Cloning and characterization of the full-length mouse Ptk7 cDNA encoding a defective receptor protein tyrosine kinase☆
Introduction
Defective receptor protein tyrosine kinases (RPTKs) are a subgroup of RPTKs with inactive catalytic activity of the protein tyrosine kinase (PTK) (Kroiher et al., 2001). These include PTK7 (Park et al., 1996), ErbB3 (Carraway et al., 1994), HEP/MEP Matsuoka et al., 1997, Gurniak and Berg, 1996, and RYK Stacker et al., 1993, Tamagnone et al., 1993. In spite of their absence of catalytic activity, they participate in signal transduction by dimerizing with or acting as a substrate of other RPTKs Alimandi et al., 1995, Sliwkowski et al., 1994, Trivier and Ganesan, 2002.
PTK7 was first identified by the cloning of its cDNA fragment that was obtained from reverse transcription of normal melanocyte mRNAs and PCR with the degenerate primer pairs corresponding to the conserved subdomains VIb to IX of PTKs (Lee et al., 1993). The full-length cDNA of colon carcinoma kinase-4 (CCK-4), which is identical to PTK7, was cloned from a colon carcinoma tissue (Mossie et al., 1995). The PTK7 polypeptide deduced from its cDNA encodes a receptor tyrosine kinase-like molecule that has an extracellular domain with 7 immunoglobulin (Ig)-like loops and a catalytic domain of PTK containing genetic alterations in the PTK consensus sequences Park et al., 1996, Mossie et al., 1995. In addition, it was shown that PTK7/CCK-4 is a human homologue of the chick kinase-like gene (KLG) Park et al., 1996, Mossie et al., 1995, the cDNA of which was previously identified (Chou and Hayman, 1991). Moreover, based on phylogenetic analysis, Miller and Steele proposed that Hydra Lemon (Miller and Steele, 2000) and Drosophila Dtrk (Pulido et al., 1992) are orthologues of the human PTK7. Therefore, the PTK7 family appears to be evolutionarily conserved in phylogenetically diverse groups.
It was reported that the human PTK7 mRNA is highly expressed in fetal colon (Mossie et al., 1995) and that Hydra Lemon mRNA is upregulated in the developmental stages of spermatogenesis, oogenesis, and embryogenesis (Miller and Steele, 2000). In addition, the human PTK7 mRNA expression increased in metastatic colon carcinoma Mossie et al., 1995, Saha et al., 2001. However, it decreased in metastatic melanoma (Easty et al., 1997). Deletions of chromosome 6p, where the human PTK7 gene is located (Banga et al., 1997) (6p21.1, http://www.ncbi.nlm.nih.gov/mapview/), were frequently found in a number of cancers, including breast cancer (Piao et al., 2001) and malignant melanoma (Baudrier-Regnier et al., 2000). These observations suggest that PTK7 may play a role in the signaling mechanism during the developmental process, cancer development, and metastasis. However, the exact function of PTK7 still remains unclear.
In an attempt to better understand the roles of PTK7 in the mammalian system, we have cloned the full-length mouse Ptk7 cDNA and determined the gene organization of the mouse Ptk7 gene. We have also investigated whether mouse PTK7 is able to autophosphorylate by itself, to be phosphorylated by other PTKs, or to influence the phosphotyrosine level of cellular proteins. Furthermore, we have analyzed expression of the mouse Ptk7 mRNA in embryos during the developmental stages as well as in adult tissues.
Section snippets
Cloning of the mouse Ptk7 cDNA
Mouse expressed-sequence tag (EST) database (http://www.ncbi.nlm.nih.gov/BLAST/) was searched with the 4.2-kb full-length human PTK7 cDNA sequence. Two mouse ESTs corresponding mouse Ptk7 cDNAs were identified; the 968-bp EST BB609974 that contains the 835-bp N-terminal coding region and the 1076-bp EST AK018379 that contains the 215-bp C-terminal coding region. The 719-bp cDNA fragment that contains the 32-bp 5′-untranslated sequence and the 687-bp N-terminal coding sequence was amplified by
Characteristics of mouse PTK7 deduced from its cDNA
The mouse PTK7 polypeptide that was deduced from its full-length Ptk7 cDNA encodes 1062 amino acid residues with a calculated molecular mass of 117.5 kDa (Fig. 1). Cleavage of signal peptide between Ala22 and Ala23 was predicted by SignalP program and confirmed by the N-terminal sequencing of the mouse PTK7 extracellular domain (AIVFIKEPSS…). The presence of the AXA motif in amino acids 20–22 suggests that the signal peptide is cleaved by the endoplasmic reticulum signal peptidase complex
Discussion
In this study, we report the cloning and characterization of mouse Ptk7 cDNA encoding a defective RPTK. The primary structure of mouse PTK7 polypeptide was very similar to that of human PTK7 polypeptide (92.6% identity). We previously demonstrated that the human PTK7 gene has a unique gene structure that is distinguishable from other RPTKs (Jung et al., 2002). We have shown that the gene structure of the mouse Ptk7 gene is exactly the same as the human PTK7 gene. In this regard, we believe that
Acknowledgements
We thank Dr. Hong-Jian Zhu (Ludwig Institute for Cancer Research, Melbourne, Australia) for a generous gift of pcDNA3-EGFR. This work was supported by grants from the MOST/KOSEF through PNRC at Yonsei University (R11-2000-078-01001-0 to S.-T. Lee), the NRL Program of MOST NRDP (2000-N-NL-01-C-244 to S.-T. Lee), the Brain Korea 21 Project in Yonsei University (to S.-T. Lee), and the Korea Health 21 R and D Project, MOHW (01-PJ1-PG1-01CH11-0003 to J. Song), Republic of Korea.
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