Calcineurin is a Ca2+/calmodulin dependent Ser/Thr protein phosphatase. To identify the molecular targets of calcineurin action in C. elegans, yeast two-hybrid screening was performed. One of the candidates from the screening,
cnp-1 (T12D8.4), shows 25% identity over 344 residues with human TBP-2/VDUP1, which has been recently reported as a negative regulator of Thioredoxin (TRX) function. Human TRX-binding protein 2(TBP-2), also known as Vitamin D3 up-regulated protein 1(VDUP1), was originally reported as an up-regulated gene in HL-60 cells treated with 1a, 25-dihydroxyvitamin D3, and significantly downregulated in chemically induced rat mammary tumors. We have cloned and sequenced the full cDNA from cDNA library and found that the gene structure predicted by database was incorrect. Complete DNA sequencing data showed that
cnp-1 gene consists of 9 exons encoding a protein of 426 amino acid residues. Our sequencing results added one more exon, exactly 96 base pairs (32 amino acids) to the previously predicted one. To confirm in vitro interaction between calcineurin A and entire coding region of
cnp-1, GST pull-down assay was conducted. GST fused full-length CNP-1 pulled down calcineurin A from the worm lysate. GFP expression of promoter region of
cnp-1 was seen mainly in nerve cells from embryo to adult stage, which is similar to that of
cna-1. We have isolated a deletion mutant of
cnp-1 (
jh105) by UV-TMP mutagenesis methods in which this gene was deleted from the beginning of exon2 to the end. Based on this molecular evidence, we suggest that this mutant is a functionally null mutant. We are in the process of characterizing phenotypes of this mutant.