Interaction of Axl receptor tyrosine kinase with C1-TEN, a novel C1 domain-containing protein with homology to tensin

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Abstract

Axl receptor tyrosine kinase is implicated in several malignancies and is the receptor for the vitamin K-dependent growth factor Gas6. From a yeast two-hybrid screen of protein–protein interactions with the Axl cytoplasmic domain, we detected a previously uncharacterised SH2 domain-containing protein. We cloned two novel splice variants of this protein that give rise to 1409- and 1419-amino acid proteins, differing only in their N-terminal residues and yielding a 150-kDa protein product by in vitro translation. The Axl-interacting C-terminus contains a tandem SH2 and PTB domain combination homologous to the focal adhesion protein tensin. We detected interaction of Axl with both domains in mammalian cells by co-immunoprecipitation and two-hybrid analyses. In addition, the protein possesses an N-terminal putative phorbol ester-binding C1 domain as well as a central tyrosine phosphatase motif. Thus, we have named the protein C1 domain-containing phosphatase and TENsin homologue (C1-TEN). Northern blot analysis of C1-TEN in human tissues revealed highest expression in heart, kidney, and liver. In summary, we have identified a novel multi-domain intracellular protein that interacts with Axl and which may furthermore be involved in other signal transduction pathways.

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Materials

Human Axl cDNA was provided by Dr. ET Liu, NIH, Bethesda, MD, USA. This contained the sequence for Axl transcript variant 1 (GenBank Accession No. NM_021913), which possesses nine amino acids more than variant 2, although their cytoplasmic sequences are identical. The cDNA clone for original KIAA1075 (Accession No. AB028998) was a gift of Kazusa DNA Research Institute, Chiba, Japan. The sequence was an ORF of 4962 bp [15] housed in pBluescript II SK+ cloning vector (Stratagene). All

Yeast two-hybrid interactions with Axl-IC

We screened a human heart cDNA library in yeast to identify proteins that interact with the cytoplasmic domain of human Axl. The result of a large-scale mating yielded 64 colonies, which still exhibited activation of all reporter genes (HIS3, ADE2, lacZ, and MEL1) after three re-streaks. We PCR-amplified and sequenced the partner DNA sequences from 33 colonies, which were then identified by BLAST search (Table 1). Expression of the actual partner fusion proteins was verified by Western blotting

Acknowledgements

S.H. was supported by a Marie Curie Fellowship of the European Community Human Potential programme under Contract No. HPMF-CT-1999-000123 and by the Swedish Royal Science Academy (Per-Erik Lindahl stipend). This work was further supported by the Swedish Cancer Society project Grant No. 4413-B01-02XBB, Alfred Österlund Trust, Malmö University Hospital Trust, Malmö University Hospital Cancer Trust, Greta and Johan Kock Trust, and Crafoord Trust. The cDNA sequences for C1-TEN/KIAA1075 splice

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    Abbreviations: C1-TEN, C1 domain-containing phosphatase and TENsin homologue; RTK, receptor tyrosine kinase; Axl-IC, Axl intracellular domain; Gas6, growth arrest-specific gene 6 product; SH2, Src homology 2; PTB, phosphotyrosine-binding; PI3K, phosphatidylinositol 3-kinase; RACE, real amplification of cDNA ends.

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