Cloning and structural characterization of ECTACC, a new member of the transforming acidic coiled coil (TACC) gene family: cDNA sequence and expression analysis in human microvascular endothelial cells.

Erythropoietin (Epo) transduces mitogenic and chemoattractant signals to human endothelial cells. Identifications of Epo-responsive genes are important for understanding the molecular nature of Epo signaling in endothelial cells. The effects of Epo on differential expression of various genes were examined in human microvascular endothelial cells (HMVEC) by differential display reverse transcriptase polymerase chain reaction (RT-PCR). In the current study we obtained from Epo-treated HMVEC a cDNA fragment with characteristics of the 3' end of mRNA. Using the cDNA fragment, we then selectively isolated a full-length clone by screening an unamplified endothelial cell cDNA library followed by 5' rapid amplification of cDNA ends by polymerase chain reaction (RACE-PCR). The nucleotide sequence of the longest cDNA revealed an open reading frame of 3311 nucleotides that encodes a protein consisting of approximately 906 amino acids with a predicted MW of approximately 100 kDa. The nucleotide sequence of the cDNA is nearly identical to that of transforming acidic coiled coil-containing (TACC2) and anti-zuai-1 (AZU-1) cDNA clones except at the 5'- and 3'-ends. Northern blot analysis showed an increase in endothelial-TACC-related mRNA levels in Epo-treated cells in comparison to that of the control cells. Endothelial-TACC-related mRNA was highly expressed in heart and skeletal muscle tissue. Placenta and brain tissue exhibited low levels of expression of endothelial-TACC-related gene. Southern blot analysis of genomic DNA from somatic cell hybrids showed that endothelial-TACC-related cDNA maps to chromosome 10. Immunofluorescence microscopy and the occurrence of several putative phosphorylation and SH3 binding sites on the deduced protein suggest that endothelial-TACC-related protein may be involved in Epo signaling cascades in endothelial cells. Copyright 2001 Academic Press.