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Molecular cloning, expression and partial characterization of Xksy, Xenopus member of the Sky family of receptor tyrosine kinases.
Kishi YA
,
Funakoshi H
,
Matsumoto K
,
Nakamura T
.
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We isolated a cDNA encoding the Xenopus member of Sky/Axl/Mer receptor tyrosine kinase family (referred as Sky family), termed Xksy. The predicted Xksy protein has conserved structural characteristics of the Sky family: an unique extracellular domain of two immunoglobulin (Ig)-like repeats, two fibronectin type III (FNIII)-like repeats and an intracellular tyrosine kinase. Homology analysis of Xksy showed the highest identity to mammalian Sky protein. In contrast to the predominant expression of sky mRNA in the adult mammalian nervous system, Northern blot analysis showed ubiquitous expression of a single 5.2-kb Xksy mRNA in tissues of the adult Xenopus. RNase protection assays revealed that, during development, Xksy mRNA is expressed from mid neurulation stage. Levels increase through the tadpole stage and become restricted to the head region in embryos by stage 40. Whole-mount in situ hybridization analyses revealed that expression of Xksy is localized to the nervous system of the tadpole stage, including origins of sensory organs and branchial arches. When a chimeric receptor (EGFR-Xksy), composed of the extracellular region of epidermal growth factor (EGF) receptor and the transmembrane/intracellular regions of Xksy, was expressed in a doxycycline repressive manner in HEK 293 cells, EGF-stimulus without doxycycline induced tyrosine phosphorylation of the chimeric receptor and evoke morphological changes. EGF treatment also induced growth modifications of EGFR-Xksy cells. And doxycycline pre-treatment eliminated these activities. These findings suggest that Xksy may play an important role in growth, differentiation and the accurate migration of cells during embryogenesis and early neural development.
Fig. 1. Sequence analysis of the Xksy protein. Sequence of Xksy cDNA (DDBJ/EMBL/GenBank accession no. AB022787) and its predicted protein product. Nucleotides and amino acids are numbered on the left and right, starting with the upstream putative initiator codon. Indicated in the coding sequence are the putative initiation site (underlined), the signal sequence (boxed), the organization of structural domains, the potential N-linked glycosylation sites (N-GLY), the potential autophosphorylation sites (asterisks), the putative SH2-binding sites (boldface type), and glutamic acid-rich sequence (double underlined). A putative polyadenylation signal, AATAAA, is printed in italic boldface type. TK, tyrosine kinase domain.
Fig. 2. Homology between Xksy and members of the Sky family of RTKs, and their phylogenic tree. (A) Schematic representation of Xksy and homol- ogy of amino acid sequences between Xksy and other Sky family members. The degree of identity of amino acid residues within each domain is expressed as a percentage. (B) Phylogenic tree of Sky family RTKs. A phylogenic tree was determined using the DNASIS program (Hitachi Soft- ware Engineering Co.) The proteins analyzed were: mouse Sky (Tyro3, I48860), rat Sky (D37880), human Sky (D17517), chicken Rek (U7005), zebrafish Dtk (AAD01694), mouse Axl (NM009465), human Axl (M76125), mouse c-Mer (NM008587), and human c-Mer (U08023).
Fig. 3. Northern blot analysis of Xksy mRNA expression in adult Xenopus tissues. The upper panel shows an autoradiogram of a Northern blot for Xksy. The lower panel shows ribosomal 28S.
Fig. 4. RNase protection assays of Xksy mRNA expression in Xenopus embryos during development. (A) The expression of Xksy mRNAs during embryogenesis. RPA was done using a 32P-labeled 250-bp anti-sense cRNA probe specific for the carboxyl-terminus of Xksy. Ten micrograms of total RNA were loaded per lane. The numbers (4, 7, 9, 12, 16, 19, 23, 28, 35 and 41) indicate developmental stages. (B) Relative levels of expression of Xksy mRNA in dissected parts of stage 32 embryos (tailbud). h, head; d, dorsal; v, ventral; t, tail. In both (A) and (B), upper panels show RPA signals and lower panels show relative levels of Xksy mRNA in percent.
Fig. 5. Whole-mount in situ hybridization of Xksy mRNA during Xenopus embryogenesis. Embryos were hybridized overnight to the DIG-labeled Xksy anti- sense cRNA probe, incubated with anti-digoxigenin-AP antibody, and subjected to color development (A,C,E,F,G). Xksy sense probe was included as a control for non-specific hybridization (B,D). (A) Stage 6.5 (blastula); lateral view. Asterisks, vegetal hemisphere. (B,C) Stage 11–11.5 (gastrula); lateral view. Asterisks, yolk plug. (D,E) Stage 40 (tadpole); lateral view. (F,G) Head region of stage 40; (F) lateral view, (G) dorsal view. fb, forebrain; mb, midbrain; hb, hindbrain; sc, spinal cord; np, nasal placodes; ov, optic vesicles; ls, lens; br, branchial arches.
Fig. 6. Immunochemical and cytoskeletal analyses of the chimeric receptor EGFR-Xksy in transiently transfected Tet-Off cells. (A) EGFR-Xksy is composed of the extracellular domain of human EGFR and transmembrane and intracellular domains of Xksy. (B,C) Forty-eight hours after transfection of pBI-EGFR- Xksy or the mock vector, Tet-Off cells were immunostained with anti-EGFR antibody (red) (B) or stained with rhodamineâphalloidin (red) (C). Transfected cells show EGFP fluorescence (green). Cells were counterstained with Hoechst 33342 (blue). Fluorescence signal was captured, using a confocal microscope.
Fig. 7. Biochemical and biological responses of Tet-Off cells stably expressing EGFR-Xksy upon EGF-stimulus. (A) Phosphorylation assay. EGFR-Xksy- or mock-cells were pre-cultured with or without doxycycline for 1 week. Cells were replated, serum-starved for 14 h, and stimulated with or without 100 ng/ml EGF at 37 8C for 5 min. Proteins were extracted, immunoprecipitated using the anti-EGFR antibody, and subjected to Western blot analyses, using the anti- phosphotyrosine antibody or the anti-EGFR antibody. (B) Morphological change of Tet-Off cells expressing EGFR-Xksy in the presence of serum. Left column: Morphological change induced by EGF. Cells were pre-cultured with or without doxycycline for 1 week, and then replated in the presence or absence of 100 ng/ml EGF. Photos were taken 27 h after replating. Right column: Reversal of EGF-induced morphology by elimination of EGF. Thirty hours after the pre-culture of EGFR-Xksy cells with 100 ng/ml EGF, the medium was replaced with EGF containing medium (control) or EGF free medium (for reverse). Photos in the same grid location were taken 0 and 27 h after the medium replacement. (C) Left graph: Altered growth of Tet-Off cells expressing EGFR-Xksy in the presence of serum. Cells were plated with or without 100 ng/ml EGF, and MTT assay was performed after 4 days of culture. Each value represents the mean ^ SD of triplicate measurements. *p , 0.0005 by Studentâs t-test. Right graph: Altered growth of Tet-Off cells expressing EGFR-Xksy in the absence of serum. Cells were plated with the indicated dose of EGF, and MTT assay was done after 4 days of culture. Each value represents the mean ^ SD of triplicate measurements. *p , 0.005 by Studentâs t-test.
