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 (MBP) 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 Co(2+)-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.
Moriyama
Shunsuke
S
Department of Agricultural and Biological Chemistry, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa 252-8510.
Ogihara
Jun
J
Kato
Jun
J
Hori
Tetsuya
T
Mizuno
Shigeki
S
D000595:Amino Acid Sequence; D000818:Animals; D001717:Birds / Q000235:genetics*; D002874:Chromosome Mapping; D019281:Dimerization; D006867:Hydrolases / Q000737:chemistry / Q000235:genetics*; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics
D011993:Recombinant Fusion Proteins; D006867:Hydrolases; C510383:AMP-lysine hydrolase, chicken