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• W2006491932 abstract "Rek (retina-expressed kinase) has been identified as a putative novel receptor-type tyrosine kinase of the Axl/Tyro3 family with a potential role in neural cell development. rek clones were isolated from a chick embryonic brain cDNA library with a DNA probe obtained by reverse transcriptase-polymerase chain reaction of mRNA from Müller glia-like cells cultured from chick embryonic retina. Sequence analysis indicated that Rek is a protein of 873 amino acids with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains with eight predicted N-glycosylation sites. Two consensus src homology 2 domain binding sites are present in the cytoplasmic domain, suggesting that Rek activates several signal transduction pathways. Northern analysis of rek mRNA revealed a 5.5-kilobase transcript in chick brain, retina, and kidney and in primary cultures of retinal Müller glia-like cells. Rek protein was identified by immunoprecipitation and immunoblotting as a 140-kDa protein expressed in the chick retina at embryonic days 6-13, which corresponded to the major period of neuronal and glial differentiation. Transfection of rek cDNA into COS cells resulted in transient expression of a putative precursor of 106 kDa that autophosphorylated in immune complex protein kinase assays. Overexpression of rek cDNA in mouse NIH3T3 fibroblasts resulted in activation of the 140-kDa rek kinase and induction of morphologically transformed foci. These properties indicated that Rek has oncogenic potential when overexpressed, but its normal function is likely to be related to cell-cell recognition events governing the differentiation or proliferation of neural cells. Rek (retina-expressed kinase) has been identified as a putative novel receptor-type tyrosine kinase of the Axl/Tyro3 family with a potential role in neural cell development. rek clones were isolated from a chick embryonic brain cDNA library with a DNA probe obtained by reverse transcriptase-polymerase chain reaction of mRNA from Müller glia-like cells cultured from chick embryonic retina. Sequence analysis indicated that Rek is a protein of 873 amino acids with an extracellular region composed of two immunoglobulin-like domains followed by two fibronectin type III domains with eight predicted N-glycosylation sites. Two consensus src homology 2 domain binding sites are present in the cytoplasmic domain, suggesting that Rek activates several signal transduction pathways. Northern analysis of rek mRNA revealed a 5.5-kilobase transcript in chick brain, retina, and kidney and in primary cultures of retinal Müller glia-like cells. Rek protein was identified by immunoprecipitation and immunoblotting as a 140-kDa protein expressed in the chick retina at embryonic days 6-13, which corresponded to the major period of neuronal and glial differentiation. Transfection of rek cDNA into COS cells resulted in transient expression of a putative precursor of 106 kDa that autophosphorylated in immune complex protein kinase assays. Overexpression of rek cDNA in mouse NIH3T3 fibroblasts resulted in activation of the 140-kDa rek kinase and induction of morphologically transformed foci. These properties indicated that Rek has oncogenic potential when overexpressed, but its normal function is likely to be related to cell-cell recognition events governing the differentiation or proliferation of neural cells. Receptor tyrosine kinases are widely expressed in the developing nervous system, where they play important roles in development of neurons and glia (1Maness P.F. Cox M.E. Semin. Cell Biol. 1992; 3: 117-126Google Scholar). Upon binding of membrane-bound or diffusable ligands to their extracellular domains, these enzymes autophosphorylate on cytoplasmic tyrosine residues, which serve as docking sites for src homology 2 (SH2) 1The abbreviations used are: SH2src homology 2Igimmunoglobulin-likeFNfibronectinPCRpolymerase chain reactionRACErapid amplification of cDNA endskbkilobase pair(s)PAGEpolyacrylamide gel electrophoresisRIPAradioimmune precipitation assayRLBRek lysis bufferEGFepidermal growth factor. domain-containing signal transduction proteins. In the developing fly eye, the receptor tyrosine kinase encoded by the sevenless gene is activated by a neighboring cell surface protein encoded by the boss gene (2Kramer H. Cagan R.L. Zipursky S.L. Nature. 1991; 352: 207-212Google Scholar), causing precursor cells to differentiate into photoreceptors (3Hafen E. Basler K. Edstrom J.-E. Rubin G.M. Science. 1987; 236: 55-63Google Scholar, 4Tomlinson, A., Ready, D. F., (1987) 123, 264–275.Google Scholar). In vertebrates, activation of receptor tyrosine kinases of the trk family by nerve growth factor, brain-derived neurotrophic factor, or neurotrophin-3 and neurotrophin-4/5 induces differentiation and survival of different neuronal populations (5Glass D.J. Yancopoulos G.D. Trends Cell Biol. 1993; 3: 262-268Google Scholar). In the peripheral nervous system, glial growth factor/heregulin causes multipotent neural crest progenitors to differentiate into glia rather than neurons (6Marchionni M.A. Goodearl A.D.J. Chen M.S. Bermingham-McDonogh O. Kirk C. Hendricks M. Danehy F. Misumi D. Sudhalter J. Kobayashi K. Wroblewski D. Lynch C. Baldassare M. Hiles I. Davis J.B. Hsuan J.J. Totty N.F. Otsu M. McBurney R.N. Waterfield M.D. Stroobant P. Gwynne D. Nature. 1993; 362: 312-318Google Scholar) by activating the erbB2/c-neu/HER2 receptor tyrosine kinase in the presence of the erbB4/HER4 tyrosine kinase (7Plowman G.D. Green J.M. Culouscou J.-M. Carlton G.W. Rothwell V.M. Buckley S. Nature. 1993; 366: 473-475Google Scholar). src homology 2 immunoglobulin-like fibronectin polymerase chain reaction rapid amplification of cDNA ends kilobase pair(s) polyacrylamide gel electrophoresis radioimmune precipitation assay Rek lysis buffer epidermal growth factor. Receptor tyrosine kinases were initially identified as homologs of retroviral oncogene products (8Downward J. Yarden Y. Mayes E. Scrace G. Totty N. Stockwell P. Ullrich A. Schlessinger J. Waterfield M.D. Nature. 1984; 307: 521-527Google Scholar); thus, it is not surprising that mutations in proto-oncogenes that result in constitutive activation of normal receptor tyrosine kinases render these proteins oncogenic. Such activating mutations can occur within the coding region of the extracellular domain, for example in the retroviral oncogene v-erbB (8Downward J. Yarden Y. Mayes E. Scrace G. Totty N. Stockwell P. Ullrich A. Schlessinger J. Waterfield M.D. Nature. 1984; 307: 521-527Google Scholar) and trk/nerve growth factor receptor gene (9Martin-Zanca D. Hughes S.H. Barbacid M. Nature. 1986; 319: 743-748Google Scholar). Alternatively, the mutation can be an amino acid substitution in the transmembrane region, as shown for the erbB/c-neu/HER2 gene in chemically induced rat glioblastomas (10Bargmann C.I. Hung M.-C. Weinberg R.A. Cell. 1986; 45: 649-657Google Scholar). Another means by which a receptor tyrosine kinase can become constitutively activated is by overexpression, which may result in forced receptor dimerization within the plasma membrane (11Heldin C.-H. Cell. 1995; 80: 213-223Google Scholar). This is one mechanism that may contribute to the deregulation of growth in glial cell tumors. For example, the erbB2/c-neu/HER2 gene is overexpressed in certain human glioblastomas (12Schwechheimer K. Laufle R.M. Schmahl W. Knodlseder M. Fisher H. Hofler H. Hum. Pathol. 1994; 25: 772-780Google Scholar), while the epidermal growth factor receptor gene, c-erbB, is amplified to various degrees in human glioblastomas with the highest levels of expression correlating with poor prognosis (13Libermann T.A. Nusbaum H.R. Razon N. Kris R. Lax I. Soreq H. Whittle N. Waterfield M.D. Ullrich A. Schlessinger J. Nature. 1985; 313: 144-147Google Scholar). Protein tyrosine phosphorylation in the developing neural retina is unusually active in Müller glia (14Sorge L.K. Levy B.T. Maness P.F. Cell. 1984; 36: 249-257Google Scholar, 15Ingraham C.A. Cooke M.P. Chuang Y.-N. Perlmutter R.M. Maness P.F. Oncogene. 1992; 7: 95-100Google Scholar, 16Shores C.G. Maness P.F. J. Neurosci. Res. 1989; 24: 59-66Google Scholar, 17Biscardi J.S. Shores C.G. Maness P.F. Curr. Eye Res. 1991; 10: 1121-1128Google Scholar). The Müller glial cell is the principal glial cell type in the vertebrate eye, where its chief function is to buffer the microenvironment following neuronal firing (18Newman E.A. Federoff S. Vernadakis A. The Müller Cell in Astrocytes. Vol. 1. Academic Press, London1986: 149-171Google Scholar). The normally quiescent Müller cell proliferates in an unregulated manner in several pathological situations, including retinal detachment, diabetic retinopathy, proliferative vitreoretinopathy, and macular pucker (19Wiedemann P. Survey Opthalmol. 1992; 36: 373-384Google Scholar). At least one receptor tyrosine kinase, the basic fibroblast growth factor receptor, is up-regulated in Müller glia that have been induced to proliferate in animal models of injury (20Lewis G.P. Erickson P.A. Guerin C.J. Anderson D.H. Fisher S.K. J. Neurosci. 1992; 12: 3968-3978Google Scholar). Müller glia and all types of retinal neurons differentiate from a common progenitor by a poorly understood process that depends on growth factors and other local environmental cues such as cell-cell contact (21Turner D.L. Cepko C.L. Nature. 1987; 328: 131-136Google Scholar, 22Wetts R. Fraser S.E. Science. 1988; 239: 1142-1145Google Scholar, 23Anchan R.M. Reh T.A. Angello J. Balliet A. Walker M. Neuron. 1991; 6: 923-936Google Scholar). Because receptor tyrosine kinases are important in growth factor and cell contact recognition, it is likely that they regulate retinal cell differentiation. Protein tyrosine phosphorylation in the developing chick retina increases strikingly during the period of differentiation of retinal neurons and Müller glia and is most abundant in regions where Müller glial processes contact their neighbors (16Shores C.G. Maness P.F. J. Neurosci. Res. 1989; 24: 59-66Google Scholar, 17Biscardi J.S. Shores C.G. Maness P.F. Curr. Eye Res. 1991; 10: 1121-1128Google Scholar, 24Biscardi J.S. Cooper N.G.F. Maness P.F. Exp. Eye Res. 1993; 56: 281-289Google Scholar). Immunoelectron microscopy of the outer chick retina with phosphotyrosine antibodies revealed that phosphotyrosine-modified proteins accumulate predominantly in Müller glia and that they are located in the Müller glial plasma membrane at sites of contact with adjacent Müller glial processes and photoreceptors (24Biscardi J.S. Cooper N.G.F. Maness P.F. Exp. Eye Res. 1993; 56: 281-289Google Scholar). To identify protein-tyrosine kinases that might be responsible for the elevated protein tyrosine phosphorylation in developing Müller cells, we used reverse transcriptase-polymerase chain reaction (PCR) with primers specific to highly conserved regions shared within the catalytic domain of receptor and nonreceptor-class protein-tyrosine kinases to amplify partial cDNAs encoding tyrosine kinases expressed in Müller glia-enriched cultures from embryonic chick retina. Such an approach has been used with success to identify novel tyrosine kinase genes expressed in nonneural as well as neural cells (25Wilks A.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1603-1607Google Scholar, 26Lai C. Lemke G. Neuron. 1991; 6: 691-704Google Scholar). Here we describe the discovery of a novel receptor-type tyrosine kinase, termed Rek (retina-expressed kinase), a new member of the Axl/Tyro3 family of receptor tyrosine kinases. The Axl/Tyro 3 family includes receptor tyrosine kinases encoded by axl (ufo, ark) (27O'Bryan J.P. Frye R.A. Cogswell P.C. Neubauer A. Kitch B. Prodop C. Espinosa III, R. Le Beau M.M. Earp H.S. Liu E.T. Mol. Cell. Biol. 1991; 11: 5016-5031Google Scholar, 28Rescigno J. Mansukhani A. Basilico C. Oncogene. 1991; 6: 1909-1913Google Scholar, 29Faust M. Ebensperger C. Schulz A.S. Schleithoff L. Hameister H. Bartram C.R. Janssen J.W.G. Oncogene. 1992; 7: 1287-1293Google Scholar, 73Janssen J.W.G. Schulz A.S. Steenvoorden A.C.M. Schmidberger M. Strehl S. Ambros P.F. Bartram C.R. Oncogene. 1991; 6: 2113-2120Google Scholar, 85O'Bryan J.P. Fridell Y.-W. Koski R. Varnum B. Liu E.T. J. Biol. Chem. 1996; 270: 551-557Google Scholar), tyro3 (sky, brt, rse, tif) (26Lai C. Lemke G. Neuron. 1991; 6: 691-704Google Scholar, 30Ohashi K. Mizuno K. Kuma K. Miyata T. Nakamura T. Oncogene. 1994; 9: 699-703Google Scholar, 31Fujimoto J. Yamamoto T. Oncogene. 1994; 9: 693-698Google Scholar, 32Dai W. Pan H. Hassanain H. Gupta S.L. Murphy Jr., M.J. Oncogene. 1994; 9: 975-979Google Scholar, 33Mark M.R. Scadden D.T. Wang Z. Gu Q. Goddard A. Godowski P.J. J. Biol. Chem. 1994; 269: 10720-10728Google Scholar), c-eyk (34Jia R. Hanafusa H. J. Biol. Chem. 1994; 269: 1839-1844Google Scholar), and c-mer (35Graham D.K. Dawson T.L. Mullaney D.L. Snodgrass H.R. Earp H.S. Cell Growth & Differ. 1994; 5: 647-657Google Scholar). There is evidence that these kinases have transforming potential. Axl was originally identified as a protein encoded by a transforming gene from primary human myeloid leukemia cells (27O'Bryan J.P. Frye R.A. Cogswell P.C. Neubauer A. Kitch B. Prodop C. Espinosa III, R. Le Beau M.M. Earp H.S. Liu E.T. Mol. Cell. Biol. 1991; 11: 5016-5031Google Scholar). Axl is overexpressed in a number of different tumor cell types and transforms mouse NIH3T3 fibroblasts (27O'Bryan J.P. Frye R.A. Cogswell P.C. Neubauer A. Kitch B. Prodop C. Espinosa III, R. Le Beau M.M. Earp H.S. Liu E.T. Mol. Cell. Biol. 1991; 11: 5016-5031Google Scholar). Experimental overexpression of Tyro3 causes anchorage-independent growth of Rat-2 fibroblasts (36Lai C. Gore M. Lemke G. Oncogene. 1994; 9: 2567-2578Google Scholar). Also, the murine homolog of sky has been shown to be expressed at elevated levels in mouse mammary tumors (37Taylor I.C.A. Roy S. Yaswen P. Stampfer M.R. Varmus H.E. J. Biol. Chem. 1995; 270: 6872-6880Google Scholar). c-eyk is a chicken proto-oncogene that was first identified as the retroviral transforming gene v-ryk (34Jia R. Hanafusa H. J. Biol. Chem. 1994; 269: 1839-1844Google Scholar). A close relative, c-mer, is a human proto-oncogene expressed in malignant B- and T-lymphocytic cell lines (35Graham D.K. Dawson T.L. Mullaney D.L. Snodgrass H.R. Earp H.S. Cell Growth & Differ. 1994; 5: 647-657Google Scholar). The hallmark of the Axl/Tyro3 family is an extracellular region consisting of two immunoglobulin-like (Ig) and two fibronectin III (FN) domains. These domains are found in cell recognition molecules such as the neural cell adhesion molecules L1 and NCAM (38Williams A.F. Barclay A.N. Annu. Rev. Immunol. 1988; 6: 381-405Google Scholar) and certain receptor tyrosine phosphatases (39Gebbink M.F.B.G. van Etten I. Hateboer G. Suijkerbuijk R. Beijersbergen R.L. van Kessel A.G. Moolenaar W.H. FEBS Lett. 1991; 290: 123-130Google Scholar, 40Sap J. Jiang Y.-P. Grumet M. Schlessinger J. Mol. Cell. Biol. 1994; 14: 1-9Google Scholar). Homotypic and heterotypic binding involving extracellular determinants have been demonstrated for some Axl/Tyro3 family members. Homotypic binding has been shown to activate the Axl tyrosine kinase (41Bellosta P. Costa M. Lin D.A. Basilico C. Mol. Cell. Biol. 1995; 15: 614-625Google Scholar), an event that may be important in signaling cell adhesion. Axl (42Varnum B.C. Young C. Elliot G. Garcia A. Bartley T.D. Fridell Y.-H. Hunt R.W. Trail G. Clogston C. Toso R.J. Yanagihara D. Bennett L. Sylber M. Merewether L.A. Tseng A. Escobar E. Liu E.T. Yamane H.K. Nature. 1995; 373: 623-626Google Scholar) and, to a lesser extent, Tyro3 (43Godowski P.J. Mark M.R. Chen J. Sadick M.D. Raab H. Hammonds R.G. Cell. 1995; 82: 355-358Google Scholar) are also activated by a heterophilic ligand, Gas6, a vitamin K-dependent protein that is up-regulated during growth arrest in confluent fibroblast cultures. Protein S, which bears significant homology to Gas6, was found to be a heterophilic ligand for Tyro3 (44Stitt T.N. Conn G. Gore M. Lai C. Bruno J. Radziejewski C. Mattsson K. Fisher J. Gies D.R. Jones P.F. Masiakowski P. Ryan T.E. Tobkes N.J. Chen D.H. DiStefano P.S. Long G.L. Basilico C. Goldfarb M.P. Lemke G. Glass D.J. Yancopoulos G.D. Cell. 1995; 80: 661-670Google Scholar). Protein S is an anticoagulant in serum and a mitogen for smooth muscle cells (45Gasic G.P. Arenas C.P. Gasic T.B. Gasic G.J. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 2317-2320Google Scholar), but its up-regulation in Schwann cells following nerve injury suggests that it may also serve as a neural growth or differentiation factor (44Stitt T.N. Conn G. Gore M. Lai C. Bruno J. Radziejewski C. Mattsson K. Fisher J. Gies D.R. Jones P.F. Masiakowski P. Ryan T.E. Tobkes N.J. Chen D.H. DiStefano P.S. Long G.L. Basilico C. Goldfarb M.P. Lemke G. Glass D.J. Yancopoulos G.D. Cell. 1995; 80: 661-670Google Scholar). The molecular cloning, structural analysis, and expression of the Rek receptor tyrosine kinase in developing chick neural tissues is described here. It is also demonstrated that overexpression and activation of Rek in mouse NIH3T3 fibroblasts leads to cell transformation, indicating that the rek gene has oncogenic potential that might contribute to malignant growth of nervous system tumors. Primary cultures enriched for Müller glia were prepared from embryonic day 10 chicken retinas as described previously (17Biscardi J.S. Shores C.G. Maness P.F. Curr. Eye Res. 1991; 10: 1121-1128Google Scholar, 46Adler R. Methods Neurosci. 1990; 2: 134-150Google Scholar). Retinas were dissected free of sclera-choroid-pigmented epithelium and cells seeded on tissue culture dishes in DMEM, 10% fetal calf serum. By 7-10 days, a mixed culture developed consisting of a monolayer of flat cells underlying a network of neuronal fascicles and cell clumps, which were removed by gentle mechanical agitation. As described previously, the identification of the flat cells as Müller glia is supported by abundant intermediate filaments (47Li H.P. Sheffield J.B. Tissue Cell Res. 1984; 16: 843-857Google Scholar), staining with vimentin antibodies (48Lemmon V. Rieser B. Brain Res. 1983; 313: 191-197Google Scholar), lack of binding to tetanus toxin, [3H]thymidine incorporation, GABA and glutamate uptake (49dePomerai D.I. Carr A. Exp. Eye. Res. 1982; 34: 553-563Google Scholar, 50dePomerai D.I. Carr A. Hyndman A.G. Adler R. Dev. Brain Res. 1982; 2: 303-314Google Scholar), expression of a filamin-related protein (51Lemmon V. J. Neurosci. 1986; 6: 43-51Google Scholar), and the presence of carbonic anhydrase and glutamine synthetase (52Linser J.D. Moscona A.A. Dev. Biol. 1983; 96: 529-534Google Scholar). The glial-like cells do not show induction of glutamine synthetase by hydrocortisone, a response characteristic of Müller cells in vivo (52Linser J.D. Moscona A.A. Dev. Biol. 1983; 96: 529-534Google Scholar), and are negative for expression of glial fibrillary acidic protein, perhaps due to the absence of necessary intercellular interactions in the monolayers. They will be referred to as Müller glia-like cultures, but they may not represent fully differentiated Müller cells or their precursors. Hatchling chicks (White Leghorn, NC State) were anesthetized with Ketamine and sacrificed by decapitation. Staging of chick embryos was done according to the guidelines of Hamilton and Hamburger (53Hamburger V. Hamilton H.L. J. Morphol. 1951; 88: 49-92Google Scholar). For RNA, tissues were rapidly excised, frozen in liquid nitrogen, and stored at −80°C until use. Poly(A)+-selected RNA was isolated using the FastTrack RNA isolation kit (Invitrogen). Poly(A)-selected RNA (1 μg) from Müller glial cell cultures was reverse transcribed using random hexamers as described by O'Bryan et al. (27O'Bryan J.P. Frye R.A. Cogswell P.C. Neubauer A. Kitch B. Prodop C. Espinosa III, R. Le Beau M.M. Earp H.S. Liu E.T. Mol. Cell. Biol. 1991; 11: 5016-5031Google Scholar) in 10 mM Tris, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 0.001% gelatin, 625 μM each deoxynucleoside triphosphate, 20 units of RNAsin (Promega), 10 mM of dithiothreitol, and 200 units of Moloney murine leukemia virus reverse transcriptase (final volume, 20 μl). The reaction was incubated for 10 min at room temperature and then for 45 min at 45°C. This first strand cDNA (1 μl) was amplified in a 100-μl reaction containing 10 mM Tris HCl, pH 8.3, 50 mM KCl, 1.5 mM MgCl2, 200 μM each dNTP, 0.001% gelatin, 1 μg each primer, and 1.25 units of Taq polymerase (Promega). The degenerate oligonucleotide primer sequences of Wilks (25Wilks A.F. Proc. Natl. Acad. Sci. U. S. A. 1989; 86: 1603-1607Google Scholar) were used corresponding to the following amino acid sequences: PTK1, CGG ATC CAC (A/C)GN GA(C/T) (C/T)T; PTK2, CT(G/A)CA(G/C) ACC AGG A(A/T)A CCT TAA GG. Reaction conditions were as follows: 1.5 min at 95°C (denaturing), 2 min at 37°C (annealing), and 3 min at 63°C (elongation). The PCR products were ligated into the plasmid pBluescript (Stratagene) and transfected into DH5 Escherichia coli cells (Life Technologies, Inc.), and colonies were selected after induction with 5-bromo-4-chloro-3-indoyl β-D-galactoside and isopropyl-1-thio-β-D-galactopyranoside. To produce a DNA fragment of suitable length for a hybridization probe, a modification of the 3′-RACE method (54Frohman M.A. Dush M.K. Martin G.R. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 8998-9002Google Scholar) was used to generate a 1.4-kb fragment representing the 3′-end of rek cDNA extending from the sequence encoding the IHRDL motif in the catalytic domain to the poly(A) tail. For this purpose, an oligo(dT)-primed λZAPII cDNA library was generated from Müller glia-enriched cultures using a cDNA cloning kit (Stratagene). A nondegenerate primer specific to rek (seal-1, ATG CTG GAT GAG AAC ATG AAT; corresponding to amino acids MLDENMN; residues 649-655) was used as the sense primer, and an oligonucleotide (pBlu5) specific to the pBluescript phagemid between the XhoI site and the T7 promoter (ATA GGG CGA ATT GGG TAC) was the antisense primer. PCR was carried out as above with 0.75 mM MgCl2, using the following reaction conditions: 5 min at 94°C (denaturing), 5 min at 60°C (annealing), and 4 min at 72°C (extending) for 1 cycle, followed by 45 s at 94°C, 45 s at 60°C, and 4 min at 72°C for 35 cycles. The PCR product was directly ligated into the pCR plasmid (TA cloning vector, Stratagene), DH10 E. coli cells were transformed, and colonies were selected. The insert sequence from one of the 3′-RACE clones was sequenced, verifying that it specified the Rek catalytic domain. An oligo(dT)-primed cDNA library in λ gt10 from chick embryonic brain (day 13) (Barbara Ranscht, Burnham Cancer Research Foundation) was screened at high stringency using the 32P-radiolabeled 3′-RACE clone (1.4 kb) as probe. Out of the 28 positive primary clones obtained, 15 clones survived plaque purification through secondary and tertiary screening. Subclones of the longest clone (approximately 4 kb) were generated by exonuclease III digestion, and DNA sequencing was completed for both strands. This clone contained a long open reading frame, a 3′-untranslated region containing an internal A-rich sequence, and a poly(A) addition site, but it lacked a start codon and signal peptide. The additional 5′-sequence was obtained by rescreening the brain library using as probe a PCR fragment amplified from the 5′-region of the 4-kb clone. Twelve clones were plaque-purified through tertiary screening, and their 5′-regions were sized by PCR. The clone with the longest 5′-region was sequenced and found to contain the full rek coding sequence, including a putative start codon and signal peptide sequence. The remainder of the sequence was identical to the 4-kb cDNA clone. Interestingly, the clone specifying the entire protein sequence (3.3 kb) represented a cDNA that had been internally primed at an A-rich sequence in the 3′-untranslated region of the mRNA. Two methods of DNA sequence analysis were used. Manual DNA sequencing by the dideoxy chain termination method was carried out using Sequenase T7 polymerase (U.S. Biochemical Corp.) with T3 and T7 primers. Automated sequencing was performed in the UNC-Chapel Hill Automated DNA Sequencing Facility (Dr. Laura Livingstone, Director), which employs a model 373A DNA sequencer (Applied Biosystems). Both sense and antisense strands were sequenced. Sequences were compared by the FASTA program to the GenBank™/EMBL and SwissProt data bases using the GCG software package. Contiguous clones were aligned using the GAP program and then merged using the ASSEMBLE program. The PILEUP program was used to compare the Rek amino acid sequence with other members of the Axl/Tyro3 family. The phylogenetic tree was generated by the computer program Phylogenetic Analogy Using Parsimony (PAUP)(55). RNA was separated on denaturing 1% agarose, 2% formaldehyde gels and subjected to blot hybridization on Hybond N membranes at high stringency (83Huff J.L. Jelinek M.A. Borgman C.A. Lansing T.J. Parsons J.T. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 6140-6144Google Scholar). Hybridization using a 32P-radiolabeled DNA probe generated by PCR amplification of the 1.4-kb 3′-RACE clone was carried out at 42°C overnight in 5 × SSC, 40% formamide, 5 × Denhardt's solution, 0.1% SDS, 1 mM NaH2PO4, and 200 μg/ml boiled salmon sperm DNA. Washes were as follows: 6 × SSC, 0.1% SDS for 30 min at 42°C, 2 × SSC, 0.1% SDS for 30 min at 42°C, and 1 × SSC, 0.1% SDS for 20 min at 55°C. The filters were exposed to film at −80°C for 6 days with intensifying screens. Normalization of the amount of RNA loaded was confirmed by hybridizing to an actin probe, which was generated by PCR amplification of Müller glial cDNA with actin specific primers as described in O'Bryan et al. (27O'Bryan J.P. Frye R.A. Cogswell P.C. Neubauer A. Kitch B. Prodop C. Espinosa III, R. Le Beau M.M. Earp H.S. Liu E.T. Mol. Cell. Biol. 1991; 11: 5016-5031Google Scholar). Chicken genomic DNA was partially digested with EcoRI or HindIII, and fragments were separated by agarose gel electrophoresis and blotted to nitrocellulose. Filters were hybridized to two EcoRI/BamHI fragments (0.4 and 0.9 kb) encoding the entire extracellular and transmembrane domains (nucleotides 1-1602), which were 32P-labeled by random priming. Filters were hybridized overnight in 50% (v/v) formamide, 4 × SSC, 5% (w/v) Denhardt's solution, 20 mM sodium phosphate, pH 7.5, at 42°C and washed two times for 20 min each in 2 × SSC, 0.5% SDS at room temperature, once in 2 × SSC, 0.1% SDS for 20 min at room temperature, and once in 2 × SSC, 0.1% SDS for 30 min at 50°C. Filters were exposed to x-ray film for 24 h. Two different BamHI-HindIII fragments of the 3.3-kb rek cDNA clone were used to express Rek fusion proteins as antigens. One (for antibody A) encoded 305 amino acids of the kinase domain (residues 459-764), and the other (for antibody B) encoded the carboxyl-terminal 100 residues. The fragments were subcloned into the pLC24 vector (56Derynck R. Remaut E. Saman E. Stanssens P. De Clercq B. Content J. Fiers W. Nature. 1980; 287: 193-197Google Scholar), and fusion proteins with 98 amino acids of the MS2 polymerase protein were expressed in E. coli upon temperature shift from 28 to 42°C. The proteins were purified by preparative SDS-PAGE and used to inoculate rabbits. Rek fusion proteins (150 μg) in complete Freund's adjuvant were used for the primary injection and for each of three boosts in Freund's incomplete adjuvant. Antisera were screened by Western blotting against the fusion proteins. Where indicated, antibodies were purified from IgG preparations by immunoaffinity on an Affi-Gel column (Bio-Rad) to which the fusion proteins were covalently coupled. Antibodies were eluted with 50 mM diethylamine and dialyzed against phosphate-buffered saline. The entire 3.3-kb cDNA encoding the complete Rek protein was cloned into the EcoRI site of the vector pSG5 (57Green S. Issemann I. Sheer E. Nucleic Acids Res. 1988; 16: 369-373Google Scholar) and transfected into bacteria. Clones were selected with rek cDNA in either the sense or antisense orientation with respect to the SV40 promoter of pSG5. COS-7 cells (in 100-mm dishes) were transfected for 6 h in OptiMEM medium (Life Technologies, Inc.) with the sense or antisense construct (5 μg) using lipofectamine (36 μl; Life Technologies, Inc.). Cells were washed and incubated in fresh medium and then passaged 24 h later onto new 100-mm dishes. Metabolic labeling was carried out for 14 h with an 35S-protein labeling solution (EXPRE35S35S, DuPont NEN) containing radiolabeled L-methionine and L-cysteine in DMEM (without methionine and cysteine), 10% dialyzed fetal bovine serum. Cells were lysed in RIPA buffer and immunoprecipitated with preimmune serum or rek antiserum (350 μl of lysate, 12 μl of serum) or Rek antiserum preadsorbed of Rek-specific antibodies by passing IgG through an Affi-Gel column to which the antigen-fusion protein was coupled. Proteins were separated by SDS-PAGE and visualized by fluorography on x-ray film (1-week exposure). Rek was immunoprecipitated from either RIPA (24Biscardi J.S. Cooper N.G.F. Maness P.F. Exp. Eye Res. 1993; 56: 281-289Google Scholar) or Brij 97 detergent extracts of transfected COS cells or Rek lysis buffer (RLB) extracts of transfected NIH3T3 cells using antibodies prepared as described above and protein A-Sepharose. The composition of Brij 87 buffer was 1% Brij-97, 10 mM Tris, pH 7.4, 150 mM NaCl, 1 mM NaEDTA, 1 mM NaEGTA, 10 mM NaF, 200 μM Na3VO4, 500 μg/ml Pefabloc, 0.01% leupeptin, 0.11 trypsin inhibitory units/ml aprotinin. The composition of RLB was 50 mM HEPES, pH 6.5, 150 mM NaCl, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100, 1 mM Na-EGTA, 200 μM sodium orthovanadate, 10 mM NaF, 0.11 trypsin inhibitory units/ml aprotinin, 0.01% leupeptin. Immune complexes were washed in RIPA, Bri" @default.
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• W2006491932 date "1996-11-01" @default.
• W2006491932 modified "2023-09-26" @default.
• W2006491932 title "rek, a Gene Expressed in Retina and Brain, Encodes a Receptor Tyrosine Kinase of the Axl/Tyro3 Family" @default.
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• W2006491932 doi "https://doi.org/10.1074/jbc.271.46.29049" @default.
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