XB-IMG-78351
Xenbase Image ID: 78351
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Figure 5. Tiki Inactivates Wnt3a via Amino-Terminal Cleavage(A) Triton X-114 phase separation. Control Wnt3a partitioned in detergent (De), but Wnt3a from TIKI2-expressing cells partitioned in aqueous (Aq) phase. T, total.(B) TIKI2 did not affect Wnt3a acylation (C77 plus S209) in metabolic labeling.(C) Amino-terminal cleavage of Wnt3a and Xwnt8 by TIKI2. The big and small arrowheads indicate major and minor cleavage sites, respectively (see Figure S5). Wnt3aδN was engineered to be identical to TIKI-cleaved Wnt3a.(D) TIKI cleaved the HA tag from HA-Wnt3a. TIKI1 cDNA was transfected at a 10-fold higher dose than that of TIKI2 for similar protein levels.(E) TIKI did not cleave Rspo1. ∗nonspecific.(F) Endogenous TIKI2 cleaved Wnt3a in HEK293T cells. TIKI2 siRNAs were as in Figures 4B and 4C.(G) Wnt3a(S25D/L26D) and Wnt3a(S25D/L26D/S33D/S34D) were partially and fully resistant to TIKI2N cleavage, respectively (lanes 4 and 6 versus 2). Both mutants exhibited slower mobility.(H) Wnt3aδN was inactive as examined by cDNA transfection in 2-fold dilutions. Error bars represent SD in triplicates.(I) Wnt3aδN in CM partitioned in the aqueous phase.(J–L) TIKI2 cleaved the HA tag from HA-Xwnt8-FLAG and HA-Wnt5a but not HA-Xwnt11-FLAG. Levels of KRM2N and TIKI2N (both FLAG-tagged) were monitored.(M) HA-Wnt3a was cleaved by TIKI2N, but not by KRM2N or TIKI2Nδ (FLAG-tagged), in HEK293T cells.(N) Silver staining of purified KRM2N, TIKI2N, or TIKI2Nδ.(O) Purified TIKI2N, but neither KRM2N nor TIKI2Nδ, cleaved recombinant HA-Wnt3a in vitro.(P) TIKI2N cleavage of HA-Wnt3a was inhibited by 1, 10-Phenanthroline but not Bestatin. Sixteen micromoles of 1, 10-Phenanthroline is about 100 times lower than manufacturer's recommended doses.(Q) TIKI2N cleavage of HA-Wnt3a was enhanced by Co2+ or Mn2+ but inhibited by Ni2+, Cu2+, or Zn2+.(R) TIKI2N inhibition by 1, 10-Phenanthroline was rescued by Co2+ or Mn2+ (lanes 6 and 8).See also Figures S4, S5, S6, and S7 and Table S4. Image published in: Zhang X et al. (2012) Copyright © 2012. Image reproduced with permission of the Publisher, Elsevier B. V. Larger Image Printer Friendly View |