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• W2068530092 abstract "The fasciculation and elongation protein ζ1 (FEZ1) is a mammalian orthologue of the Caenorhabditis elegans protein UNC-76, which is necessary for axon growth in that nematode. In previous studies FEZ1 has been found to interact with protein kinase Cζ, DISC1, the agnoprotein of the human polyomavirus JC virus, and E4B, a U-box-type ubiquitin-protein isopeptide ligase. We reported previously that FEZ1 and its paralogue FEZ2 are proteins that interact with NEK1, a protein kinase involved in polycystic kidney disease and DNA repair mechanisms at the G2/M phase of the cell cycle. Here we report the identification of 16 proteins that interact with human FEZ1-(221–396) in a yeast two-hybrid assay of a human fetal brain cDNA library. The 13 interacting proteins of known functions take part either in transcription regulation and chromatin remodeling (6 proteins), the regulation of neuronal cell development (2 proteins) and cellular transport mechanisms (3 proteins) or participate in apoptosis (2 proteins). We were able to confirm eight of the observed interactions by in vitro pull-down assays with recombinant fusion proteins. The confirmed interacting proteins include FEZ1 itself and three transcription controlling proteins (SAP30L, DRAP1, and BAF60a). In mapping studies we found that the C-terminal regions of FEZ1, and especially its coiled-coil region, are involved in its dimerization, its heterodimerization with FEZ2, and in the interaction with 10 of the identified interacting proteins. Our results give further support to the previous speculation of the functional involvement of FEZ1 in neuronal development but suggest further that FEZ1 may also be involved in transcriptional control. The fasciculation and elongation protein ζ1 (FEZ1) is a mammalian orthologue of the Caenorhabditis elegans protein UNC-76, which is necessary for axon growth in that nematode. In previous studies FEZ1 has been found to interact with protein kinase Cζ, DISC1, the agnoprotein of the human polyomavirus JC virus, and E4B, a U-box-type ubiquitin-protein isopeptide ligase. We reported previously that FEZ1 and its paralogue FEZ2 are proteins that interact with NEK1, a protein kinase involved in polycystic kidney disease and DNA repair mechanisms at the G2/M phase of the cell cycle. Here we report the identification of 16 proteins that interact with human FEZ1-(221–396) in a yeast two-hybrid assay of a human fetal brain cDNA library. The 13 interacting proteins of known functions take part either in transcription regulation and chromatin remodeling (6 proteins), the regulation of neuronal cell development (2 proteins) and cellular transport mechanisms (3 proteins) or participate in apoptosis (2 proteins). We were able to confirm eight of the observed interactions by in vitro pull-down assays with recombinant fusion proteins. The confirmed interacting proteins include FEZ1 itself and three transcription controlling proteins (SAP30L, DRAP1, and BAF60a). In mapping studies we found that the C-terminal regions of FEZ1, and especially its coiled-coil region, are involved in its dimerization, its heterodimerization with FEZ2, and in the interaction with 10 of the identified interacting proteins. Our results give further support to the previous speculation of the functional involvement of FEZ1 in neuronal development but suggest further that FEZ1 may also be involved in transcriptional control. FEZ1 (fasciculation and elongation protein ζ-1) was initially identified as a mammalian orthologue of the Caenorhabditis elegans UNC-76 protein, which is necessary for normal axonal outgrowth, bundling, and elongation in this nematode (1Bloom L. Horvitz H.R. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 3414-3419Crossref PubMed Scopus (146) Google Scholar). FEZ1 mRNA is expressed abundantly in the rat adult brain and throughout all developmental stages of the brain in mouse embryos (2Kuroda S. Nakagawa N. Tokunaga C. Tatematsu K. Tanizawa K. J. Cell Biol. 1999; 114: 403-411Crossref Scopus (84) Google Scholar, 3Fujita T. Ikuta J. Hamada J. Okajima T. Tatematsu K. Tanizawa K. Kuroda S. Biochem. Biophys. Res. Commun. 2004; 313: 738-744Crossref PubMed Scopus (25) Google Scholar). The human FEZ1 includes 392 amino acid residues, and its predicted structural organization shows that the protein possesses three glutamine-rich regions and a coiled-coil region (4Suzuki T. Okada Y. Semba S. Orba Y. Yamanouchi S. Endo S. Tanaka S. Fujita T. Kuroda S. Nagashima K. Sawa H. J. Biol. Chem. 2005; 280: 24948-24956Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar) (Fig. 3). A mammalian homologue of FEZ1, the protein FEZ2, which shows ubiquitous tissue expression, was described in rat and human and has 48% amino acid sequence identity to FEZ1 (3Fujita T. Ikuta J. Hamada J. Okajima T. Tatematsu K. Tanizawa K. Kuroda S. Biochem. Biophys. Res. Commun. 2004; 313: 738-744Crossref PubMed Scopus (25) Google Scholar). Interestingly, FEZ1 was identified as an interacting protein partner in several yeast two-hybrid screens with independent protein baits. The first bait shown to interact with FEZ1 was the regulatory domain of the protein kinase C ζ (PKCζ) 4The abbreviations used are: PKC, protein kinase C; GST, glutathione S-transferase; 3-AT, 3-amino 1,2,4-triazole; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride. 4The abbreviations used are: PKC, protein kinase C; GST, glutathione S-transferase; 3-AT, 3-amino 1,2,4-triazole; PBS, phosphate-buffered saline; PVDF, polyvinylidene difluoride. (2Kuroda S. Nakagawa N. Tokunaga C. Tatematsu K. Tanizawa K. J. Cell Biol. 1999; 114: 403-411Crossref Scopus (84) Google Scholar). It was further found that FEZ1 is a cellular substrate for phosphorylation through PKCζ and that phosphorylated FEZ1 promotes neurite extension of PC12 cells in the absence of nerve growth factor. In a second study, using the C terminus of DISC1 (disrupted-in-schizophenia 1) as bait, FEZ1 was also identified as an interacting protein. The DISC1 gene has been implicated as a candidate gene for the etiology of schizophrenia (5Millar J.K. Wilson-Annan J.C. Anderson S. Christie S. Taylor M.S. Semple C.A.M. Devon R.S. Clair D.M. Muir W.J. Blackwood D.H. Porteous D.J. Hum. Mol. Genet. 2000; 9: 1415-1423Crossref PubMed Scopus (1072) Google Scholar, 6Miyoshi K. Honda A. Baba K. Taniguchi M. Oono K. Fujita T. Kuroda S. Katayama T. Tohyama M. Mol. Psychiatry. 2003; 8: 685-694Crossref PubMed Scopus (260) Google Scholar, 7Kamiya A. Kubo K. Tomoda T. Takaki M. Youn R. Ozeki Y. Sawamura N. Park U. Kudo C. Okawa M. Ross C.A. Hatten M.E. Nakajima K. Sawa A. Nat. Cell Biol. 2005; 7: 1167-1178Crossref PubMed Scopus (444) Google Scholar). The interaction of FEZ1 and DISC1 was found to be up-regulated in PC12 cells during neuronal differentiation, and this caused an enhanced extension of neurites in the presence of nerve growth factor (6Miyoshi K. Honda A. Baba K. Taniguchi M. Oono K. Fujita T. Kuroda S. Katayama T. Tohyama M. Mol. Psychiatry. 2003; 8: 685-694Crossref PubMed Scopus (260) Google Scholar). Third, the agnoprotein of the human polyomavirus JC virus, the causative agent of a fatal demyelinating disease, showed direct interaction with FEZ1 and microtubules (4Suzuki T. Okada Y. Semba S. Orba Y. Yamanouchi S. Endo S. Tanaka S. Fujita T. Kuroda S. Nagashima K. Sawa H. J. Biol. Chem. 2005; 280: 24948-24956Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). It was further verified that FEZ1 associated with the microtubules and that the agnoprotein induced FEZ1 dissociation from the microtubules leading to inhibited neurite outgrowth in PC12 cells. Furthermore, FEZ1 was found to interact with E4B, a U-box-type ubiquitin-protein isopeptide ligase, again via yeast two-hybrid system studies (8Okumura F. Hatakeyama S. Matsumoto M. Kamura T. Nakayama K. J. Biol. Chem. 2004; 279: 53533-53543Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar). This interaction is enhanced in the presence of PKCζ, and phosphorylation and/or ubiquitination of FEZ1 may contribute to neurite extension. We found FEZ1, as well as its paralogue FEZ2, as NEK1 protein kinase interacting proteins in a previous study (9Surpili M.J. Delben T.M. Kobarg J. Biochemistry. 2003; 42: 15369-15376Crossref PubMed Scopus (61) Google Scholar). Members of the NEKs (Nima-related kinases) take part in the regulation of the cell cycle and meiosis and constitute the kinase family so far less well characterized functionally (10Quarmby L.M. Mahjoub M.R. J. Cell Sci. 2005; 118: 5161-5169Crossref PubMed Scopus (120) Google Scholar). Further NEK1 interactors include kinesin family member 3A (KIF3A), which had been described to also interact with FEZ1 (4Suzuki T. Okada Y. Semba S. Orba Y. Yamanouchi S. Endo S. Tanaka S. Fujita T. Kuroda S. Nagashima K. Sawa H. J. Biol. Chem. 2005; 280: 24948-24956Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). The FEZ1 orthologue UNC-76 has also been reported to interact with kinesin (11Gindhart J.G. Chen J. Faulkner M. Gandhi R. Doerner K. Wisniewski T. Nandlestadt A. Mol. Biol. Cell. 2003; 14: 3356-3365Crossref PubMed Scopus (72) Google Scholar). Together, this may suggest that UNC-76/FEZ1 could play a role in kinesin-mediated transport pathways (4Suzuki T. Okada Y. Semba S. Orba Y. Yamanouchi S. Endo S. Tanaka S. Fujita T. Kuroda S. Nagashima K. Sawa H. J. Biol. Chem. 2005; 280: 24948-24956Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar). We set out to use FEZ1 itself as bait in a yeast two-hybrid assay and screened a human fetal brain cDNA library for potential FEZ1-interacting proteins. We found that FEZ1 interacts with itself and were able to confirm both FEZ1 homodimerization as well as its heterodimerization with FEZ2 by a series of in vitro experiments. In total we identified 16 FEZ1-interacting proteins that are either involved in transcriptional regulation (6 proteins), neuronal cell development (2 proteins), intracellular transport processes (3 proteins) or apoptosis (2 proteins), or are of unknown function (3 proteins), and we were also able to confirm 8 of these interactions by in vitro pull-down assays with recombinant proteins. In summary, our results further support previous findings that FEZ1 may be a regulatory protein with important functions during neuronal development, possibly through its involvement in intracellular transport processes. However, our new finding that FEZ1 interacts with nuclear proteins, six of which are functionally involved in transcription regulation and chromatin remodeling, opens the intriguing new possibility that FEZ1 may in addition to the previous predicted functions also have regulatory functions in the nucleus. Plasmid Constructions—Several sets of oligonucleotides were designed for PCR amplification of complete FEZ1 or deletion constructs thereof, which were then inserted in vector pBTM116 in fusion with the LexA DNA binding domain or in vectors pACT2 or pGAD424 (Clontech) in fusion with the Gal4 activation domain (Fig. 1A). The nucleotide sequence coding residues 207–353 of human FEZ2 were PCR-amplified using a specific primer set, 5′-AGGAATTCGGCAGTTATGAAGAGAGAGTG-3′ and 5′-AGGTCGACGTTACACTCTCTCTTCATAACT-3′, and then cloned into EcoRI and SalI restriction site in vector pBTM116. To express full-length FEZ1 or its different indicated deletions fused to a His tag, the corresponding nucleotide sequences were amplified by PCR and inserted into bacterial expression vector pET28a (Novagen/EMD Biosciences, San Diego, CA). FEZ1-(131–392) represents a FEZ1 clone obtained in a yeast two-hybrid screening using the regulatory domain of NEK1 (9Surpili M.J. Delben T.M. Kobarg J. Biochemistry. 2003; 42: 15369-15376Crossref PubMed Scopus (61) Google Scholar) and was subcloned and expressed using the vector pProEX-HTc (Invitrogen). For expression of BAF60a-(404–515) and complete FEZ1 as well as its different deletion constructs fused to GST, the corresponding nucleotide sequences were cloned into a modified vector pET28a-GST that codifies GST protein upstream of the protein to be inserted. All nucleotide sequences encoding the proteins identified to interact with the FEZ1-(221–392), except that encoding BAF60a-(404–515), were subcloned from the vector pACT2 to the bacterial expression vector pGEX-4T-2 (GE Healthcare, Waukesha, WI), which allows the expression of the proteins in the form of a GST fusion. The orientation, frame, and correctness of sequence of each insert DNA were confirmed by restriction endonuclease analysis and automated DNA sequencing. Yeast Two-hybrid Screen and DNA Sequence Analyses—The yeast two-hybrid screen (12Chien C.T. Bartel P.L. Sternglanz R. Fields S. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 9578-9582Crossref PubMed Scopus (1225) Google Scholar) of a human fetal brain cDNA library (Clontech) was performed by using the yeast strain L40 (trp1-901, his3Δ200, leu2-3, ade2 LYS2::(lexAop)4-HIS3 URA3::(lexAop)8-lac GAL4) and human FEZ1-(221–392) as a bait fused to the yeast LexA DNA binding domain in vector pBTM116 (13Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (302) Google Scholar). This fragment of FEZ1 does not autoactivate the yeast reporter genes (see Fig. 1 and “Results”). The autonomous activation test for HIS3 was performed in minimal medium plates without tryptophan and histidine but containing 0, 5, 10, 20, 30, or 50 mm of 3-amino-1,2,4-triazole (3-AT). Furthermore, the autonomous activation of LacZ was measured by the β-galactosidase filter assay described below. Yeast cells were transformed according to the protocols supplied by Clontech. The screening was performed in minimal medium plates without tryptophan, leucine, and histidine. Half of the transfected cells were plated in selective medium containing 10 mm 3-AT, and the other half was plated on selective medium without addition of 3-AT. Recombinant pACT2 plasmids of positive clones were isolated and their insert DNAs sequenced with a DNA sequencer model 377S (Applied Biosystems, Foster City, CA). The obtained DNA sequence data were translated using the TRANSLATE Tool of ExPASy (Expert Protein Analysis System), available online, and compared with sequences in the NCBI data bank using the BLASTP 2.2.12 program (14Altschul S.F. Madden T.L. Schäffer A.A. Zhang J. Zhang Z. Millerm W. Lipman D.J. Nucleic Acids Res. 1997; 25: 3389-3402Crossref PubMed Scopus (59762) Google Scholar). The prediction of coiled-coil structures in the analyzed protein sequences was performed by the software COILS available on line (Swiss Institute for Experimental Cancer Research). Assay for β-Galactosidase Activity in Yeast Cells—β-Galactosidase activity in yeast cells was determined by the filter assay method. Yeast transformants (Leu+, Trp+, and His+) were transferred onto nylon membranes, permeabilized in liquid nitrogen, and placed on Whatman 3MM paper previously soaked in Z buffer (60 mm Na2HPO4, 40 mm NaH2PO4, 10 mm MgCl2, 50 mm 2-mercaptoethanol, pH 7.0) containing 1 mg/ml 5-bromo-4-chloro-3-indolyl-β-d-galactoside (X-gal). After incubation at 37 °C for 1 h, the yeast cells forming dark blue colonies were taken from replica plates for further analysis. Mapping the Protein Interaction Sites—One of the deletion constructs of FEZ1-(269–392) had the coiled-coil region removed. This construct was co-transformed in Saccharomyces cerevisiae strain L40 with the “bait”-plasmid DNAs isolated from the two-hybrid screening. After transformation, yeast clones were streaked on minimal medium plates without tryptophan, leucine, and histidine for testing their growth capacity under interaction-selective conditions. The presence of both types of plasmids was controlled by growth on plates with minimal medium plates without tryptophan and leucine (15Vojtek A.B. Hollenberg S.M. Methods Enzymol. 1995; 255: 331-342Crossref PubMed Scopus (236) Google Scholar). To further test if the paralogue of FEZ1, FEZ2, interacts with the proteins that were isolated in the yeast two-hybrid screen using FEZ1, the corresponding bait-plasmid DNAs were co-transformed in L40 with the construct pBTM116-FEZ2-(207–353). FEZ2-(207–353) alone does not transactivate the reporter genes, and the co-transfected L40 clones were selected on minimal medium without tryptophan, leucine, and histidine. Protein Expression and Purification—The nucleotide sequences in the library vector pACT2, which are inserted between restriction sites EcoRI and XhoI and code for the interacting proteins identified in the yeast two-hybrid system screen, were subcloned into the bacterial expression vector pGEX-4T-2 (GE Healthcare, Waukesha, WI) to allow expression of recombinant GST fusion proteins in Escherichia coli BL21 (DE3) cells. Soluble FEZ1 (complete or deletions), fused to the His tag or the GST tag, was purified for in vitro analyses from 1 liter of culture of E. coli BL21 (DE3) cells that were induced for 3 h to protein expression at 30 °C using 0.4 mm isopropyl 1-thio-β-d-galactopyranoside. For size exclusion chromatography, the nucleotide sequence of the pACT2 clone (Fig. 1A), containing FEZ1-(131–392), was subcloned into expression vector pProExHTb (Invitrogen) using the restriction sites EcoRI and XhoI, as described (9Surpili M.J. Delben T.M. Kobarg J. Biochemistry. 2003; 42: 15369-15376Crossref PubMed Scopus (61) Google Scholar). All His-tagged proteins used in this study were purified using a HiTrap chelating column in anÄKTA™ FPLC™ (GE Healthcare) as follows. Cells were harvested by centrifugation at 4,500 × g for 10 min, and the cell pellet was resuspended and incubated for 30 min with 10 volumes of lysis buffer (137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4, pH 7.4, 1 mg/ml lysozyme, 1 mm phenylmethylsulfonyl fluoride, and 0.05 mg/ml DNase). After three cycles of sonication, soluble and insoluble fractions were separated by centrifugation at 28,500 × g for 30 min at 4 °C. The cleared supernatant was then loaded onto a HiTrap chelating column (GE Healthcare) pre-equilibrated with lysis buffer (lacking lysozyme and DNase), followed by extensive wash of the column with the same buffer. Bound proteins were eluted in a gradient of 0–100% of elution buffer (137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4, 1 mm phenylmethylsulfonyl fluoride, and 500 mm imidazole, pH 7.4). Aliquots of each eluted fraction obtained were analyzed by SDS-PAGE, and peak fractions containing FEZ1 were dialyzed with buffer (137 mm NaCl, 2.7 mm KCl, 10 mm Na2HPO4·7H2O, 1.8 mm KH2PO4, 0.5 mm dithiothreitol, and 5% glycerol, pH 7.4). The FEZ1 proteins (complete or deletions) fused to GST were induced for expression as described above. After sonication the lysate was cleared by centrifugation at 28,500 × g for 30 min at 4 °C. The resulting supernatant was incubated with glutathione-Uniflow resin (Clontech) and used for in vitro binding assay. The cDNA coding the proteins identified by yeast two-hybrid assay were cloned into the vector pGEX-4T-2 in fusion with GST, and fusion proteins were expressed in E. coli BL21 (DE3) cells at 37 °C using 0.5 mm isopropyl 1-thio-β-d-galactopyranoside for 4 h. In Vitro Binding Assay—Expressed GST, GST-KIBRA, GST-SAP30L, GST-CLASP2, GST-RAI14, GST-Bamacan, GST-DRAP1, and GST-BAF60a proteins were allowed to bind to 25 μl of glutathione-Uniflow resin (Clontech) in 1 ml of total bacterial protein extract in PBS for 1 h at 4 °C. After incubation, the beads containing bound recombinant proteins were washed three times with PBS (0.14 m NaCl, 2.7 mm KCl, 10 mm Na2HPO4, 1.8 mm KH2PO4, pH 7.4) at 4 °C. 25 μg of purified full-length 6xHis-FEZ1 fusion protein were added to the resins containing GST or GST fusion proteins and incubated in 0.1 ml of PBS 1× for 4 h at 4 °C to allow protein-protein interactions to occur. The beads were then washed three times with 0.5 ml of PBS, followed by three washings with 0.5 ml of PBS containing 0.1% Triton X-100, then three washes with 0.5 ml of PBS only. Resin-bound proteins were resolved on two separate 10% SDS-polyacrylamide gels. After electrophoresis, the proteins were transferred to PVDF membranes by semi-dry electroblotting. After saturation with unspecific protein (5% bovine serum albumin) in TBS (0.15 m NaCl, 20 mm Tris-HCl, 0.05% Tween 20, pH 7.2), one of the membranes was incubated with a mouse anti-His tag (1:5000) and the other with mouse monoclonal anti-GST antibody 5.3.3 (hybridoma supernatant 1:5) for 1 h each. The anti-GST monoclonal antibody 5.3.3 had been generated by immunizing BALB/c mice with a GST-CGI-55 recombinant fusion protein (16Lemos T.A. Passos D.O. Nery F.C. Kobarg J. FEBS Lett. 2003; 533: 14-20Crossref PubMed Scopus (49) Google Scholar). Selection of hybridoma producing anti-CGI-55 or anti-GST antibodies was tested by enzyme-linked immunosorbent assay with purified recombinant 6xHis-CGI-55 or GST protein. Specificity of the recloned hybridoma 5.3.3 was confirmed by anti-GST Western blot. After three washes with TBS, 0.05% Tween 20, the membranes were incubated with the secondary horseradish peroxidase-conjugated anti-mouse IgG antibody (1:5000; Santa Cruz Biotechnology) for 1 h and washed again three times with TBS. The membranes were then developed by chemiluminescence using the reagent Luminol (Santa Cruz Biotechnology) for detection of His-tagged or GST fusion proteins. To confirm the in vitro interaction of FEZ1 with itself, GST-FEZ1-(1–392), GST-FEZ1-(1–227), GST-FEZ1-(221–392), or GST-FEZ1-(269–392), which lacks the coiled-coil region, were all allowed to bind to glutathione-Uniflow resin as described above. For each preparation of loaded beads, we added in separate reactions 25 μg of each of the following three different purified 6xHis-FEZ1 fusion proteins, 6xHis-FEZ1-(1–392), 6xHis-FEZ1-(1–227), or 6xHis-FEZ1-(221–392), which were incubated, washed, and analyzed for protein interaction by Western blot as described above. Size Exclusion Chromatography—Five milligrams of purified 6xHis-FEZ1-(131–392) were loaded on a Superdex™ 75 10/30 Prep Grade column (GE Healthcare) that had been equilibrated previously with 20 mm Tris, pH 7.5, 150 mm NaCl and calibrated with the following standard proteins: aldolase (158 kDa), bovine serum albumin (67 kDa), ovalbumin (43 kDa), and chymotrypsinogen A (25 kDa). 0.5-ml fractions were collected, and 10-μl samples of each fraction of the different peaks observed were analyzed by 12.5% SDS-PAGE. Analysis of the Auto-activation of Different FEZ1 Constructs in the Yeast Two-hybrid System—Before the yeast two-hybrid screening of a human fetal brain cDNA library, the construction encoding full-length FEZ1 was tested for autonomous activation of the reporter gene HIS3, i.e. capacity of growth in minimal medium (lacking tryptophan and histidine) and with addition of 0, 5, 10, 20, 30, or 50 mm 3-AT, an inhibitor of HIS3, that suppresses background growth of the yeast on minimal medium lacking histidine. To further investigate the autonomous activation of the second reporter gene lacZ, we also performed the β-galactosidase filter assay. The results of both assays revealed a strong and autonomous activation of the reporter genes HIS3 and lacZ by the full-length protein FEZ1. To solve this problem, a set of oligonucleotides was designed to subclone different regions of FEZ1 into the pBTM116 vector and to test them for autoactivation. Only the two constructions FEZ1-(221–392) and FEZ1-(269–392), which lack the coiled-coil region, did not show autonomous activation of the HIS3 and lacZ reporter genes (Fig. 1B). The construction FEZ1-(221–392) was chosen for the screening of the human fetal brain cDNA library. Interestingly, all N-terminal constructs of FEZ1 showed strong autoactivation (1–129, 122–227, 122–392, and 1–227). This may indicate that FEZ1 could be a transcriptional activation protein, and its putative transcriptional activation domain located at its N terminus seems to have a conserved function in respect to the activation of the reporter genes in yeast. It is noteworthy that the three glutamic acid-rich motifs are found in the N-terminal region of FEZ1 (Fig. 3B), the second and third of which are partially conserved also in FEZ2. Identification of Proteins that Interact with FEZ1—To identify proteins interacting with FEZ1, we employed the yeast two-hybrid system (13Bartel P.L. Fields S. Methods Enzymol. 1995; 254: 241-263Crossref PubMed Scopus (302) Google Scholar) and screened a human fetal brain cDNA library. FEZ1-(221–392) was used as bait, and a total of about 1.5 × 106 co-transformed clones were assayed in two groups. Although the first half of transformants was plated on selective minimal medium plates (without tryptophan, leucine, and histidine), the another half was plated on selective minimal medium (without tryptophan, leucine, and histidine) with the addition of 10 mm 3-AT. All grown colonies that showed a strong blue color in the subsequent β-galactosidase filter assay had their plasmid DNA extracted and sequenced. A total of 101 plasmid DNAs from clones positive for both HIS3 and LacZ reporters were sequenced. 16 different proteins were identified using FEZ1-(221–392) as bait (Table 1), which can be organized into the following groups according the major described function attributed to them: 1) proteins involved in transcriptional control and chromatin organization (6 proteins); 2) proteins that take part in the regulation of neural cell development, microtubule dynamics, and transport (5 proteins); 3) proteins taking part in apoptosis processes (2 proteins) or tumorigenesis; and 4) proteins with little or no functional information available as yet (3 proteins). Table 1 summarizes the domain organization and functional characteristics of the proteins found to interact with FEZ1.TABLE 1Human FEZ1-interacting proteins identified by the yeast two-hybrid system screenProtein interacting with FEZ1C (aliases)Coded protein residues (complete sequence/retrieved)Accession no.Domain composition (native protein)aOther domains may be present.FunctionbOther functions may be known.Ref.DRAP1 (NC2α)205/1-205NP_006433Histone domain, coiled-coil regionTranscriptional control23Kamada K. Shu F. Chen H. Malik S. Stelzer G. Roeder R.G. Meisterernest M. Burley S.K. Cell. 2001; 106: 71-81Abstract Full Text Full Text PDF PubMed Scopus (126) Google ScholarBAF60a (SMARCD1)515/82-515 and 515/404-515AAH09368SWIB domain, coiled-coil regionTranscriptional control; recruitment of chromatin-remodeling complex by specific transcription factors27Hsiao P.W. Fryer C.J. Trotter K.W. Wang W. Archer T.K. Mol. Cell. 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Biol. 2004; 24: 9059-9069Crossref PubMed Scopus (59) Google ScholarTlk2830/33-830AAH44925Catalytic domain of a serine/threonine kinase; coiled-coil regionTranscriptional control; chromatin remodeling; DNA damage checkpoint17Silljé H.H.W. Takahashi K. Tanaka K. Van Houwe G. Nigg E.A. EMBO J. 1999; 18: 5691-5702Crossref PubMed Scopus (103) Google ScholarZinc finger protein 251293/25-293AAH06258ZnF C2H2Transcriptional control?18Strausberg R.L. Feingold E.A. Grouse L.H. Derge J.G. Klausner R.D. Collins F.S. Wagner L. Shenmen C.M. Schuler G.D. Altschul S.F. Zeeberg B. Buetow K.H. Schaefer C.F. Bhat N.K. Hopkins R.F. Jordan H. Moore T. Max S.I. Wang J. Hsieh F. Diatchenko L. Marusina K. Farmer A.A. Rubin G.M. Hong L. Stapleton M. Soares M.B. Bonaldo M.F. Casavant T.L. Scheetz T.E. Brownstein M.J. Usdin T.B. Toshiyuki S. Carninci P. Prange C. Raha S.S. Loquellano N.A. Peters G.J. Abramson R.D. Mullahy S.J. Bosak S.A. McEwan P.J. McKernan K.J. Malek J.A. Gunaratne P.H. Richards S. Worley K.C. 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