Biochemical and Biophysical Research Communications
Identification of Dok-4b, a Dok-4 splice variant with enhanced inhibitory properties
Section snippets
Methods
Cells and transfections. Caco-2 human colon cancer cells were cultured in α-MEM containing 10% FBS. Caco-2 cells stably overexpressing Dok-4bMyc were generated by selection in G-418 after transfection with pCND3.1-Dok-4bMyc/His. COS-1, T47D, HeLa, and 293 cells were cultured in DMEM/high glucose containing 10% FBS. Transfections were performed with the Lipofectamine 2000 reagent (Invitrogen).
cDNA’s. The following expression constructs were described previously [5]: pCDNA3.1-Ret9 (human c-Ret),
Evidence of a human splice variant of Dok-4
We had previously generated an antiserum directed specifically at the C-terminal region of Dok-4 and noted that, in addition to the expected 37 kDa band, it also detected an approximately 45 kDa band in Caco-2 intestinal epithelial cells [5]. Using this same antiserum, we performed anti-Dok-4 immunoprecipitation and immunoblotting in T47D and HeLa human epithelial cells. As shown in Fig. 1A, the same 45 kDa band was observed in these cells. High levels of this protein were also detected in TT
Discussion
In the current study, we demonstrate the existence of a major splice variant of Dok-4, Dok-4b, characterized by the insertion of a 39 amino acid sequence in the C-terminal region of the molecule. We detected this variant protein in several human epithelial cell lines and also identified corresponding sequences in the genomes of monkeys and cow, but not in rodents or other species, suggesting that the appearance of Dok-4b is a relatively recent event in mammalian evolution. Compared to Dok-4,
Acknowledgments
We thank Drs. J. Nunès, L. Mulligan, M. Park, C. Stanners, and T. Yamamoto for gift of reagents. This work was supported by grants from the Kidney Foundation of Canada and Canadian Institutes of Health Research (CIHR) to S.L. and T.T. S.L. is a CIHR New Investigator. T.T. is supported by a scholarship from the Fonds de la recherche en santé du Québec.
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