ANTICANCER RESEARCH 26: 4539-4548 (2006) The Prion-like Protein Doppel (Dpl) Interacts with the Human Receptor for Activated C-Kinase 1 (RACK1) Protein ALBERTO AZZALIN, IGOR DEL VECCHIO, LUCA FERRETTI and SERGIO COMINCINI Dipartimento di Genetica e Microbiologia, Università di Pavia, via Ferrata 1, 27100 Pavia, Italy Abstract. Background: Doppel (Dpl) is a homologue of the expressed in the testis, especially in Sertoli cells and in prion protein (PrPC). In contrast to PrPC, Dpl is dispensable for spermatozoa, and its involvement in male fertility has been prion disease, but appears to have an essential function in male recently proposed (4, 5). NMR studies of Dpl have revealed a spermatogenesis. Recently, Dpl has been found to be aberrantly high structural similarity with the prion protein (PrPC) (6, 7), expressed in astrocytic and leukaemic tumor specimens, showing which supported the possibility that the two proteins share a peculiar cytosolic cellular localization. The aim of this study similar functions in vivo. Despite a multitude of studies, the was to clarify some of the putative Dpl interacting proteins. cellular functions of Dpl and PrPC are still unknown. However, Materials and Methods: A yeast two hybrid system was employed current data suggested that Dpl, unlike PrPC, is probably not and the results were verified by co-immunoprecipitation using required for the pathogenesis of prion diseases (8, 9) and is not transfected cells. Results: Several potential Dpl-binding converted into a PrPSc-like isoform (10, 11). Importantly, Dpl candidates were identified and, among them, the receptor for can cause Purkinje cell death and ataxia when over-expressed activated C-kinase (RACK1) protein was further investigated. in Prnp-ablated mice (12). These mice are rescued from RACK1 deletion mutants showed that some of its WD neuronal degeneration by the re-introduction of a Prnp containing domains were directly involved in the binding with transgene, arguing that the absence of PrPC is necessary for Dpl. Our data showed that Dpl interacts with RACK1 by means Dpl to induce cell death (13) and that PrPC and Dpl may of its structured globular carboxyl-terminal region. Conclusion: compete for a common ligand (14). This possible PrPC-Dpl This new Dpl interacting partner might suggest functional interplay has attracted research towards the identification of hypotheses about the role of this protein in an astrocytoma common interacting proteins in order to highlight their cellular context where Dpl was found ectopically expressed. functions. In particular, several efforts have been made in order to investigate the function of PrPC, identifying many Doppel (Dpl) is an N-glycosylated, glycosylphosphati- protein interactants, although several appear to be located in dylinositol (GPI) membrane-anchored prion-like protein cellular compartments that are not compatible to sustain encoded by the prion gene (PRND) gene, which has been interactions (15). Pivotal studies of Dpl interactants started found downstream of the PRNP in several species, including with the evidence of common biochemical and topological human, mouse, cattle, sheep and goat (1-3). Dpl is highly features between Dpl and PrPC and moved towards the identification of common protein partners. To date, only one PrPC interacting protein, namely the 37 kDa/67 kDa laminin receptor (LRP) has been reported to interact also with Dpl Abbreviations: AD, activation domain; BD, binding domain; (16); besides, our group has recently reported the absence of DMEM, Dulbecco’s modified Eagle’s medium; ONPG, 2- Dpl interaction with two cytoplasmic PrPC interacting proteins, nitrophenyl ‚-D-galactopyranoside; PBS, phosphate-buffered namely GFAP and Grb2 (17), while others have reported the saline; SDS-PAGE, sodium dodecyl sulfate polyacrylamide gel C electrophoresis; X-Gal, 5-bromo-4-chloro-3-indolyl-beta-D- failure of Dpl-Dpl and Dpl-PrP interactions (18). Due to the galactopyranoside; WD, tryptophan-aspartic acid; GPI, glucosyl- incompleteness of these data, it would be useful to perform a phosphatidylinositol; LRP, laminin receptor, GFAP, glial fibrillary wider screening of Dpl interacting proteins. Thus, in this acidic protein; Grb2, growth factor receptor-bound protein2. contribution a yeast two hybrid assay is described that suggests the binding of Dpl to the receptor for activated Correspondence to: Dr. Sergio Comincini, Dipartimento di C-kinase 1 (RACK1) protein. Our results may provide new Genetica e Microbiologia, via Ferrata 1, Università di Pavia, 27100 insights into the identification of Dpl cellular functions, Pavia, Italy. Tel: +39 0382 985539, Fax: +39 0382 528496, e-mail: [email protected] particularly in some types of cancer, such as the astrocytomas, where we recently described an aberrant accumulation of Dpl Key Words: Doppel, RACK1, protein interaction. in the cellular cytoplasm (19). 0250-7005/2006 $2.00+.40 4539 ANTICANCER RESEARCH 26: 4539-4548 (2006) Table I. The table shows all the oligonucleotides designed and employed in this work. The bases flanking the gene-specific sequences are in bold; restriction sites (underlined) and endonucleases are indicated. PCR thermal profile was: 94ÆC for 3 min, 31 cycles of 94ÆC for 20 sec, 62ÆC for 30 sec, 72ÆC for 1 min; final extension at 72ÆC for 10 min. Name Sequence (5'-3') Restriction enzyme Yeast Two Hybrid Assay: YDpl26E-U CCG GAA TTC ACG AGG GGC ATC AAG CAC A EcoRI YDpl152B-L CGG GAT CCT TAG CCC CTC TCC AAC CAA AAC BamHI RACK-U TTC CAT ATG ATG ACT GAG CAG ATG ACC CTT NdeI RACK-L CGA GCT CGG CGT GTG CCA ATG GTC AC SacI Rwd4-U TTC CAT ATG GTG TGC AAA TAC ACT GTC CAG NdeI Rwd5-U TTC CAT ATG AAC TGC AAG CTG AAG ACC AAC NdeI Rwd6-U TTC CAT ATG AAC GAA GGC AAA CAC CTT TAC AC NdeI Rwd7-U TTC CAT ATG CTG AAG CAA GAA GTT ATC AGT AC NdeI Rwd5-L CGA GCT CGG AGA TCC CAT AAC ATG GCC T SacI Rwd4-L CGA GCT CGA GCC AGG TTC CAT ACC TTG A SacI Rwd3-L CGA GCT CGA CCC AGG GTA TTC CAT AGC T SacI Rwd2-L CGA GCT CGT GTG AGA TCC CAG AGG CG SacI Rwd1-L CGA GCT CGA TCC CTG GTC AGT TTC CAC A SacI Co-immunoprecipitation: Dpl26-U GGA ATT CCC ACG AGG GGC ATC AAG CAC A EcoRI Dpl152-L ATT TGC GGC CGC TTA GCC CCT CTC CAA CCA AAA C NotI Dpl53-U CCG CTC GAG CGG AGA ACC GCC CGG GAG XhoI Dpl53-L ATT TGC GGC CGC CTC AGC CAC CTG GGC C NotI RACK1-U GGG GTA CCA TGA CTG AGC AGA TGA CCC TT KpnI RACK1-L ATT TGC GGC CGC CTA GCG TGT GCC AAT GGT C NotI Materials and Methods streaked on -Leucine, -Tryptophan, -Histidine, -Adenine quadruple dropout (QDO) medium (Clontech). Single colonies Yeast two hybrid assay. The "BD Matchmakerì Library were subjected to PCR amplification, using the Matchmaker AD Construction & Screening Kit" (Clontech, Palo Alto, CA, USA) was LD-Insert Screening flanking primers, to analyze the sizes of the employed for the yeast two hybrid assay. The bait plasmid inserts contained into the prey plasmids. Transformants were containing the PRND coding region, encoding the 26-152 amino assayed for lacZ reporter transcription by a ‚-galactosidase filter acids fragment, was amplified from human genomic DNA using assay and the positive clones were subjected to a liquid 2- YDpl26E-U and YDpl152B-L primers (Table I). PCR fragments nitrophenyl ‚-D-galactopyranoside (ONPG) assay, following the were sub-cloned into the pGBKT7 plasmid, thus, generating the "Yeast Protocol Handbook" (Clontech). Finally, clones were pDpl(26-152)-bait vector. inoculated overnight at 30ÆC on TDO liquid medium: the culture Prey plasmids were derived from a cDNA library, pellets were treated with 50 U of lyticase (Sigma-Aldrich, St. retrotranscribed from 2 Ìg of total RNA, isolated from a pool of Louis, MO, USA) for 1 h at 30ÆC and the prey plasmids were 12 glioblastoma multiforme resections (19). Four hundred ng extracted with the GeneEluteì Plasmid Miniprep kit (Sigma- cDNA were amplified and co-transformed with 3 Ìg of SmaI- Aldrich). Sequencing of the DNA inserts was performed with linearized pGADT7-Rec AD Cloning Vector (Clontech) into the BigDye® Terminator Cycle Sequencing kit v 1.1 (Applied S. cerevisiae yeast strain AH109 (MATa, trp1-901, leu2-3, 112, ura3- Biosystems, Foster City, CA, USA), subjected to automated ® 52, his3-200,gal4¢, gal80¢, LYS2 :: GAL1UAS-GAL1TATA-HIS3, sequencer analysis (ABI PRISM 310 Genetic Analyzer, Applied GAL2UAS -GAL2TATA-ADE2, URA3 :: MEL1UAS-MEL1TATA-lacZ, Biosystems). The sequences were finally analyzed for nucleotide MEL1), following the manufacturer’s instructions. similarities with Genbank database entries using the BLAST After an in vivo recombination-mediated cloning, algorithm (http://www.ncbi.nlm.nih.gov/BLAST/). transformants were plated on a -Leucine single dropout (SD) Different portions of the RACK1 sequence were amplified from medium (Clontech), recovered and transformed again into the the cDNA library with the primers containing NdeI and SacI AH109 strain with 5 Ìg of the pDpl(26-152)-bait plasmid. restriction sites, shown in Table I; the primer pairs used for each Transformants were plated on -Leucine, -Tryptophan, -Histidine construct are shown in Figure 1. PCR products were sequenced, triple dropout (TDO) medium (Clontech). Colonies appeared sub-cloned into pGADT7 plasmids and subjected to ONPG liquid after 5-6 days of incubation at 30ÆC and were subsequently assays, in the presence of the pDpl(26-152)-bait plasmid. 4540 Azzalin et al: Dpl Interacts with RACK1 Figure 1. ONPG liquid assays of protein extracts from cells containing the bait construct, BD/Dpl(26-152), and the RACK1 deletion mutants (AD/RACK1¢). The figure shows, from the left, the employed primer pairs used to obtain mutants, the name of the AD/RACK1¢ fusion proteins produced, the schematic representation of the deleted proteins with the indication of RACK1 residues (between square brackets) and the ‚-galactosidase activity.