Molecular identification and characterization of Xenopus egg uroplakin III, an egg raft-associated transmembrane protein that is tyrosine-phosphorylated upon fertilization.
Here we describe mass spectrometric identification, molecular cloning, and biochemical characterization of a lipid/membrane raft-associated protein that is tyrosine-phosphorylated upon Xenopus egg fertilization. This protein is homologous to mammalian uroplakin III, a member of the uroplakin family proteins (UPs) that constitute asymmetric unit membranes in the mammalian urothelial tissues, thus termed Xenopus uroplakin III (xUPIII). xUPIII contains N-linked sugars and is highly expressed in Xenopus eggs, ovary, urinary tract, and kidney. In unfertilized eggs, xUPIII is predominantly localized to the lipid/membrane rafts and exposed on the cell surface, as judged by surface biotinylation experiments and indirect immunofluorescent studies. After fertilization or hydrogen peroxide-induced egg activation, xUPIII becomes rapidly phosphorylated on tyrosine residue-249, which locates in the carboxyl-terminal cytoplasmic tail of the molecule. Raft localization and tyrosine phosphorylation of xUPIII can be reconstituted in HEK293 cells by coexpression of xUPIII, and Xenopus c-Src, a tyrosine kinase whose fertilization-induced activation in egg rafts is required for initiation of development. In mammals, UPIII is forming a complex with a tetraspanin molecule uroplakin Ib. As another tetraspanin, CD9, is known to be a critical component for sperm-egg fusion in the mouse, we have assumed that xUPIII is involved in sperm-egg interaction. An antibody against the extracellular domain of xUPIII blocks sperm-egg interaction, as judged by the occurrence of egg activation and first cell cleavage. Thus, xUPIII represents an egg raft-associated protein that is likely involved in sperm-egg interaction as well as subsequent Src-dependent intracellular events of egg activation in Xenopus.
Sakakibara
Keiichi
K
Graduate School of Science and Technology, Kobe University, Nada, Japan.
Sato
Ken-ichi
K
Yoshino
Ken-ichi
K
Oshiro
Noriko
N
Hirahara
Shino
S
Mahbub Hasan
A K M
AK
Iwasaki
Tetsushi
T
Ueda
Yasushi
Y
Iwao
Yasuhiro
Y
Yonezawa
Kazuyoshi
K
Fukami
Yasuo
Y
D000595:Amino Acid Sequence; D000818:Animals; D015703:Antigens, CD / Q000737:chemistry; D019921:Biotinylation; D000081247:CSK Tyrosine-Protein Kinase; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002499:Centrifugation, Density Gradient; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D018076:DNA, Complementary / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D020224:Expressed Sequence Tags; D005260:Female; D005306:Fertilization; D019084:Fluorescent Antibody Technique, Indirect; D005982:Glutathione Transferase / Q000378:metabolism; D006801:Humans; D006861:Hydrogen Peroxide / Q000737:chemistry; D015151:Immunoblotting; D007150:Immunohistochemistry; D047468:Immunoprecipitation; D050356:Lipid Metabolism; D008297:Male; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000737:chemistry / Q000378:metabolism*; D021962:Membrane Microdomains / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013084:Sperm-Ovum Interactions; D060245:Tetraspanin 29; D014018:Tissue Distribution; D014443:Tyrosine / Q000737:chemistry*; D060171:Uroplakin III; D060169:Uroplakin Ib; D014981:Xenopus; D019061:src-Family Kinases
D015703:Antigens, CD; C554869:CD9 protein, human; C554870:Cd9 protein, mouse; D018076:DNA, Complementary; D008562:Membrane Glycoproteins; D011993:Recombinant Fusion Proteins; D060245:Tetraspanin 29; C086452:UPK1B protein, human; C554660:UPK3A protein, human; C554661:Upk3a protein, mouse; D060171:Uroplakin III; D060169:Uroplakin Ib; D014443:Tyrosine; D006861:Hydrogen Peroxide; D005982:Glutathione Transferase; D011505:Protein-Tyrosine Kinases; D000081247:CSK Tyrosine-Protein Kinase; D019061:src-Family Kinases; C000631346:CSK protein, human