[CANCER RESEARCH 58. 4766-4770. November I. 1998] Advances in Brief Hiimartin, the Product of the Tuberous Sclerosis l (TSC1} Gene, Interacts with Tuberin and Appears to Be Localized to Cytoplasmic Vesicles1 Tracey L. Plank, Raymond S. Yeung, and Elizabeth Petri Henske2 Dcfiat'tinenl of Medical Oncology, Fo.\ Chase Cancer Center. Philadelphia Pennsylvania Will ¡T.L P., E. P. H.j. and Department cf Surgery, University of Washington. Seattle. Wuxhinxtiin IK. S. Y.¡ Abstract Tuberous sclerosis is an inherited syndrome associated with mutations in two tumor suppressor genes: TSCI and TSC2. Tuberin, the product of TSC2, appears to be localized to the Golgi apparatus and may have a function in vesicular transport. The function of hamartin, the product of TSCI, is not known. In this report, we demonstrate an interaction between hamartin and tuberin, which is detectable at endogenous protein levels. Hamartin is present in a cell line derived from the Eker rat that lacks functional tuberin, indicating that the stability of hamartin is not depend ent on its interaction with tuberin. Hamartin is localized to the membrane/ particulale (PI(H)) fraction of cultured cells. The I"I(10 localization is unchanged in the Eker cells. Finally, we show that at endogenous expres sion levels, hamartin has a punctate pattern of immunofluorescence in the cytoplasm. Taken together, the presence of hamartin in the membrane/ participate fraction and its pattern of cytoplasmic staining suggest that it is localized to cytoplasmic vesicles. If altered vesicular trafficking leads to tumorigenesis in tuberous sclerosis, TSCI and TSC2 may have a novel mechanism of tumor suppression. Introduction TSC3 is an autosomal dominant tumor suppressor gene syndrome characterized by seizures, mental retardation, autism, and tumors of multiple organ systems (1, 2). The tumors, which are primarily be nign, arise in the brain, heart, kidney, skin, and retina. Two genes are associated with TSC: (a) TSCÃŒ on chromosome 9q34 (3); and (b) TSC2 on chromosome 16pl3 (4). Germ-line TSCÃŒandTSC2 muta tions appear to be inactivating, and loss of heterozygosity at either the TSCI or the TSC2 region occurs in TSC tumors (5-8), indicating that TSCI and TSC2 are tumor suppressor genes. The protein product of the TSC2 gene, tuberin, is expressed in a variety of different cell types (9, 10). Sequence analysis has revealed a region near the COOH terminus of tuberin with homology to the catalytic domain of the GAP RaplGAP. Tuberin has GAP activity for Rapi (11) and Rab5 (12). Tuberin has been localized to the perinuclear region of cultured cells and has been shown at endogenous expression levels to colocalize with Rapi in the stacks of the Golgi apparatus (13). The TSCI gene was identified in 1997 (3). Its protein product, hamartin, is primarily hydrophilic. with a putative coiled-coil domain near the COOH terminus. Hamartin has no homology to tuberin or other known vertebrate proteins. It does have a possible homologue in Schizoxaccltanmiyces pombe with no identified function. Recently, a coiled-coil domain near the COOH terminus of hamartin was shown Received 8/14/98; accepted 9/17/98. The costs of publication of this article were defrayed ¡npart by the payment of page charges. This article must therefore he hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicale this fact. 1 Supported in part by grants from the National Tuberous Sclerosis Association. The Lymphangiomyomatosis Foundation, and the W. W. Smith Charitable Trust. 2 To whom requests for reprints should be addressed, at Department of Medical Oncology, Fox Chase Cancer Center. 7701 Burholme Avenue. Philadelphia. PA 19111. Phone: (2151 728-2428: Fax: (215) 728 2741: E-mail: EP_Henske<s>fccc.edu. 1The abbreviations used are: TSC. tuberous sclerosis complex; GAP. GTPase-acti- valing protein: GST. glutathione 5-transferasc. to bind specifically to a coiled-coil domain near the NH2 terminus of tuberin (14). suggesting that tuberin and hamartin are partners in a common cellular pathway. To gain insight into the function of hamartin, we have raised a rabbit polyclonal antibody against a COOH-terminal peptide from hamartin that specifically recognizes a Mr 130,000 protein in both human and rat cells. In this report, we independently demonstrate the interaction of hamartin with tuberin in vivo at endogenous protein levels. We also show that hamartin is localized to the membrane/ paniculate (100.000 X g) fraction in cells containing tuberin and also in Eker rat tumor-derived cells lacking tuberin. Hamartin has a punc tate pattern of cytoplasmic immunofluorescence, which is consistent with vesicular localization. These findings suggest that hamartin is localized to cytoplasmic vesicles. This pattern of localization, together with data suggesting that tuberin plays a role in vesicular transport, may indicate that tumorigenesis in TSC is the result of aberrant vesicular trafficking. Materials and Methods Antibodies. A polyclonal antibody to hamartin referred to as Ham-C was generated against a peptide from the COOH terminus of the TSCI polypeptide (residues 1146-1164). The peptide was conjugated to keyhole limpet hemo- cyanin and used as an immunogen. Two rabbits were immuni/.ed with 0.1 mg of the peptide in an emulsion with Freund's adjuvant. Boosts with O.I mg of peptide were given during weeks 2. 6, and 8. Antiserum was prepared after ammonium sulfate precipitation (50% saturation) by immunoat'finity purifica tion using an AminoLink column with the COOH-terminal peptide as the immobili/.ed ligand (Pierce Chemical Co.). Fractions from the column con taining antibodies to hamartin were identified by dot-blot analysis with the peptide immobili/.ed on nitrocellulose. The Ham-C antibody was used at a 1:100 dilution for Western blotting. The C20 tuberin antibody (Santa Cruz Biotechnology. Inc.) was generated against a peptide mapping at the COOH terminus of tuberin. The C20 antibody was used at a 1:1500 dilution for Western blotting. For immunoprecipitation. 3 ;nl of the C20 antibody were added to 500 ¡JL\ of cell lysate. The L3 tuberin antibody was generated against a GST fusion protein containing the COOH-terminal of tuberin ( 12). A 1:2500 dilution of the L3 antibody was used for Western blotting. For immunopre cipitation, 1 ¡i\of the L3 antibody was added to 500 ¿¿1 of cell lysate. The anti-/3-tubulin antibody (Boehringer Mannheim) was used at a 1:400 dilution for immunoblotting. Cell Lines and Culture Conditions. The human embryonic kidney cell line 293. the rat tlbroblast cell line Rat I-R12. and the rat normal kidney epithelial cell line NRK-52E were obtained from the American Type Culture Collection. Ratl-R12 cells, which are transf'ected with a plasmid containing a tetracycline transactivator gene and the neomycin resistance gene, were cul tured in DMEM containing 10% fetal bovine serum and 0.4 mg/ml G418. NRK-52E cells were maintained in DMEM containing 0.1 mM nonessential amino acids and 5% calf serum. The 293 cell line was cultured in DMEM with 10% horse serum. The Eker tumor cell line ERC18M (12). which was the gift of Alfred Knudson (Fox Chase Cancer Center. Philadelphia, PA), was derived from an Eker rat renal cell carcinoma with the subsequent passage of the cell line through a SCID mouse. Passage of ERC18M cells was performed as described previously (12). 4766 Research. on November 28, 2021. © 1998 American Association for Cancer cancerres.aacrjournals.org Downloaded from