The Serine Protease Htra1 Specifically Interacts and Degrades the Tuberous Sclerosis Complex 2 Protein

Published OnlineFirst July 29, 2010; DOI: 10.1158/1541-7786.MCR-09-0473 Published OnlineFirst on August 24, 2010

Molecular Signaling and Regulation Cancer Research The Serine Protease HtrA1 Specifically Interacts and Degrades the Tuberous Sclerosis Complex 2 Protein

Mara Campioni1, Anna Severino5, Lucrezia Manente2, Ioana L. Tuduce1, Stefano Toldo5, Michele Caraglia1, Stefania Crispi3, Michael Ehrmann6, Xiaoping He7, Jacie Maguire7, Maria De Falco4, Antonio De Luca2, Viji Shridhar7, and Alfonso Baldi1

Abstract Hamartin and tuberin are products of the tumor suppressor genes TSC1 and TSC2, respectively. Mutations affecting either gene result in the tuberous sclerosis syndrome, a neurologic genetic disorder characterized by the formation of multiple benign tumors or hamartomas. In this study, we report the identification of TSC2, but not TSC1, as a substrate of HtrA1, a member of the human HtrA family proteins of serine proteases. We show the direct interaction and colocalization in the cytoplasm of HtrA1 and TSC2 and that HtrA1 cleaves TSC2 both in vitro and in vivo. Finally, we show that alterations in HtrA1 expression cause modifications in phosphorylation status of two downstream targets of TSC2: 4E-BP1 and S6K. Our data suggest that, under particular physiologic or pathologic conditions, HtrA1 degrades TSC2 and activates the downstream targets. Considering that HtrA1 levels are significantly increased during embryogenesis, we speculate that one of the targets of HtrA1 activity during fetal development is the TSC2-TSC1 pathway. Mol Cancer Res; 8(9); OF1–13. ©2010 AACR.

Introduction motif. Therefore, the human HtrA1 protein is a secreted serine protease that exhibits endoproteolytic activity, including HtrA1 is the first identified member of the human HtrA autocatalytic cleavage. It is also known that conversion by family proteins, a group of heat shock–induced serine pro- site-directed mutagenesis of the putative conserved active teases involved in several aspects of protein quality control site Ser328 to alanine eliminates this enzymatic activity (4). and cell fate. This family consists of four members (HtrA1, Until now, several targets of HtrA1 have been identified, HtrA2, HtrA3, and HtrA4), which share a highly con- most of which are extracellular proteins. These include served trypsin-like serine protease domain and one PDZ components of extracellular matrix, such as type III colla- domain at the COOH terminus (1), and specific distinct gen (5) and fibronectin (6), or components of cartilage, regulation domains, in relation with their physiologic func- such as aggrecan, decorin, and fibromodulin (7). Moreover, tions, at the NH2 terminus. it has been described that purified HtrA1 degrades various β Structurally, the NH2 terminus of HtrA1 shares a signal amyloid precursor protein fragments, including amyloid- sequence for secretion and two homology domains: the and its precursor, the C99 peptide, both involved in a first region of the protein is homologous to mac25, a gene metabolic balance able to prevent plaque formation in the product related to insulin-like growth factor–binding pro- brain of Alzheimer's disease patients (8). tein (2), and to follistatin, an activin-binding protein (3), In accordance with its proteolytic activity and extracellu- and the second domain shows homology >40% to bacterial lar localization, HtrA1 wasinitiallydescribedasagene serine protease and allows its proteolytic activity (4). downregulated in SV40-trasformed human fibroblasts (9) Among these two regions, there is a Kazal-type inhibitor and overexpressed in osteoarthritic cartilage (4). Subse- quently, it has been reported that the expression of HtrA1 is also decreased in progression and invasion of melanomas, Authors' Affiliations: 1Department of Biochemistry, Section of Pathology and 2Department of Medicine and Public Health, Second University of ovarian cancers, lung cancer, and mesotheliomas (10-13). Naples; 3Gene Expression Core, Human Molecular Genetics Laboratory, It has been shown that its overexpression inhibits cell Institute of Genetics and Biophysics, Consiglio Nazionale delle Ricerche; growth and proliferation in vitro and in vivo, thus suggest- 4Department of Evolutive and Comparative Biology, University of Naples “Federico II,” Naples, Italy; 5Institute of Cardiology, Catholic University, ing a possible role as a tumor suppressor (10). Moreover, it Rome, Italy; 6Centre for Medical Biotechnology, University of Duisburg- seems that molecular alterations associated with the expres- Essen, Essen, Germany; and 7Department of Experimental Pathology, sion of this HtrA1 serine protease may play an important Mayo Clinic College of Medicine, Rochester, Minnesota role in tumor behavior and response to chemotherapy (14). Corresponding Author: Alfonso Baldi, Department of Biochemistry, Section of Pathology, Second University of Naples, Via L. Armanni 5, Recently, several studies revealed the involvement of HtrA1 80138 Naples, Italy. Phone: 39-081-5666003; Fax: 39-081-5569693. also in age-related macular degeneration. Genome-wide E-mail: [email protected] linkage analyses showed a strong association of a variant doi: 10.1158/1541-7786.MCR-09-0473 of HtrA1 gene with increased susceptibility to age-related ©2010 American Association for Cancer Research. macular degeneration in different populations (15-19).

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To identify new HtrA1-binding partners and thereby Materials and Methods obtain new clues toward the functions of this protein, we have used the enzymatically inactive form of HtrA1, called Plasmids and DNA constructs HtrA1-S328A, as a bait in a yeast two-hybrid screening Human wild-type HtrA1 cDNA was a kind gift of experiment. Among the proteins identified as possible part- Dr. Trueb (University of Bern, Bern, Switzerland). The ner of HtrA1, we found one of the two proteins involved truncated expression constructs encoding the mac25 in the pathogenesis of tuberous sclerosis, a 1,807–amino domain (amino acids 22-155), the KI domain (amino acids acid/220-kDa protein called TSC2. 97-155), and the COOH-terminal HtrA (amino acids Tuberous sclerosis complex (TSC) is a rare inheritable 150-480) were generated by PCR. disorder characterized by the development of hamartomas The HtrA1-S328A variant expression cDNA was in the brain and in other vital organs such as kidneys, obtained by site-directed mutagenesis of the original HtrA1 heart, eyes, lungs, and skin. The growth of benign tumors expression construct using the Stratagene QuikChange kit is frequently associated with skin rashes, seizures, mental as previously described (4). retardation, and renal dysfunction (20-22). For transfection experiments, the full-length HtrA1 and Two tumor suppressor genes responsible for TSC have its variant HtrA1-S328A cDNAs were cloned as EcoRI/ been identified: TSC1 located on chromosome 9q34 and BamHI fragments into pcDNA3T7Tag mammalian TSC2 on 16p13, encoding hamartin and tuberin, respec- expression vector (Invitrogen). tively (23, 24). For production of the glutathione S-transferase (GST)– It has been elucidated that TSC1 and TSC2 physically HtrA1 recombinant fusion protein, the full-length HtrA1 interact to form an intracellular heterodimer (TSC1/TSC2 (HtrA1-S328A) and the COOH-terminal truncated frag- complex) participating in the control of cell growth and ments (HtrA, mac25, and KI) were cloned as EcoRI/BamHI division (25, 26). This TSC complex acts upstream of fragments into the pGEX2TK vector (Invitrogen). the mammalian target of rapamycin (mTOR), a serine- The full-length TSC1-pcDNA3.1 and TSC2-pcDNA3.1 threonine kinase that increases cell growth and prolife- expression constructs were kindly provided by Dr. Halley ration. Activation of mTOR leads to increased protein (Erasmus Medisch Centrum, Rotterdam, the Netherlands). synthesis through phosphorylation of two effector mole- Truncated TSC2 cDNAs were obtained by PCR using cules: p70S6K and 4E-BP1. p70S6K, the ribosomal protein TSC2-pcDNA3.1 as template and specific primers: TSC2 S6 kinase I, can phosphorylate translational regulators, such (1-607), 5′-GATCGGATCCATGGCCAAACCAACAAG- as eukaryotic initiation factor eIF4B, to enhance the trans- CAAAGAT-3′ (forward) and 5′-GATCTCGAGCT- lational efficiency of several mRNAs (27, 28). 4E-BP1, the CACGCGATTGGCAGGGTGTAGCT-3′ (reverse); eukaryotic initiation factor 4E-binding protein 1, is able to TSC2 (607-1099), 5′-GATCGGATCCATGGCGAG- interact with the eukaryotic translation initiation factor CAGCATCCGGCTG-3′ (forward) and 5′-GATCTC- eIF4E. Binding of the 4E-BPs to eIF4E is regulated by GAGCTCACCCGGGGCTGGAGCTCGA-3′ (reverse); phosphorylation: Hypophosphorylated 4E-BP1 isoform TSC2 (1099-1807), 5′-GATCGGATCCATGGGGGTG- interacts strongly with eIF4E to prevent translation initia- CATGTGAGACAGACC-3′ (forward) and 5′-GATC- tion. On phosphorylation by the TOR kinase, 4E-BP1 TCGAGCTCACACAAACTCGGTGAAGTCCTC-3′ releases eIF4E and translation initiation ensues (29). When (reverse). the TSC complex is active, usually under conditions that All these truncated TSC2 cDNAs were cloned as EcoRI/ are adverse for proliferation, it is able to inhibit mTOR SacI fragments into pcDNA3.1 expression vector. All of through the inhibition of Rheb, a member of the Ras super- the constructs were sequenced completely. family of GTPases, important for the progression from G0 to G1 phase. Therefore, inhibition of mTOR activity Antibodies leads to prolonged G1 phase or arrest in G1 (27). On insulin The TSC1 and TSC2 polyclonal antibodies used in this or growth factor stimulation, TSC2 is phosphorylated by study were purchased from Santa Cruz Biotechnology and several kinases, including Akt/PKB (a serine-threonine used at the manufacturer's recommended dilutions. The kinase involved in cellular survival pathways) and RSK1 anti-T7Tag monoclonal antibody was from Novagen. (p90 ribosomal S6 kinase 1), thereby suppressing its The anti–4E-BP1 antibody was from Cell Signaling. GTPase-activating protein activity (30). As a consequence, Anti–phospho-ribosomal S6 kinase (p-S6K) and anti–total the TSC complex is no more functional, and downstream S6K antibodies were from Santa Cruz Biotechnology. The targets of mTOR act together to increase protein synthesis rabbit polyclonal antiserum raised against HtrA1 has been and cell growth. Mutations in TSC1 or TSC2 genes lead to previously described (10). To normalize each Western blot constitutive activation of this pathway; as a consequence, experiment, we used the anti–β-actin antibody from Santa mTOR uncontrolled activity triggers cell cycle progression Cruz Biotechnology. and tumor formation (27). To the best of our knowledge, TSC2 is the first cytoplas- Yeast two-hybrid selection mic target of HtrA1 identified until now. In this article, The HtrA1-S328A cDNA was cloned into the EcoRI/ we have characterized in vitro and in vivo the interaction BamHI sites of vector pGBKT7 (BD Clontech) in frame between these two proteins. with the GAL4-binding domain. The yeast strain AH109,

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carrying UAS-His3, UAS-LacZ, and UAS-ADE2 reporter After washing, the beads were analyzed on 6% acrylamide genes, was cotransformed with the pGBKT7-HtrA1- SDS-PAGE. S328A bait and with a human fetal brain cDNA library To do a GST pull-down assay with endogenous TSC2 (BD Clontech) fused to the GAL4 activation domain in and TSC1 from cell extracts, an equal amount of GST the pACT2 vector (BD Clontech). Transformation was or GST-HtrA1wt protein (5 μg) and 500 μg of Phoenix carried out using the lithium acetate method. Cells were cell lysate were used for the assay. The mixture of GST- plated on minimal synthetic defined medium (BD Clon- HtrA1wt beads and cell lysate was incubated with rotation tech) supplemented with the required bases and amino at 4°C for 3 hours. Then, the beads were washed three acids and lacking tryptophan (Trp), leucine (Leu), histidine times with lysis buffer. The complexes were separated on (His), and adenine (Ade). Plates were incubated for 7 days 6% SDS-PAGE. Western blot was done with specific at 30°C, and then His+ Ade+ transformants were isolated. anti-TSC2 or anti-TSC1 antibodies. The His+ Ade+ colonies, replica plated on SD-Leu-Trp- His-Ade medium and LacZ+, were identified by a filter- Cell culture, transfection, and Western blot analysis lifting assay for β-galactosidase activity. Plasmid DNA Phoenix cells were kept in DMEM supplemented with was prepared from candidate clones and transformed into L-glutamine, 10% heat-inactivated fetal bovine serum, and Escherichia coli XL1-Blue cells (Stratagene). Recovered penicillin-streptomycin. A375P and A375M cell lines were library-derived plasmids were analyzed by DNA sequencing. cultured in RPMI 1640 supplemented with L-glutamine, 10% heat-inactivated fetal bovine serum, and penicillin- In vitro binding assay, GST pull-down assay, and streptomycin. LM cell line was cultured in RPMI 1640 mapping experiment as described elsewhere (31). GST fusion protein expression was induced for 3 hours For transfection experiments, Phoenix cells were seeded in a 500-mL culture of E. coli BL21 by the addition of into 10-cm culture dishes and grown overnight to a con- 1 mmol/L isopropyl-L-thio-B-D-galactopyranoside. The fluence of 60% to 80%. Expression constructs were trans- bacterial pellet was resuspended in 20 mL of NENT buffer fected into the cells using standard calcium phosphate [20 mmol/L Tris-Cl (pH 8.0), 100 mmol/L NaCl, protocols. The LM, A375P, and A375M cells were seeded 1 mmol/L EDTA, 0.5% NP40, 1 mmol/L phenylmethyl- into 10-cm culture dishes and grown overnight to a con- sulfonyl fluoride, 10 μg/mL leupeptin] or NENT buffer fluence of 80% to 90%. Expression constructs were trans- added with 2% Sarkosyl (for insoluble protein) and soni- fected using Lipofectamine 2000 transfection reagent cated thoroughly (10 × 30 seconds). After gentle mixing (Invitrogen) following the protocol instructions. for 30 minutes at 4°C, the lysate was spun at 12,000 × g Twenty-four hours after transfection, the cells were har- for 10 minutes at 4°C. The induced GST fusion protein vested and lysed in lysis buffer [50 mmol/L Tris-Cl (pH in the supernatant fraction was bound to 250 mL of pre- 7.4), 5 mmol/L EDTA, 250 mmol/L NaCl, 50 mmol/L washed glutathione-Sepharose beads (Amersham Pharma- NaF, 0.1% Triton X-100, 0.1 mmol/L Na3VO4, cia Biotech) for 60 minutes at 4°C. The beads were 1 mmol/L phenylmethylsulfonyl fluoride, 10 μg/mL washed extensively with NENT buffer to remove nonspecif- leupeptin] for 30 minutes on ice. Lysates were centrifuged ically bound bacterial proteins, and the quantity and purity at 14,000 × g for 10 minutes at 4°C. Protein concentra- of the GST fusion protein bound to the glutathione- tion was determined by the Bradford assay (Bio-Rad) at Sepharose beads were analyzed by SDS-PAGE. 595 nm. Proteins were denatured by boiling in Laemmli For the in vitro binding assay, 1 μg of pcDNA3.1 encod- sample buffer (Bio-Rad) and resolved in SDS-PAGE. ing TSC1 or TSC2 was used to program a TnT rabbit Gels were transferred to Immobilon P membrane (polyvi- reticulocyte lysate (Promega) under the control of the T7 nylidene difluoride; Millipore) and probed with the indi- polymerase in the presence of [35S]methionine (Amersham cated antibodies. Pharmacia Biotech). Aliquots of the reaction mixture were A2780 ovarian cancer cell line was used to produce sta- added to glutathione-Sepharose beads (Amersham Pharma- ble clones expressing HtrA1 as described elsewhere (32); cia Biotech) coupled with 5 μg of GST-HtrA1wt, GST- SKOV3 and TOV21G ovarian cancer cell lines were used mac25, GST-KI, and GST-HtrA. Incubation was carried to produce HtrA1 short hairpin RNA (shRNA) stable out in NENT buffer for 60 minutes at 4°C with gentle clones as described elsewhere (33). rocking. Beads were washed three times in NENT buffer, and electrophoresis was done on 6% acrylamide SDS- Coimmunoprecipitation assay PAGE. Gels were dried and exposed at −70°C using Kodak For the immunoprecipitation experiment, 800 μgof Biomax MS films. Phoenix cell lysate transfected with pcDNA3T7Tag-HtrA1 For the mapping experiment, 1 μg of plasmid encoding were incubated with 1 μg of anti-T7Tag monoclonal for the different truncated expression constructs of TSC2 antibody (to pull down HtrA1) or 1 μg of anti-TSC2 poly- [TSC2 (1-607), TSC2 (607-1099), and TSC2 (1099- clonal antibody (to pull down TSC2) on ice for 15 hours 1807)] was used for in vitro translation as described above. before the addition of 30 μL of 50% protein G–Sepharose The same amount of each in vitro–translated constructs suspension (Amersham Pharmacia Biotech). As a control, was incubated with GST-HtrA1wt (5 μg) or with GST 800 μg of lysate were incubated with a monoclonal anti- alone as control for 60 minutes at 4°C with gentle rocking. body against p21 protein (BD Pharmingen) or a polyclonal

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antibody against Rb protein (Abcam). After gentle rotation lindole (DAPI; 0.1 g/mL in distilled water) and mounted for 90 minutes at 4°C, the beads were washed three times in Vectashield (Vector Laboratories). Images were taken with lysis buffer before being analyzed by immunoblotting. using an Olympus AX70 microscope configured for epi- A specific anti-TSC2 was used in Western blot to detect fluorescence and equipped with a cooled camera device. TSC2 protein bound to HtrA1 after immunoprecipitation with T7Tag antibody. To detect the amount of HtrA1 Protein phosphatase treatments pulled down with anti-TSC2 antibody in Western blot, Phoenix cells were lysed in protein phosphatase 1 (PP1) we used ExactaCruz IP/Western B kit (Santa Cruz Biotech- lysis buffer [50 mmol/L Tris-HCl (pH 7.4), 5 mmol/L nology) to eliminate the detection of heavy and light chains EDTA, 250 mmol/L NaCl, 0.1% Triton X-100] as de- of the immunoprecipitation antibody (the size of HtrA1 is scribedabove.PP1(10units;NewEnglandBiolabs, the same of the heavy chains of immunoglobulin). Inc.) and MnCl2 (1 mmol/L final concentration) were added to the lysate, which was subsequently incubated Subcellular localization of HtrA1 with immunoelectron for 60 minutes at 30°C. As a control, the lysate was incu- microscopy bated for 60 minutes at 30°C without PP1. Full-term placentas (n = 15) were collected, cut into To investigate whether the phosphatase activity prevents pieces of <1 mm3, and fixed in 2.5% glutaraldehyde in the interaction between HtrA1 and TSC2, 500 μg of trea- phosphate buffer (pH 7.3) for 2 hours. Tissues were ted cell lysates were added to 5 μg of GST-HtrA1 beads postfixed in osmium tetroxide (4%), dehydrated through (or GST beads as control) and mixed by gentle rotation graded concentrations of ethanol and propylene oxide, for 1 hour at 4°C. The beads were recovered by centrifu- and subsequently embedded in Epon 812. For the immu- gation, washed three times with lysis buffer, and dissolved nogold technique, tissues were fixed only in 2.5% glutar- in SDS-PAGE loading buffer for the immunoblot analysis. aldehyde in phosphate buffer (pH 7.3). Ultrathin sections were cut from blocks and mounted on copper grids for the Purification of recombinant HtrA1 and proteolytic inspection of morphology or on nickel grids for immuno- enzyme assessment gold. All sections were etched on drops of 3% hydrogen Purified recombinant HtrA1 was produced as described peroxidase for 10 minutes to permeabilize the resin. Tissue (8). One microgram of TSC2-pcDNA3.1 was transcribed sections were washed in microfiltered distilled water and and translated in vitro in the presence of [35S]methionine floated for 30 minutes on a drop of background blocking with the TNT Coupled Reticulocyte Lysate Systems (Pro- solution [0.05 mol/L Tris-HCl (pH 7.4), 0.1% bovine mega) as instructed by the manufacturer. serum albumin (BSA)]. Grids were then incubated at The translation product was buffered with the digestion 4°C overnight with a rabbit polyclonal antiserum raised buffer [50 mmol/L Tris-HCl (pH 8.5), 150 mmol/L NaCl] against HtrA1 (10) at 1:10 dilution. After several rinses using Microcon Y10 filter (Millipore). Purified HtrA1 to remove excess antibody, the grids were incubated with (5 μg) was added to the labeled TSC2 35Seluted(Vf= an anti-rabbit antibody conjugated with 10-nm gold parti- 50 μL), and the digestion was done in digestion buffer cles used as a secondary antibody at 1:100 dilution in at 37°C for 16 hours. As a positive control, 5 μg of purified diluent for gold-absorbed antibody [0.05 mol/L Tris-HCl HtrA1 were incubated with 10 μg of purified fibronectin buffer (pH 8.2), 1% BSA] for 1 hour. The grids were con- in the same condition of the sample. As a negative control, trasted with lead citrate and uranyl acetate as for conven- 0.5 μg of BSA (Promega) was incubated with 5 μg HtrA1. tional electron microscopy. A preimmune serum was used The proteins were resolved in SDS-PAGE and stained as a negative control. The sections were observed by means with Coomassie Brilliant Blue R-250. Finally, the gel was of a Siemens Elmiskop IA electron microscope. dried and exposed for autoradiography to detect the digestion products. Colocalization analysis with immunofluorescence Phoenix cells transfected with pcDNA3T7Tag-HtrA1 Expression analysis of different factors involved in were grown on glass coverslips in Petri dishes. Cells were TSC2 pathway fixed in 3.7% formaldehyde for 15 minutes at 4°C and Two different types of melanoma cell lines, A375 (pri- permeabilized in PBS containing 0.1% Triton X-100 for mary and metastatic of the same cell type) and LM, were 5 minutes. Fixed cells were preincubated in 20% FCS transfected with pcDNA3T7Tag-HtrA1, pcDNA3T7Tag- for 30 minutes at 37°C in a humidified chamber. The fol- HtrA1-S328A, and empty vector as a control as described lowing primary antibodies were used: mouse anti-T7Tag above. Lysate of transfected cells was run on gel to make (Novagen) and rabbit anti-TSC2 (Santa Cruz Biotech- Western blot using different antibodies against proteins nology). After 1-hour incubation, cells were washed three involved in TSC pathway. Each experiment has been done times in 0.1% Tween/PBS and further incubated for at least three times and individually normalized using the 30 minutes with anti-mouse Texas red–conjugated anti- antibody against β-actin. body (Vector Laboratories) to detect HtrA1T7Tag and Expression levels of HtrA1, TSC2, p-S6K, and total S6K anti-rabbit fluorescein-conjugated antibody (Jackson were analyzed by immunoblot analysis in stable clones ImmunoResearch Laboratories) to detect TSC2. Cell overexpressing HtrA1 (A2780 cells) or in stable clone spreads were counterstained with 4′,6-diamidino-2-pheny- with HtrA1 shRNA (SKOV3 and TOV21G cells). Empty

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vector–transfected cells and nontargeted control shRNA– the binding of TSC2 on HtrA1 is into the mac25 domain transduced cells served as controls. The protein in the insol- of the serine protease. Finally, no TSC1 was retained by uble fraction was used for the analysis of the proteins. GST-HtrA1wt in this experiment (Fig. 1C), thus excluding SKOV3 or TOV21G batch clonal cells with HtrA1 shRNA the hypothesis of a possible interaction of HtrA1 with the were seeded at 5 × 105 per well into six-well plates and cul- other member of the TSC complex. tured until cells became confluent. Following washing with To further investigate the association between HtrA1 1.5 mmol/L Na3VO4 in PBS twice, the cells were placed on and TSC2, a GST pull-down assay to precipitate TSC2 ice and the protein lysate was extracted with 0.5 mL of lysis from cell extracts was carried out. To this end, a cleared buffer [10 mmol/LTris (pH 8.0), 150 mmol/L NaCl, 0.2% lysate from Phoenix cells, expressing endogenous levels of NP40, 1.5 mmol/L Na3VO4], and cocktail protein inhibi- TSC2, was applied to glutathione-Sepharose beads alone tor was added into each well. Two minutes later, soluble or to glutathione-Sepharose beads bound with the GST- fraction was removed by centrifugation and the insoluble HtrA1wt chimeric construct. As shown in Fig. 2A, endog- fraction was subjected to Western blot analysis. enous TSC2 retained by the GST-HtrA1wt beads was detected by immunoblot analysis with anti-TSC2 poly- Results clonal antibody. Tuberin bound to the GST-HtrA1wt beads, but not to glutathione-Sepharose beads containing Identification of TSC2 as a binding partner of HtrA1 only the GST protein, thereby confirming that there is a To identify new proteins able to interact with HtrA1, we specific interaction between HtrA1 and TSC2. We repeated did a yeast two-hybrid screening experiment on ∼5×105 the same experiment using a specific anti-TSC1 polyclonal clones from the human fetal brain cDNA library (Clon- antibody, but no TSC1 was detectable by immunoblot tech). As a bait, we used the construct encoding full-length analysis, so it was confirmed that there is not direct inter- HtrA1-S328A, which lacks proteolytic activity and does action between HtrA1 and TSC1 (Fig. 2B). not exhibit autoproteolysis (4), to avoid the digestion of possible targets of our protease during the screening in HtrA1 interacts with the NH2-terminal region of TSC2 yeast. From the 77 colonies that grew on selective medium, To better characterize the binding between HtrA1 and 52 were positive in a β-galactosidase selection assay, and TSC2, we next decided to do a mapping experiment to 25 clones were recovered from yeast and then transformed determine the exact regions of TSC2 involved in the inter- into XL1-Blue–competent cells. Individual clones were action with HtrA1. screened and sequenced using the primers from vector. Figure 3A shows the structure of full-length TSC2 and Among rescued plasmids, we found two clones with the of the deletion constructs used for mapping experiment. cDNA of the tumor suppressor gene TSC2. The region ob- Each fragment was in vitro translated, [35S]methionine tained within the positive plasmids was the NH2-terminal labeled, and used for in vitro pull-down assay using part of TSC2; this finding suggested that the interaction GST-HtrA1wt fusion protein. As shown in Fig. 3B, the between TSC2 and HtrA1 might take place in this region. only fragment precipitated by the GST-HtrA1wt recombinant protein was the TSC2 (1-607) domain, which is the in vitro Confirmation of HtrA1/TSC2 binding first part of the NH2-terminal region of the protein. To verify the interaction between HtrA1 and TSC2 and to determine whether this association was specific, we did In vivo interaction between HtrA1 and TSC2 an in vitro binding assay. The activity of TSC2 in the cell is To investigate whether the interaction between HtrA1 strictly dependent by its interaction and binding with and TSC2 occurs also in vivo, coimmunoprecipitation TSC1, the other member of the TSC complex; the dimer experiments were done using lysate from Phoenix cells acts as a tumor suppressor only when both the proteins are transfected with pcDNA3T7Tag-HtrA1. As shown in functional and are able to interact with each other (34). Fig. 4A, a consistent amount of endogenous TSC2 was Therefore, we decided to investigate whether HtrA1 is able coimmunoprecipitated by the antibody against the epitope to interact also with TSC1. tag of HtrA1-tag overexpressed protein. The same result is BothTSC1andTSC2weretranslatedin vitro in the obtained using anti-TSC2 to coimmunoprecipitate endog- presence of [35S]methionine and incubated with GST- enous TSC2- and HtrA1-interacted protein (Fig. 4B). This HtrA1wt construct and with the truncated constructs result indicates that TSC2 and HtrA1 associate in vivo, encoding three different domains of HtrA1: GST-mac25, and the data are consistent with the results of the GST- GST-KI, and GST-HtrA (Fig. 1A). We used GST alone as HtrA1wt–binding assay. a control. Washed beads were used to detect the presence of TSC2 or TSC1 on acrylamide SDS-PAGE; gels were HtrA1 localizes both in the cytoplasm and in the dried and exposed at −70°C. extracellular space of the cell As shown in Fig. 1B, TSC2 coprecipitated with GST- The subcellular localization of HtrA1 has been investi- HtrA1wt and GST-mac25, but not with GST-HtrA; more- gated by the use of immunoelectron microscopy on human over, there was a weak interaction also between TSC2 and placental tissue. As shown in Fig. 5, we detected a dif- GST-KI, the small domain included inside mac25. This fuse immunopositivity for HtrA1 scattered in the cyto- indicates that HtrA1 interacts with TSC2, and the site of plasm. Interestingly, HtrA1 immunogold particles were

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FIGURE 1. TSC2 binds specifically to the GST-HtrA1 fusion protein. A, biochemical structure of full-length HtrA1 and different construct domains used for expression of GST fusion proteins. B, the in vitro–translated TSC2 was retained by GST-HtrA1wt and by the truncated fusion proteins GST-mac25 and GST-KI, but not by the COOH-terminal region of HtrA1wt (GST-HtrA). C, the in vitro–translated TSC1 was not retained by GST-HtrA1. Input was 20% of the sample used for pull-down assay. Equal amount of GST or fusion proteins used in the experiments is confirmed by Coomassie staining.

not associated to organelles or vesicles inside the cytoplasm. before incubation with GST or GST-HtrA1wt beads. After Moreover, as already shown in a previous article (35), it was incubation of 60 minutes at 30°C either with or without clear that HtrA1 immunopositivity was present in the extra- the addition of PP1, TSC2 was retained by the GST- cellular spaces of the same human tissues. HtrA1wt beads but not by GST alone (Fig. 7). These data clearly show that the binding between HtrA1 and TSC2 is HtrA1 and TSC2 colocalize in the cytoplasm of independent from the phosphorylation status of TSC2. Phoenix cells To further examine whether HtrA1 and TSC2 colocalize Delta-ss-mac25-HtrA1 digests TSC2 in vitro and in the cells, Phoenix cells overexpressing HtrA1 were in vivo stained using DAPI, mouse anti-T7Tag, or rabbit anti- To explore whether TSC2 is a substrate of HtrA1, we TSC2. As shown in Fig. 6A, TSC2 localizes in the cyto- did a digestion assay using purified recombinant human plasm as well as in HtrA1. Thus, in transfected Phoenix HtrA1 and human TSC2 translated in vitro and labeled 35 cells, colocalization of HtrA1 and TSC2 was evident in with L-[ S]methionine. Further, we used fibronectin the cytoplasm. In Fig. 6B, the same cells were transfected (as a positive control) to test the protease activity in our with the empty vector as a control. No positive staining condition and BSA (as a negative control) to check the was detectable in this case. specificity of HtrA1 substrate recognition (Fig. 8B). After an overnight incubation, HtrA1 completely digests TSC2 TSC2 phosphorylation is not necessary for the (Fig. 8A). As TSC2 disappeared, a lot of bands of lower interaction with HtrA1 mass in the second lane become visible, corresponding To investigate whether phosphorylation of TSC2 is to TSC2 digestion products. Probably, the formation of necessary for the HtrA1/TSC2 interaction, the effect of defined proteolytic bands is due to HtrA1 recognition of phosphatase activity on the GST-HtrA1–binding assay specific TSC2 sequences. As expected, BSA was not was analyzed. Phoenix cells lysates were treated with PP1 digested by HtrA1.

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FIGURE 2. Interaction between GST-HtrA1 and endogenous TSC2. A, Phoenix cell lysates were incubated with GST-HtrA1 beads. The amount of tuberin in the lysates and retained by HtrA1 was determined by immunoblotting. B, no hamartin was retained in the same experiment. Equal amount of GST or GST- HtrA1wt used in the experiments is confirmed by Coomassie staining.

To investigate if the degradation of TSC2 is physiologi- cancer cells with HtrA1 shRNA. As shown in Fig. 9B, cally relevant, we decided to analyze the protein level of overexpression of HtrA1 is able to induce degradation of endogenous tuberin in A2780 ovarian cancer cell line over- endogenous TSC2 in A2780 cell line; otherwise, downre- expressing HtrA1 and in SKOV3 or TOV21G ovarian gulation of HtrA1 by shRNA in SKOV3 and TOV21G

FIGURE 3. Mapping experiment. A, biochemical structure of full-length TSC2 and truncated TSC2 fragments. GAP, GTPase- activating protein. B, the in vitro– translated truncated TSC2 proteins were incubated with GST-HtrA1wt beads. The only domain retained by the beads was the NH2-terminal region of tuberin. Input was 20% of the sample used for pull-down assay. Equal amount of GST or GST-HtrA1wt used in the experiment is confirmed by Coomassie staining.

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to an increase in the endogenous levels of TSC2 with a concomitant decrease of p-S6K form.

Discussion

HtrA1 is a serine protease ubiquitously expressed in normal human adult tissues, which is found either as a secreted protein or in the cytoplasm of the cells (35). The transcription of this gene is highly regulated both during development and in the adult (35, 36), and in particular, it has been shown that HtrA1 is upregulated during the progress of human pregnancy, suggesting an important role of this protein in placental formation and function (37, 38). It is known that HtrA1 expression is decreased in progression and invasion of ovarian and endometrial cancers, melanomas, lung cancer, and mesotheliomas (10-13, 39, 40), and it is also involved in chemotherapy- FIGURE 4. Coimmunoprecipitation of TSC2 and HtrA1. A cleared induced apoptosis (32). Moreover, it has been shown lysate was prepared from Phoenix cells transfected with HtrA1 has the ability to promote degeneration of extracel- pcDNA3T7Tag-HtrA1wt. A, immunoprecipitation was done with an antibody specific for the epitope tag of the overexpressed HtrA1 protein lular matrix components, including fibronectin (6, 41), (HtrA1 Ip). Endogenous TSC2 associated with HtrA1-tag and was collagen (7), and amyloid precursor protein fragments coimmunoprecipitated. B, immunoprecipitation was done with a specific involved in Alzheimer's disease (8). anti-TSC2 antibody (TSC2 Ip). Overexpressed HtrA1 associated with To obtain additional information about the role of endogenous TSC2 and was coimmunoprecipitated. In both experiments, HtrA1 in the cell, a yeast two-hybrid system was used to a different antibody (monoclonal antibody against p21 protein) was used as a control (ctrl Ip). identify new possible targets of our protease. We decided to use the point mutant of full-length HtrA1 as a bait to cell lines leads to an increase in the endogenous levels of avoid the degradation of possible targets of our protease. TSC2 as expected. Collectively, these results indicate that It is known that the expression of HtrA1 in human em- TSC2 is a substrate of HtrA1. bryos is dramatically upregulated from the first to the third trimester of gestation (36). Because of the high expression 4E-BP1 is hyperphosphorylated in cells of HtrA1 in the brain during embryo development, we overexpressing HtrA1 decided to screen a human fetal brain cDNA library. We decided to overexpress both the full-length HtrA1 One of the proteins detected in the screening was TSC2, and the point mutant of our protein (HtrA1-S328A) into a tumor suppressor gene associated with tuberous sclerosis two different types of melanoma cell lines: LM (metastatic disease. Tuberous sclerosis is characterized by a variety of melanoma cell line) and A375 (both primary melanoma hamartomatous growths in different organs and tissues. cell line, A375P, and metastatic melanoma cell line of The defects in cell proliferation, migration, and differenti- the same cellular type, A375M). ation that these lesions display indicate that the TSC1 and As shown in Fig. 9A, the eukaryotic initiation factor 4E-BP1 seems to be hyperphosphorylated in all three types of cells overexpressing HtrA1 wild-type, whereas, in the cells transfected with the functional mutant of HtrA1, 4E-BP1 is clearly dephosphorylated if compared with the cells overexpressing HtrA1. Moreover, in the LM cells, 4E-BP1 seems to be hypophosphorylated also compared with the cells used as a control, which express the physiologic level of HtrA1.

S6K is hyperphosphorylated in stable clones overexpressing HtrA1 and hypophosphorylated in stable clone with downregulation of HtrA1 Immunoblot analysis, shown in Fig. 9Ba, shows that enhanced expression of HtrA1 in A2780 ovarian cancer cell line leads to diminished levels of TSC2. Consistent FIGURE 5. HtrA1 subcellular localization by immunoelectron microscopy assay. A wide and diffuse immunopositivity (arrows) for HtrA1 is localized with this downregulation, there was an increase of p-S6K in the cytoplasm (C) of placental cells. To note, HtrA1 immunogold protein level. Conversely, stable downregulation of HtrA1 particles were not associated to organelles or vesicles inside the in SKOV3 (Fig. 9Bb) and TOV21G (Fig. 9Bc) cells leads cytoplasm. N, nucleus. Magnification, ×40,000.

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FIGURE 6. Colocalization of TSC2 and HtrA1 in Phoenix cells. A, Phoenix cells transfected with pcDNA3T7Tag-HtrA1. Blue, cells stained with DAPI; red, cytoplasm localization of HtrA1; green, cytoplasm localization of TSC2. Colocalization (yellow) of HtrA1 and TSC2 in the cytoplasm. B, Phoenix cells transfected with pcDNA3T7Tag empty vector. No yellow staining was detectable.

TSC2 gene products participate in the control of cell by TSC1-TSC2 complex overexpression and is activated growth and division (40), and it is now recognized that in cells lacking the TSC1-TSC2 dimer (43-46). As a con- the primary function of the TSC1-TSC2 complex is a cri- sequence of TSC gene disruption, the mTOR signaling tical negative regulation of the mTOR activation (27). becomes robustly activated in a growth factor–independent mTOR plays an evolutionarily conserved role in the con- manner; moreover, elevated mTOR activity has also been trol of cell growth (i.e., an increase in cell size), but it has detected in all rodent and human tumors and tumor- also been found to regulate aspects of cell proliferation, derived cell lines lacking TSC1 or TSC2 (47-50). survival, and metabolism in specific settings. In particular, The direct interaction between HtrA1 and TSC2 was mTOR acts through two well-characterized classes of confirmed by in vitro binding experiments and by GST downstream targets: the ribosomal S6 kinases S6K1 and pull-down assays. These binding experiments indicate that S6K2, and 4E-BP1 and 4E-BP2 (42, 43). Genetic experi- HtrA1 is able to interact with TSC2 but not with the other ments in mammalian cells showed that mTOR-mediated component of the TSC complex, TSC1, thereby confirm- phosphorylation of both S6K1 and 4E-BP1 is inhibited ing that there is a specific interaction between HtrA1 and

FIGURE 7. Tuberin phosphorylation is not necessary for binding to HtrA1. A cleared Phoenix lysate was incubated at 30°C for 60 min either with (+) or without (−) PP1 before incubation with GST or GST-HtrA1 beads. The lysate was also run on gel without incubation at 30°C. TSC2 retained by the GST-HtrA1 was detected by immunoblotting. Equal amount of GST or GST-HtrA1wt used in the experiment is confirmed by Coomassie staining.

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FIGURE 8. HtrA1 digests TSC2 in vitro. A, autoradiography of 35S-labeled TSC2 incubated overnight at 37°C alone or in combination with HtrA1. In the presence of HtrA1 are detectable lower mass bands of TSC2. B, Coomassie-stained HtrA1, fibronectin, and BSA. Fibronectin, used as a control substrate of HtrA1, is digested as shown by the presence of a lower band of 30 kDa. BSA was used as a negative control of the protease activity of HtrA1.

TSC2. It is well known that the PDZ domain is the prime (8), it is possible to suggest that this proteolytic event is site of interactions for HtrA1 (41, 51-53). Nevertheless, important for HtrA1 function, as it could reduce the abi- our study shows the first interaction in which HtrA1 binds lity of HtrA1 to interact with other proteins. Interestingly, to another protein through the mac25 domain. Because in our experimental conditions, full-length HtrA1 is able this domain sometimes is cleaved off during HtrA1 activity to interact with TSC2, but the activated form of the

FIGURE 9. Effect of HtrA1 on downstream effectors of TSC2. A, 4E-BP1 is hyperphosphorylated in 3 different melanoma cell lines overexpressing HtrA1wt but not in the same cells overexpressing the point mutant of the protein. B, stable clone overexpressing HtrA1 (a) or with downregulation of HtrA1 (b, c) were analyzed to detect protein levels of TCS2 and S6K; moreover, the phosphorylation status of S6K was investigate in relation to the presence of HtrA1 and TSC2. Abbreviation: V1, empty vector; WT20, A2780 overexpressing HtrA1; sh1, clonal cells with HtrA1 shRNA.

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FIGURE 10. Schematic representation of TSC pathway and the possible role of HtrA1. A, following the activation of the pathway, insulin receptor substrate, activated by insulin-like growth factor, recruits phosphoinositide 3-kinase to the membrane. Phosphoinositide 3-kinase converts phosphatidylinositol 4,5-bisphosphate to phosphatidylinositol 3,4,5,-trisphosphate and activates the serine/threonine kinase Akt. Active Akt inhibits TSC2 activity through direct phosphorylation; thus, Akt-driven TSC1/TSC2 complex inactivation allows Rheb to accumulate in a GTP-bound state. Rheb-GTP then activates the protein kinase activity of mTOR. Downstream targets of mTOR include S6K and 4E-BP1. Once phosphorylated by mTOR, 4E-BP1 and S6K can initiate translation. B, hypothetical role of HtrA1. protein, lacking ss-signal peptide and mac25 domain, is indeed detected in the cytoplasm of the cells and that HtrA1 still able to proteolyse TSC2. immunogold particles were not associated to organelles or Evidences suggested that HtrA1 is essentially a secreted vesicles inside the cytoplasm. Moreover, endogenous protein; most of it is located in the extracellular space and TSC2 associated with HtrA1 and was coimmunoprecipi- only a small percentage of the protein remains into the cells tated in Phoenix cell lysate. Finally, colocalization analysis (54). It is also true, however, that several evidences show with immunofluorescence showed that HtrA1 and TSC2 the presence of this protease in the cytoplasm. Clawson interact into the cytoplasm of the cells. and colleagues (55) have shown that processed forms TSC2 is phosphorylated at multiple amino acid residues, of HtrA1 are found intracellularly and intranuclearly, and this phosphorylation is important for the properties of and the active intranuclear form of HtrA1 shows an binding of TSC2. Therefore, we investigated if the binding ∼ Mr 29,000. Chien and colleagues (33) have reported that between HtrA1 and TSC2 is independent by the phos- HtrA1 localized to the cytoplasm and associates with tubu- phorylation state of TSC2. Indeed, our data indicate that lins within the cell. They have shown that HtrA1 coloca- this posttranscriptional regulation of TSC2 does not affect lizes with intracellular microtubules and degrades tubulins the binding of HtrA1. into the cytoplasm. Moreover, they observed coimmuno- The role of HtrA1 as a protease capable to cleave extra- precipitation of α-, β-, and γ-tubulins when endogenous cellular proteins such as fibronectin, collagen, or compo- HtrA1 is immunoprecipitated. In a different study, He nents of cartilage is known (5, 7). In the present report, and colleagues (56) showed, with immunoprecipitation we identified the intracellular protein TSC2 as a substrate and confocal assays, that HtrA1, as well as its 35-kDa of HtrA1. By using the purified activated form of HtrA1, active form, can bind to epidermal growth factor receptor called delta-ss-mac25-HtrA1, we have shown that HtrA1 on the cell membrane. Interestingly, in a few cells, the effectively degraded in vitro–translated TSC2, generating colocalization of HtrA1 and epidermal growth factor recep- fragments of various sizes with low molecular weight. tor in the nucleus was also observed. Thesamephenomenonwasfoundin vivo using three Therefore, to confirm that the binding between our pro- different cellular systems engineered to overexpress or tease and TSC2 takes place in the cytoplasm of the cells, silence HtrA1. we decided to show this interaction also in vivo. First, by As previously described, we found that the binding site immunoelectron microscopy, we have shown that HtrA1 is of HtrA1 on TSC2 is in the same region in which TSC1

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interacts with TSC2 during the dimer formation; further- In conclusion, in our study, we have shown for the first more, our protease is able to digest TSC2. The observation time an interaction involving TSC2 and HtrA1; moreover, that TSC2, when phosphorylated, translocates to the cyto- we have shown that HtrA1 is able to digest TSC2 both plasm (57) further supports the idea that HtrA1 interacts in vitro and in vivo. The functional role of this interaction with TSC2 predominantly within cells due to this mech- is still unclear, but we can speculate that, in particular anism of phosphorylated TSC2 becoming cytosolic. physiologic or pathologic conditions, HtrA1 degrades It might be that in particular physiologic conditions in TSC2 and activates the downstream targets. Considering which HtrA1 is present in high level in the cells, TSC2 that HtrA1 levels are significantly increased during is degraded, and the downstream factors of the pathway embryogenesis, we speculate that one of the targets of are activated as depicted in the cartoon in Fig. 10. HtrA1 activity during fetal development is the TSC2- To confirm our hypothesis, we decided to investigate if TSC1 pathway. Further experiments are indeed required upregulation or downregulation of HtrA1 in vitro is able to to further define this interaction and to investigate its role cause altered expression of the factors involved in the TSC during differentiation and tumor progression. pathway. In the first experiment, we used two different types of melanoma cell lines (A375 and LM). In our experimental conditions, 4E-BP1 was hyperphosphorylated in Disclosure of Potential Conflicts of Interest cells overexpressing HtrA1, whereas the level of the same No potential conflicts of interest were disclosed. molecule was lower when we used the point mutant form of HtrA1 (HtrA1-S328A) to transfect the cells, thus confirming that the proteolytically active form of HtrA1 is able Grant Support to interfere with the expression of downstream targets of Ministry of the University, Futura-onlus, and Second University of Naples TSC2. Therefore, we decided to investigate in a different (A. Baldi). experimental setting the effects of HtrA1 upregulation or The costs of publication of this article were defrayed in part by the payment of downregulation on S6K phosphorylation status. Indeed, page charges. This article must therefore be hereby marked advertisement in we also found that this downstream effector of TSC2 accordance with 18 U.S.C. Section 1734 solely to indicate this fact. is hyperphosphorylated when HtrA1 is upregulated and, Received 10/25/2009; revised 07/15/2010; accepted 07/24/2010; published coherently, hypophosphorylated when HtrA1 is silenced. OnlineFirst 07/29/2010.

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The Serine Protease HtrA1 Specifically Interacts and Degrades the Tuberous Sclerosis Complex 2 Protein

Mara Campioni, Anna Severino, Lucrezia Manente, et al.

Mol Cancer Res Published OnlineFirst July 29, 2010.

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