Uroplakin III, a novel Src substrate in Xenopus egg rafts, is a target for sperm protease essential for fertilization.
In a previous study, we identified Xenopus egg uroplakin III (xUPIII), a single-transmembrane protein that localized to lipid/membrane rafts and was tyrosine-phosphorylated upon fertilization. An antibody against the xUPIII extracellular domain abolishes fertilization, suggesting that xUPIII acts not only as tyrosine kinase substrate but also as a receptor for sperm. Previously, it has been shown that the protease cathepsin B can promote a transient Ca2+ release and egg activation as seen in fertilized eggs (Mizote, A., Okamoto, S., Iwao, Y., 1999. Activation of Xenopus eggs by proteases: possible involvement of a sperm protease in fertilization. Dev. Biol. 208, 79-92). Here, we show that activation of Xenopus eggs by cathepsin B is accompanied by tyrosine phosphorylation of egg-raft-associated Src, phospholipase Cgamma, and xUPIII. Cathepsin B also promotes a partial digestion of xUPIII both in vitro and in vivo. A synthetic xUPIII-GRR peptide, which contains a potential proteolytic site, inhibits the cathepsin-B-mediated proteolysis and tyrosine phosphorylation of xUPIII and egg activation. Importantly, this peptide also inhibits sperm-induced tyrosine phosphorylation of xUPIII and egg activation. Protease activity that digests xUPIII in an xUPIII-GRR peptide-sensitive manner is present in Xenopus sperm. Several protease inhibitors, which have been identified to be inhibitory toward Xenopus fertilization, are shown to inhibit sperm-induced tyrosine phosphorylation of xUPIII. Uroplakin Ib, a tetraspanin UP member, is found to be associated with xUPIII in egg rafts. Our results highlight novel mechanisms of fertilization signaling by which xUPIII serves as a potential target for sperm protease essential for fertilization.
Mahbub Hasan
A K M
AK
Graduate School of Science and Technology, Kobe University, Kobe 657-8501, Japan.
Sato
Ken-Ichi
K
Sakakibara
Keiichi
K
Ou
Zhize
Z
Iwasaki
Tetsushi
T
Ueda
Yasushi
Y
Fukami
Yasuo
Y
D000818:Animals; D002401:Cathepsin B / Q000378:metabolism; D004527:Egg Proteins / Q000378:metabolism; D005306:Fertilization; D008562:Membrane Glycoproteins / Q000378:metabolism*; D021962:Membrane Microdomains; D010063:Ovum / Q000378:metabolism; D010447:Peptide Hydrolases / Q000502:physiology*; D051966:Phospholipase C gamma / Q000378:metabolism; D010766:Phosphorylation; D060171:Uroplakin III; D014981:Xenopus; D029867:Xenopus Proteins; D019061:src-Family Kinases / Q000378:metabolism
D004527:Egg Proteins; D008562:Membrane Glycoproteins; D060171:Uroplakin III; D029867:Xenopus Proteins; D019061:src-Family Kinases; D051966:Phospholipase C gamma; D010447:Peptide Hydrolases; D002401:Cathepsin B