2004a,b; Pan et al. 2004; Long et al. 2005). It is postulated that formation of hamartomas in TSC is a result of abnormal regulation of RHEB GTPase. A loss of TSC1/2 would allow constitutive activation of the GTPase as well as its target, mTOR. Corresponding author: Radiation Biology Center, Kyoto University, Yoshida-Konoe cho, Sakyo Ku, Kyoto, Japan 606-8501. E-mail: [email protected] Genetics 173: 569–578 ( June 2006) Homologs of the mTOR kinase and RHEB can be found in lower eukaryotes. The genome of Schizosaccharomyces pombe contains two genes homologous to mTOR (tor1 and tor2) and a gene homologous to RHEB, rhb1 (Mach et al. 2000). It also contains genes tsc1 and tsc2, each of which corresponds to mammalian TSC1 and TSC2, respectively (Matsumoto et al. 2002). Although the genome of Saccharomyces cerevisiae also encodes proteins homologous to mTOR (Cafferkey et al. 1994) and RHEB GTPase (Urano et al. 2000), it does not contain any obvious homologs to TSC1/2, suggesting that RHEB GTPase may be regulated by another mechanism. In our previous study we showed that fission yeast strains lacking either tsc1 or tsc2 are viable in rich media, but exhibit several defects. First, deletion strains for tsc1 (Dtsc1) and tsc2 (Dtsc2) are defective in uptake of nutrients such as amino acids and adenine. Consistent with this defect, an amino acid permease, which is normally positioned on the plasma membrane, aggregates in the cytoplasm or is confined in vacuole-like structures inDtsc1 andDtsc2. Second,Dtsc1 andDtsc2 are unable to induce the sxa2 gene, which is usually expressed upon stimulation by a mating-type pheromone, P factor, in starved h! cells (Imai and Yamamoto 1994). On the basis of these phenotypes, we postulate that tsc1 and tsc2 are required for sensing/responding to starvation. We speculate that S. pombe Tsc1/2 regulates Tor1/2 via Rhb1 and plays a role in sensing/responding to starvation. In this study we continued to take advantage of this simple and tractable system and attempted to dissect genetic pathways to interact with Tsc1/2. Through a genetic screen of extragenic suppressors of Dtsc2, we identified a gene, cpp1, encoding a subunit of the enzyme required for protein farnesylation. MATERIALS AND METHODS Yeast strains, media, and transformation: The S. pombe strains used in this study are listed in Table 1. The yeast cells were grown in YEA and EMM with appropriate nutrient supplements as described previously (Moreno et al. 1991). All yeast transformations were carried out by lithium acetate methods (Okazaki et al. 1990; Gietz et al. 1992). Screen for an extragenic suppressor of Dtsc2: The reversion rate of the AE512 strain used for the screening was 1.253 10!6. The spontaneous revertants were grown at 26! for 4 days on EMM medium with leucine at 40 mg/ml. Sixty-five revertants obtained through the primary screen were tested for their temperature sensitivity in the secondary screen. The revertants were replicated on two YEA plates and incubated at 26! (for 3 days) or 36! (for 2 days), respectively. Among the revertants isolated through the primary screen, 11 strains exhibited a temperature sensitivity for growth at 36!. Finally, the 11 revertants were further tested for their ability to induce fnx1 and mei2 upon nitrogen starvation by Northern analysis. Two revertants satisfied the final criterion and were further examined genetically. Cloning of cpp1: The cpp1-1 mutant (YKK25) was transformed with an S. pombe genomic library containing partially digested Sau3AI DNA fragment constructed in a multicopy plasmid, pAL-KS (Tanaka et al. 2000). Plasmids were recovered from Ts Leu transformants and their nucleotide sequences were determined. BLAST search was performed for the obtained sequences, and the region covered by the inserted genomic sequence was determined. Plasmid construction: Plasmid pREP41-cpp1 was constructed as follows. The cpp1 gene was amplified by PCR using the forwardprimer F-cpp1 [59-CCCCCCGTCGAC(SalI)GATGG ATGAATTATCAGAAAC-39] and the reverse primerR-cpp1 [59CCCCCCGGATCC(BamHI)TTAGAATTTTGATGATTCTTG-39]. The resulting fragment was digested with BamHI and SalI and then cloned into pREP41 (Maundrell 1993). Plasmids pREP41-rhb1 and pREP81-rhb1 were constructed as follows. The rhb1 gene was amplified by PCRusing the forward primer F-rhb1 [59CCCCCCGTCGAC(SalI)CATGGCTCCTATTAAATC TC -39] and the reverse primer R-rhb1 [59-CCCCCCGGATCC (BamHI)TTAGGCGATAACACAACCCTTTCC-39]. The resulting fragment was digested with BamHI and SalI and then cloned into pREP41 and pREP81, respectively. pREP41-rhb1 was constructed similarly with the exception of the primer used for PCR that was the reverse primer R-rhb1 [59-CCCCCCGGAT CC(BamHI)TTACAAGATAACACAACCC-39]. Generation of anti-Rhb1 antibody: A His-tagged protein of Rhb1 produced in Escherichia coli was used to raise polyclonal antibodies. His-Rhb1 was obtained as follows: A 558-bp DNA fragment carrying the entire rhb1 coding region was amplified by PCRwith two oligonucleotides, 59-GGGGGGATCC(BamHI) GCTCCTATTAAATCTCGTAGAATTG-39 and 59 CCCCGTCG AC(SalI)TTAGGCGATAACACAACCCTTTCC-39. The amplified DNA was digested with BamHI and SalI and then inserted into the same sites of the His-tag expression vector pET-30-a to make pET(rhb1). The pET(rhb1) was transformed into E. coli Tuner. The fusion protein was purified from the MagneHis Protein Purification System (Promega,Madison,WI) and used to immunize rabbits. TABLE 1 Strains used in this study Strain Genotype Source SP6 h! leu1-32 Laboratory stock AE512 h! tsc2Tura4 ura4-D18 leu1-32 Laboratory stock AE413 h! tsc1Tura4 ura4-D18 leu1-32 Laboratory stock YKK25 h! tsc2Tura4 ura4-D18 leu1-32 cpp1-1 This work YKK59 h! tsc1Tura4 ura4-D18 leu1-32 cpp1-1 This work 972h! h! Laboratory stock YKK55 h! ura4-D18 leu1-32 cpp1-1 This work SP740 h! ura4-D18 leu1-32 Laboratory stock 570 Y. Nakase et al.