Abstract
NDRG1 (N-myc downstream regulated gene-1) is a metastasis suppressor that is down-regulated in prostate cancer. NDRG1 phosphorylation is associated with inhibition of metastasis and Western blots indicate two bands at ~41 and ~46 kDa. Previous investigations by others suggest the higher band is due to NDRG1 phosphorylation. However, the current study using a dephosphorylation assay and the Phos-tag (phosphate-binding tag) SDS/PAGE assay, demonstrated that the 46 kDa NDRG1 protein band was not due to phosphorylation. Further experiments showed that the NDRG1 protein bands were not affected upon glycosidase treatment, despite marked effects of these enzymes on the glycosylated protein, fetuin. Analysis using RT–PCR (reverse transcriptase–PCR) demonstrated only a single amplicon, and thus, the two bands could not result from an alternatively spliced NDRG1 transcript. Western-blot analysis of prostate cancer cell lysates identified the 41 kDa band to be a truncated form of NDRG1, with MS confirming the full and truncated proteins to be NDRG1. Significantly, this truncated protein was not present in normal human PrECs (prostate epithelial cells). Western-blot analysis using anti-NDRG1 raised to its N-terminal sequence failed to detect the truncated protein, suggesting that it lacked N-terminus amino acids (residues 1–49). Sequence analysis predicted a pseudotrypsin protease cleavage site between Cys49–Gly50. Such cleavage of NDRG1 in cancer cells may result in loss of NDRG1 tumour suppressive activity.
Highlights
NDRG1 (N-myc downstream regulated gene-1) is a member of the NDRG gene family [1]
NDRG1 does not have an associated splice variant MS analysis identified that the amino acid sequence coverage spanned the translated regions of exons 15 and 16 of NDRG1 (Supplementary Figure S2 available at http://www.bioscirep.org/ bsr/033/bsr033e042add.htm), confirming that the Cterminus of the 41 kDa NDRG1 isoform was intact
reverse transcriptase–PCR (RT–PCR) analysis of DU145 total RNA showed that each of the four primer sets generated only a single amplicon corresponding to that of the predicted size (Figure 4). This indicated that the 41 kDa NDRG1 protein was not a result of translation of an alternatively spliced transcript of NDRG1
Summary
NDRG1 (N-myc downstream regulated gene-1) is a member of the NDRG gene family [1]. All members of this group of proteins contain an α/β hydrolase fold, without a hydrolytic catalytic site [1]. Upon visualization of Western blots with goat polyclonal antibody against C-terminal NDRG1, two protein bands were detected in DU145 cell lysates at ∼41 kDa and ∼46 kDa respectively (Figure 1A). In the presence of the Mn2 + -Phos-tag (25 μM), there was no induced electrophoretic mobility-shift of the 46 kDa against the 41 kDa NDRG1 protein bands relative to control SDS/PAGE immunoblots conducted at the same time (Figure 1B).
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