Impairment of MAD2B–PRCC Interaction in Mitotic Checkpoint Defective T(X;1)-Positive Renal Cell Carcinomas
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Impairment of MAD2B–PRCC interaction in mitotic checkpoint defective t(X;1)-positive renal cell carcinomas Marian A. J. Weterman*†‡§, Jan J. M. van Groningen*‡, Leon Tertoolen¶, and Ad Geurts van Kessel* *Department of Human Genetics, University Medical Center Nijmegen, 6500 HB Nijmegen, The Netherlands; and ¶Hubrecht Laboratory, Netherlands Institute for Developmental Biology, 3584 CT Utrecht, The Netherlands Edited by Janet D. Rowley, University of Chicago Medical Center, Chicago, IL, and approved September 19, 2001 (received for review June 15, 2001) The papillary renal cell carcinoma (RCC)-associated (X;1)(p11;q21) were transfected with a bait plasmid carrying the PRCC coding translocation fuses the genes PRCC and TFE3 and leads to cancer by sequence, and consecutively with DNA of the target plasmids an unknown molecular mechanism. We here demonstrate that the containing cDNAs of a library of t(X;1)-positive tumor cells (13). mitotic checkpoint protein MAD2B interacts with PRCC. The PRCC- Transfected yeast cells were first selected for the presence of TFE3 fusion protein retains the MAD2B interaction domain, but this both bait and target vector, after which colonies were scraped interaction is impaired. In addition, we show that two t(X;1)- from the plates, titered, and replated on selective medium positive RCC tumor cell lines are defective in their mitotic check- (without Leu, Trp, His, or Ade) at a density at least 10-fold more point. Transfection of PRCCTFE3, but not the reciprocal product than was originally plated. These cells were then allowed to grow TFE3PRCC, disrupts the mitotic checkpoint in human embryonic for at least 5 days at 30°C. kidney cells. Our results suggest a dominant-negative effect of the PRCCTFE3 fusion gene leading to a mitotic checkpoint defect as an Deletion Constructs. Deletion constructs were made by using early event in papillary RCCs. primers 27–28 nt in length dispersed throughout the cDNA sequence. For the PRCC forward primers, the 5Ј ends are hromosomal translocations often occur as tumor-specific located at positions 1, 101, 201, 301, and 401 (13). The 5Ј-end of Cabnormalities, suggesting that the underlying molecular the reverse primers start at positions 491, 391, 291, 191, and 91. alterations are crucial for tumor development (1, 2). In a subset For MAD2B, the 5Ј-end of the forward primers used are located of renal cell carcinomas (RCCs) with chromophilic histology and at positions 1, 51, 101, 151, and 201, and at positions 211, 160, a mainly papillary growth pattern, referred to as papillary RCCs, 110, and 60 for the reverse primers. The resulting PCR products chromosomal translocations involving the Xp11 region, usually were cloned into pGEM-T (Promega), isolated by appropriate t(X;1)(p11;q21), are recurrently encountered (3–11). Positional restriction analysis for cloning in pBD-Gal4-T1c and pAD-Gal4- cloning of the Xp11 breakpoint by us and others revealed that the T3a, respectively, and checked by sequence analysis. The men- t(X;1)(p11;q21) translocation results in an in-frame fusion of the tioned positions correspond to the amino acid positions in the transcription factor TFE3 gene on the X-chromosome to the protein. PRCC gene on chromosome 1 (12–14). Consequently, two fusion genes are formed, TFE3PRCC and PRCCTFE3, both of which Tissue Culture, Constructs, and Transfection. Green monkey COS7 are expressed in t(X;1)-positive tumor cells (13). or COS1 cells were cultured and transiently transfected by TFE3 is a ubiquitously expressed transcription factor charac- electroporation as described before (18). CL89–12117 and terized by the presence of a basic region followed by helix-loop- CL89–17872 were cultured in RPMI-1640 medium (Life Tech- helix and leucine zipper domains, both of which are needed for nologies, Gaithersburg, MD) supplemented with 10% FCS, dimerization and DNA binding of the transcription factor (15– penicillin (100 units͞ml) and streptomycin (100 g͞ml). CL89– 17). The fusion protein PRCCTFE3 retains all these domains. 12117 cells are diploid with t(X;1) as sole karyotypic abnormal- PRCC is also ubiquitously expressed and characterized by a ity. CL89–17827 cells exhibit several numerical anomalies next to relatively high proline content. We have shown that the N- t(X;1) (4). HeLa cells were cultured in DMEM with the same terminal 156 amino acids of PRCC, when fused to TFE3, supplements. significantly elevate the transactivating capacity of this fusion For the localization studies, the PRCC and MAD2B cDNAs protein as compared with wild-type TFE3 (18). Moreover, were cloned into pECFP-N1 (CLONTECH) and pVSV2, re- transfection studies with conditionally immortalized mouse re- spectively. For fluorescence resonance energy transfer (FRET) nal proximal epithelial cells, from which chromophilic tumors analysis, COS cells were transfected with 4 g of the coding are thought to arise, showed that PRCCTFE3 could bypass regions of MAD2B cloned into pECFP-N1, and 16 gofthe temperature-induced growth arrest and differentiation (19). On PRCC cDNA cloned into pEYFP-N1 (CLONTECH). For im- the basis of the limited functional information available, we chose to further characterize PRCC via the identification of interacting proteins through yeast two-hybrid screening. This This paper was submitted directly (Track II) to the PNAS office. resulted in the identification of MAD2B, member of a family of Abbreviations: RCC, renal cell carcinoma; GFP, green fluorescent protein; DAPI, 4Ј,6- genes involved in processes of mitotic checkpoint control mech- diamidino-2-phenylindole; FRET, fluorescence resonance energy transfer; CFP, cyano flu- anisms (20–22). Our results indicate that PRCCTFE3 expression orescent protein; YFP, yellow fluorescent protein. may contribute to RCC development through a mechanism that †Present address: Department of Pathology, L2-255, Academic Medical Center Amsterdam, Meibergdreef 9, 1105 AZ Amsterdam, The Netherlands. affects the PRCC–MAD2B interaction. ‡M.A.J.W. and J.J.M.v.G. contributed equally to this work. Materials and Methods §To whom reprint requests should be addressed at: Department of Human Genetics 417, University Medical Center Nijmegen, P.O. Box 9101, 6500 HB Nijmegen, The Netherlands. Yeast Two-Hybrid Analysis. Yeast two-hybrid analysis and filter lift E-mail: [email protected]. assays were basically performed as described by the manufac- The publication costs of this article were defrayed in part by page charge payment. This turer (Stratagene). In short, yeast cells (pJ69–4A), kindly pro- article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. vided by Philip James (University of Wisconsin, Madison, WI), §1734 solely to indicate this fact. 13808–13813 ͉ PNAS ͉ November 20, 2001 ͉ vol. 98 ͉ no. 24 www.pnas.org͞cgi͞doi͞10.1073͞pnas.241304198 Downloaded by guest on September 24, 2021 munoprecipitation, 10 g of both MAD2B-pSG8-VSV and processed and normalized at 480 nm. For the image analysis, two PRCC-pEYFP-N1 were used. other filter sets were used, the first to measure the free donor T-REx-293 cells (Invitrogen) were cultured as described by emission (excitation 440(8), 455-nm dichroic, 490 emitter) and the manufacturer. The cells were stably transfected by using the second to measure the total acceptor fluorescence by using Fugene according to the recommended protocol (Roche Diag- a 515-nm dichroic mirror in combination with a 535(30) band- nostics) with 5 g of expression constructs carrying the coding pass filter (Chroma Technology, Brattleboro, VT). Images were sequences of PRCCTFE3 or TFE3PRCC cloned into a tetracyclin recorded (Leitz, ϫ50 n.a. 1.0 water immersion objective) by inducible expression vector (pcDNA͞TO͞myc-His; Invitrogen). using an Image Intensifier (Delft Electronic Products, Roden, The number of colonies was comparable between transfections The Netherlands) connected to a JAI CV-M10 progressive scan with PRCCTFE3 and TFE3PRCC (15–35). Vector transfection charge-coupled device camera and digitized with an AG-5 yielded more colonies (200). Colonies were pooled for further framegrabber (Scion, Frederick, MD). Intensities for each pixel experiments. were corrected for illumination intensities at both wavelengths (Spex reference photomultiplier), quantum efficiencies, and Polyclonal Antibodies, Immunolocalization, and Immunoprecipitation. molar extinction coefficients [ CFP(440) and yFP(495), respec- A polyclonal antibody directed against a PRCC synthetic peptide tively 32.5 ϫ 103 and 55.3 ϫ 103 MϪ⅐cmϪ1 (25)]. The image (GPPLGLPKPKKRKEP) was raised in rabbits (Genosys, The processing (4 ϫ 4 Gaussian filtering, arithmetic, false color Woodlands, TX), according to standard procedures. The final overlay, and the intensity distribution histogram) was performed bleed was used for purification using caprylic acid, followed by with Scion IMAGE for Windows NT, a modified version of NIH precipitation with ammonium sulfate (23, 24). A polyclonal IMAGE. To obtain equal amounts of protein expression, different antibody directed against recombinant MAD2B protein was ratios of MAD2B-CFP and PRCC-YFP were used for the raised using an expression construct encompassing the coding transfections. On the basis of Western blot analyses, the FRET ͞ region of MAD2B coupled to the His6 tag of the pET28a vector analysis was performed with 4 g of MAD2B-CFP 16 gof (Novagen). The serum was used without further purification. A PRCC-YFP. polyclonal antibody directed against green fluorescent protein (GFP), which