Isp7 is a novel regulator of amino

51 Pages·2013·1.78 MB·English

MCB Accepts, published online ahead of print on 16 December 2013
Mol. Cell. Biol. doi:10.1128/MCB.01473-13
Copyright © 2013, American Society for Microbiology. All Rights Reserved.
1 Isp7 is a novel regulator of amino acid uptake in the TOR signaling pathway
2 Dana Laor1, Adiel Cohen2, Metsada Pasmanik-Chor3,Varda Oron-Karni3, Martin Kupiec1 and
3 Ronit Weisman1,2#
4 1Department of Molecular Microbiology and Biotechnology, Tel Aviv University, Israel
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5 2Department of Natural and Life Sciences, The Open University of Israel, Raanana, Israel www
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6 3Bioinformatics Unit, Faculty of Life Sciences Tel Aviv University, Ramat-Aviv, Israel ddd
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8 Running title: Isp7 is a novel regulator of amino acid homeostasis ttt
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10 #Corresponding author. Mailing address: Dept. of Molecular Microbiology and ...
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11 Biotechnology, Green Building, Room 211, Tel-Aviv University, Ramat Aviv, 69978
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12 Tel-Aviv, Israel. Phone: (972) 03-640-9208. Fax: (972) 03-640-9407. E-mail: g/g/g/

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13 [email protected]
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16 Word count for the Material and Methods: 6,687 (characters with no spaces). 000
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17 Word count for Introduction, Results and Discussion section: 31,234 (characters with no b b b
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20 Abstract
21 TOR proteins reside in two distinct complexes, TOR complex 1 and 2 (TORC1 and
22 TORC2) that are central for the regulation of cellular growth, proliferation and survival.
23 TOR is also the target for the immunosuppressive and anti-cancer drug rapamycin. In
24 Schizosaccharaomyces pombe, disruption of the TSC complex, mutations in which can D
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25 lead to the Tuberous Sclerosis syndrome in humans, results in a rapamycin sensitive n
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26 phenotype under poor nitrogen conditions. We show here that the sensitivity to d
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27 rapamycin is mediated via inhibition of TORC1 and suppressed by overexpression of f
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28 isp7+, a member of the family of 2-oxoglutarate-Fe(II) dependent oxygenases. The
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29 transcript level of isp7+ is negatively regulated by TORC1 but positively regulated by p:
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30 TORC2. Yet, we find extensive similarity between the transcriptome of cells disrupted c
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31 for isp7+ and cells mutated in the catalytic subunit of TORC1. Moreover, Isp7 regulates s
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32 amino acid permease expression similarly to TORC1 and in contrast to TORC2. r
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33 Overexpression of isp7+ induces TORC1-dependent phosphorylation of ribosomal protein on

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34 Rps6, while inhibiting TORC2-dependent phosphorylation and activation of the AGC- n
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35 like kinase Gad8. Taken together, our findings suggest a central role for Isp7 in amino ry

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36 acid homeostasis and the presence of isp7+-dependent regulatory loops that affect both , 2
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37 TORC1 and TORC2. 9

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38 Introduction u
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39 TOR (target of rapamycin) is an atypical serine/threonine kinase that functions as t
40 a central regulator of growth (48, 64). TOR was originally identified in the budding yeast
41 in a genetic screen for mutations which conferred resistance to the growth inhibitory
42 effect of the immunosuppressant and anticancer drug rapamycin (11). Rapamycin forms a
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43 complex with the highly conserved small protein FKBP12 and as part of such a complex
44 can bind and inhibit TOR proteins. TOR proteins exist in two distinct complexes, TOR
45 complex 1 (TORC1) and TOR complex 2 (TORC2) (23, 42). In many different
46 eukaryotes TORC1 positively regulates cell growth in response to nutrients, growth
47 factors, energy signals and stress. TORC2 affects metabolism, cell survival and D
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48 proliferation, yet its cellular functions are less well understood compared with TORC1 n
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49 (48, 64). The main target of rapamycin is TORC1 but TORC2 can also be inhibited by d
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50 rapamycin following long exposures to the drug (21, 43). One of the main negative f
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51 regulators of TOR signaling is the TSC1-TSC2 heterodimer. Mutations in TSC1 or TSC2
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52 can lead to the Tuberous Sclerosis syndrome (6). The TSC complex converts the GTPase :
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53 Rheb into its inactive form and thus prevents TORC1 activation (19, 24, 47). TORC1 is c
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54 regulated by additional GTPase proteins called Rag that were found to mediate mostly s
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55 amino acid signaling (41). r
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56 In the fission yeast, Schizosaccharomyces pombe, two TOR homologues exist, n

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57 Tor1 and Tor2, which were named by the order of their discovery (58). Later it was found n
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58 that Tor1 is the catalytic subunit of TORC2, while Tor2 is the catalytic subunit of ry

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59 TORC1. TORC1 also contains the protein Mip1 (raptor in humans) and TORC2 also 2
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60 contains the proteins Ste20 (rictor in humans) and Sin1 (mSin1 in humans) (10, 12, 17, 9

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61 30, 38, 45, 58, 61). TORC1 is essential under normal growth conditions and plays major g
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62 roles in the control of cellular growth, possibly in response to nitrogen availability (1, 30, s
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63 53, 54, 60-62). Consistently, most of the genes that are upregulated in tor2ts mutant cells
64 are involved in nitrogen starvation response (30). S. pombe contains homologs for TSC1
65 and TSC2, known as tsc1+ and tsc2+, that, similarly to human cells, negatively regulate
66 TORC1 activity via inhibition of Rhb1 (Rheb in human) (51-53, 57). Disruption of tsc1+
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67 or tsc2+ leads to amino acid uptake defects and impaired sexual development or gene
68 induction upon nitrogen starvation (28, 56). Recently, Rag homologs, Gtr1 and Gtr2,
69 were characterized in fission yeast. Gtr1 and Gtr2 function as a heterodimeric complex
70 and were found to induce cellular growth and repress sexual differentiation by activating
71 TORC1 in response to the presence of amino acids (54). D
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72 S. pombe TORC2 regulates cell survival under stress conditions and is required n
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73 for starvation responses via the AGC protein kinase Gad8 (29). Disruption of TORC2 or d
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74 gad8+ results in pleiotropic defects that include inability to initiate sexual development or f
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75 acquire stationary phase physiology, severe sensitivity to a variety of stresses, a delay in
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76 entrance into mitosis, decrease in amino acid uptake, sensitivity to DNA damaging agents :
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77 and elongated telomeres (12, 17, 38, 45, 58, 61). Interestingly, TORC1 and TORC2 in S. c
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78 pombe oppositely regulate starvation responses, including sexual development and s
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79 transcription of nitrogen-starvation-induced genes (62). Moreover, TORC1 and TORC2 r
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80 play antagonistic roles with respects to mitosis (12) and longevity (39). n

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81 Rapamycin does not inhibit growth of wild type S. pombe cells, indicating that n
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82 under normal growth conditions the essential function of TORC1 is resistant to ry

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83 rapamycin. Nevertheless, rapamycin inhibits TORC1-dependent phosphorylation of the 2
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84 AGC kinases Sck1, Sck2 or Psk1 (36). A triple deletion mutant of these kinases is viable 9

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85 (36), indicating that additional TORC1 downstream effectors are yet to be discovered. g
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86 More recently, it was shown that rapamycin can inhibit TORC1 in the presence of s
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87 caffeine, which potentially lowers the kinase activity of TORC1 and induces a nitrogen-
88 starvation-like response (39, 49). Rapamycin inhibits sexual development and amino acid
89 uptake (59-61) and following long exposure reduces the expression of nitrogen-
90 starvation-induced amino acid permeases (61). Under certain conditions rapamycin can
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91 also induce sexual development (38), suggesting that rapamycin may involve inhibition
92 of either TORC1 or TORC2, depending on the experimental conditions.
93 Previously, we showed that tsc1 or tsc2 mutant cells are highly sensitive to
94 rapamycin on proline medium. No inhibition by rapamycin was observed when the
95 nitrogen source was ammonium, which is normally used in the standard growth medium, D
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96 or glutamate, which is considered a relatively good nitrogen source (62). The rapamycin- n
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97 sensitive phenotype of tsc mutant cells is dependent on the presence of the S. pombe d
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98 FKBP12 protein (62), indicating that TOR (either Tor1 or Tor2) is the target of f
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99 rapamycin. Here we show that the sensitivity of tsc mutant cells to rapamycin is mediated
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100 by TORC1 and can be suppressed by overexpression of the 2-oxoglutarate-Fe(II) :
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101 dependent oxygenase, Isp7. We show that Isp7 is a novel regulator of amino acids uptake c
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102 that acts via regulation of gene expression, both upstream and downstream of TOR s
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103 signaling. r
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104 Materials and methods J
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105 Yeast techniques. Table S4 shows the list of S. pombe strains used in this work. Unless a
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106 otherwise specified, S. pombe strains were grown at 30°C in Edinburgh minimal medium ,

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107 (EMM, 5g/L NH4Cl) as described before (32). For proline medium, the ammonium 19

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108 chloride in the minimal medium was replaced with 10mM proline. Rapamycin (R0395, y

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109 Sigma) was used at a final concentration of 100ng/ml unless otherwise specified. S. e
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110 pombe cDNA library in pREP3X multicopy plasmid was used for genetic screens (31).
111 The ORFs were identified by comparison with the S. pombe gene database, GeneDB
112 (http://www.genedb.org/genedb/pombe/).
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113 Construction of tor1SE and tor2SE mutant strains. A mutation at the FRB (FKBP12-
114 rapamycin binding) domain of tor2+, converting serine to glutamic acid (S1837E), was
115 created by site-directed mutagenesis using PCR overlap extension, as described
116 previously (61). The resulting PCR fragment was cloned into the pREP81 plasmid. A
117 plasmid carrying an equivalent mutation in the tor1+ gene, tor1SE, was previously D
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118 described (58). The tor1SE and tor2SE mutations were integrated into their respective n
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119 loci in the genome. First, a 1.5kbp kanMX6 fragment was cloned into plasmids carrying d
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120 tor1SE or tor2SE. Fragments containing the tor1SE or tor2SE and the kanMX6 cassette f
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121 were then released from the plasmids by enzymatic restrictions and introduced into the
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122 genome by homologous recombination to replace the wild type copies of the respective :
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123 genes. The presence of the mutations was confirmed by sequencing. c
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124 Construction of the isp7-H276A plasmid. Site-directed mutagenesis of pREP3X- isp7+ s
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125 was carried out by PCR amplification with ACCUZYMETM DNA Polymerase (Bioline), or
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126 using the complementary primers: 584 (5'- n

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127 CGTCTAGGTGTTCAAGAGGCCACGGATGCTGATGCGCT-3') and 585 (5'- n
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128 AGCGCATCAGCATCCGTGGCCTCTTGAACACCTAGACG-3') that contain the ry

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129 desired mutation (the underlined region). PCR products were digested with DpnI (NEB) 2
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130 to eliminate the methylated wild type template and propagated in XL1-Blue Escherichia 9

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131 coli cells. Mutant clones were identified by DNA sequencing and checked for the absence g
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132 of additional mutations in the isp7+ coding sequence. Their growth phenotype was tested s
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133 by transformation into tsc2 mutant strain.
134 Construction of isp7-HA. The C-terminus of Isp7 was tagged with triple HA. PCR was
135 used to amplify the HA tag using the pFA6a-3HA-kanMX6 plasmid as a template (25)
136 with primers 633 (5′-GAACCAATTGCTGTGGAAGACTTGTTACGCGATCATTTCC
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137 AAAACAGCTATACTTCACATACCACCTCATTGGAAGTTGCACGGATCCCCGG
138 GTTAATTAA-3′) and primer 634 (5′-CAAGACAAATATACAATAATAATAGGACA
139 ACAAACAACAACGAGCAAGTCTAAATTGAAATTTTTTTTCCTCCAAGTTCAAG
140 AATTCGAGCTCGTTTAAAC-3′). The PCR fragment was purified and transformed
141 into 972 wild type strain. The DNA fragment was introduced into the genome by D
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142 homologous recombination. Correct integration at the isp7+ loci was validated by PCR. n
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143 The expression of tagged proteins was confirmed by Western blotting. d
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144 Construction of LacZ reporter gene. The isp7+ promoter was fused into the f
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145 promoterless β-galactosidase gene of the shuttle vector pSPE356 (20). The upstream
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146 sequence of isp7+ gene was amplified by PCR using the primer 712 (5′- p:
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147 TATGGATCCTCAGTTTGGCATCTATAAAACAGGCA-3′) and primer 713 (5′- c
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148 TATGTCGACTATGCAGAATGTGAATTAAGTAGAAAAGAAAAAAT-3′) which s
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149 contained BamHI and SalI sites, respectively. The resulting PCR fragment containing the r
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150 DNA sequence from −2550 to +1 of isp7+ was cloned into pSPE356. on

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151 Northern blotting. Yeast RNA was extracted from logarithmic growing cells and RNA n
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152 was prepared using the hot phenol method and subjected to Northern blot analysis as ry

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153 described in (62). Gene-specific probes were labeled with [α-32P]dCTP using the Random , 2
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154 Primer DNA labeling kit (20-101-25A; Biological Industries). 9

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155 Uptake assays. We followed amino acid uptake assays as previously described (61). g
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156 Briefly, cells were grown to mid-log phase in minimal or proline media. One-half s
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157 milliliter of logarithmic cells was harvested and resuspended in 0.5 ml cold medium
158 containing 100µM arginine together with 3H-labeled arginine (2 μCi of [1-4,5-3H(N)]-
159 arginine, 54.6 Ci/mmol; Perkin Elmer) or 100µM proline together with 3H-labeled
160 proline (2 μCi of [1-4,5-3H(N)]-proline, 92.6 Ci/mmol; Perkin Elmer). Cells were
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161 incubated at 30°C and samples were taken after six minutes and mixed with chilled
162 medium containing 5mM arginine or proline. Cells were then washed three times before
163 being resuspended in water containing 0.5% SDS. 3H-labeled arginine/proline were
164 measured by scintillation counting. Error bars represent standard deviation calculated
165 from three independent cultures. D
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166 Microarray analysis. RNA was prepared using the hot phenol method (62). RNA was n
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167 hybridized to the Affymetrix Yeast Genome 2.0 microarray d
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168 (http://media.affymetrix.com/support/technical/datasheets/yeast2_datasheet.pdf f
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169 Affymetrix, Santa Clara, CA, USA). Three independent biological replicates were
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170 performed. Microarray analysis was performed on CEL files using Partek® Genomics :
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171 Suite TM, version 6.5 (http://www.partek.com). Data were normalized and summarized c
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172 with the robust multi-average method, followed by analysis of variance (ANOVA). Gene s
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173 expression data were sorted using cutoffs of p<0.05 under FDR (false discovery rate) and r
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174 fold-change cutoffs of 1.5 or 2. Venny was used to cross between gene lists n

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175 (http://bioinfogp.cnb.csic.es/tools/venny/index.html). n
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176 Western blotting. Proteins were extracted with TCA and resolved by SDS-PAGE using ry

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177 12% acrylamide gels. Proteins were transferred to nitrocellulose membranes, and blocked 2
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178 with 5% milk in TBST before immuneblotting. Detection was carried out using the ECL 9

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179 SuperSignal detection system (Thermo scientific). Rps6 phosphorylation was detected g
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180 using the anti-PAS antibody (Cell Signaling Technology), Rps6 by anti-Rps6 antibody s
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181 (Abcam), tubulin by anti- tubulin antibody (Sigma), Isp7-HA by anti-HA antibody (Santa
182 Cruz), Cdc2 by anti-PSTAIRE antibody (Santa Cruz), actin by anti- actin antibody (MP-
183 Biomedicals). Antibodies against Gad8 phosphorylated at Ser546 and antibodies against
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184 the C-terminus of the Gad8 were created using the peptides CRFANW-Ps-YQRPT and
185 CKSDDINTIAPGSVIR, respectively (Bio Basic Canada Inc).
186 galactosidase assays. Cells were grown to mid-log phase in minimal medium
187 supplemented with adenine, leucine and histidine before being harvested and subjected to
188 galactosidase assays (40). Error bars represent standard deviation calculated from three D
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189 independent cultures. n
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190 In vitro kinase assay. As a substrate for the Gad8 kinase assay, amino acid residues 291 d
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191 (Gln) to 411 (Pro) of Fkh2 was expressed in Escherichia coli BL21strain as GST fusion, fr
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192 using the pGEX-4T1 expression vector and purified. Cells expressing Gad8-HA were h
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193 grown in minimal or YE media and disrupted with glass beads in lysis buffer (20mM :/
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194 TRISS/HCl pH7.5, 0.5mM EDTA, 1mM DTT, 125mM Kac, 0.5mM EGTA, 0.1% b
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195 Triton-X100, 12.5% glycerol). After centrifugation at 10,000g for 10 minutes at 4ºC, m
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196 supernatants were incubated overnight with anti-HA antibodies. Protein A and protein G rg
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197 mixtures were added and immunoprecipitates were washed once with the lysis buffer, n

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198 once with lysis buffer containing 0.5M NaCl and twice in buffer A (50mM Tris-HCl n
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199 pH7.5, 0.1mM EGTA, 0.1% -mercaptoethanol). The immunoprecipitations were y

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200 incubated with 0.1mg of GST-Fkh2 in kinase buffer (10mM MgAc, 100mM ATP and 2
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201 phosphatase inhibitor cocktail, Sigma) for 10 minutes in 30ºC. The reaction was detected
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202 by Western blot analysis using anti-PAS antibody (Cell Signaling Technology). The level g
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203 of Gad8 was detected by anti-HA antibody (Santa Cruz). t
204 Microarray data accession number. The microarray data determined in this study have
205 been deposited in GEO under accession number GSE52759.
206
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207 Results
208 The sensitivity of tsc mutant cells to rapamycin is mediated by TORC1
209 The growth of tsc1 or tsc2 mutant cells is strongly inhibited by rapamycin
210 when the sole nitrogen source in the medium is proline but not ammonia [(62) and also
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211 see Fig. 1A]. We have previously shown that Δtsc1/2 mutant cells are unable to grow on o
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212 proline medium in the absence of the tor1+ gene (62). Tor1 is found mainly as part of lo
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213 TORC2 (10). Deletion of the specific component of TORC2, sin1+ or the downstream e
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214 kinase gad8+ also resulted in a similar growth inhibition in the genetic background of ro
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215 Δtsc1 or Δtsc2 (Fig. 1B). Thus, either TORC2-Gad8 or the TSC complex is required for t
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216 growth on proline. /m
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217 In order to determine whether rapamycin inhibits the growth of tsc mutant cells .
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218 via inhibition of Tor1 (TORC2) or Tor2 (TORC1), we used rapamycin-binding defective
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219 alleles of Tor1 (61) or Tor2 (see Materials and Methods). These mutated alleles contain /

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220 substitution of the conserved serine within the FRB domain of the TOR proteins. The J
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221 tor1S1834E (tor1SE) or tor2S1837E (tor2SE) alleles were integrated into their respective u
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222 genomic loci, replacing the wild type tor1+ and tor2+ genes. In tsc1 ortsc2 mutant 2
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223 cells, only the tor2SE allele, but not tor1SE, conferred rapamycin resistance (Fig. 1C). 0
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224 This result indicates that the target for rapamycin is TORC1 and not TORC2. Thus, b
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225 although TORC2 is required for growth on proline in the absence of tsc1/2, it is the u
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226 inhibition of TORC1 that is critical for rapamycin sensitivity.
227
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