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Fig. 1. Identification of XPIASy as a Smad2-interacting protein. (A) An alignment of human and Xenopus PIASy (GenBank AF077952 and AF397163). Shaded amino acids are conserved residues (75% identical). (B) Phylogenetic tree of human PIAS family members and XPIASy. (C) The C-terminal region of XSmad2 binds to XPIASy in a yeast two-hybrid assay. L40 cells were transformed with pLexA-Smad2 (amino acids 180 to 432) or pLexA-Ras(G12V) and with pACTII-XPIASy, pACTII-HK-Swift (Shimizu et al., 2001), pVP-Raf or a vector, pACTII-HK. The interaction was tested by growth on SD-Trp-Leu-His plates supplemented with 10 mM 3-AT for 3 days. (D) Full-length of XSmad2 interacts with full-length XPIASy in immunoprecipitation assay. The mRNA of Flag-tagged XPIASy was injected alone (lane 3) or with XSmad2 (lanes 1,2). (E) XPIASy interacts weakly with XSmad4{alpha} or XSmad4ß but not with XSmad1. The mRNA of Flag-tagged XPIASy was injected with the indicated Myc-tagged Xenopus Smad members, and interaction was analyzed by immunoprecipitation using anti-Flag antibody. (F) Mouse PIASy interacts strongly with XSmad2 but weakly with XSmad4{alpha} or XSmad4ß. The mRNA of T7-tagged mouse PIASy was injected with the indicated Myc-tagged Xenopus Smad members, and interaction was analyzed by immunoprecipitation using the anti-T7 antibody. (G) Structures of XPIASy deletion constructs. (H) Immunoprecipitation of XPIASy deletion constructs with full-length of XSmad2. (Upper panel) A western blot of immunoprecipitated samples. The mRNA of Flag-tagged XPIASy construct was injected with Myc-tagged XSmad2 in Xenopus embryos. After immunoprecipitation against Myc, precipitated proteins were analyzed by anti-Flag staining. (Lower two panels) Before immunoprecipitation, expressed proteins were confirmed by Flag and Myc staining. (I) The N-terminal region of XPIASy interacts with the C-terminal region of XSmad2. A yeast two-hybrid assay was performed supplemented with 5 mM 3-AT, using pLexA-Smad2 (amino acids 180 to 432) and XPIASy deletion constructs subcloned into pACTII. (J) Immunoprecipitation of RING domain deleted construct, P{Delta}R. The indicated constructs were injected into Xenopus embryos. After immunoprecipitation against Myc, western blotting was performed with anti-Flag antibody.