© 2006 The Japanese Biochemical Society.
Regular Paper |
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
1 Department of Agricultural and Biological Chemistry, College of Bioresource Sciences, Nihon University, 1866 Kameino, Fujisawa, 252-8510; and 2 National Institute of Genetics, 1111 Yata, Mishima 411-8540
* To whom correspondence should be addressed. Tel/Fax: +81-466-84-3944, E-mail: junkat{at}brs.nihon-u.ac.jp
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 Co2+-resin. The homodimer of PKCI-Z, but not PKCI-W, bound to an N6-(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.