PKCI-W forms a heterodimer with PKCI-Z and inhibits the biological activities of PKCI-Z in vitro, supporting the predicted role of PKCI-W in sex determination in birds

The two chicken genes, PKCI-W on the W chromosome and PKCI-Z on the Z chromosome, belong to the gene family encoding the Hint (histidine triad nucleotide-binding protein)branch proteins in the widely conserved HIT (histidine triad)-family. It has been speculated that PKCI-W is involved in the sex determination of birds by forming a heterodimer with PKCI-Z and inhibiting the function of PKCI-Z in female embryos. In this study, both PKCI-W and PKCI-Z were expressed in fusion [maltose-binding protein (MEP) or glutathione-S-transferase (GST)] and tagged [(His)(6) or FLAG] forms (FT-forms) in Escherichia coli and purified. Formation of homodimers of PKCI-W-containing or the PKCI-Z-containing FT-protein and the formation of a heterodimer between the PKCI-W-containing and the PKCI-Z-containing FT-proteins were demonstrated by Western blotting after GST-pulldown or binding to and elution from the Co2+-resin. The homodimer of PKCI-Z, but not PKCI-W, bound to an N-6-(3-aminopropyl) adenosine affinity column and hydrolyzed adenosine 5'-monophosphoramidate. Both of these activities were inhibited in vitro in a dominant-negative manner by the formation of a heterodimer containing PKCI-W. These in vitro experimental results support the predicted role of PKCI-W in the process of sex determination in birds.