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Science 321 (5894): 1350-1353

Copyright © 2008 by the American Association for the Advancement of Science

Wnt3a-Mediated Formation of Phosphatidylinositol 4,5-Bisphosphate Regulates LRP6 Phosphorylation

Weijun Pan1*, Sun-Cheol Choi2*, He Wang3*, Yuanbo Qin3, Laura Volpicelli-Daley4, Laura Swan4, Louise Lucast4, Cynthia Khoo5, Xiaowu Zhang6, Lin Li3, Charles S. Abrams5, Sergei Y. Sokol2, and Dianqing Wu1{dagger}

1 Department of Pharmacology, Yale University School of Medicine, New Haven, CT 06510, USA.
2 Department of Developmental and Regenerative Biology, Mount Sinai School of Medicine, New York, NY 10029, USA.
3 State Key Laboratory of Molecular Biology and Center of Cell Signaling, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 200031, China.
4 Department of Cell Biology and Howard Hughes Medical Institute, Yale University School of Medicine, New Haven, CT 06510, USA.
5 Department of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
6 Cell Signaling Technology, Danvers, MA 01923, USA.


Figure 1 Fig. 1.. Effect of depletion of PtdIns kinases on Wnt3a signaling. (A and B) Effects of PtdIns kinase siRNAs on Wnt3a-induced phosphorylation of LRP6 at Ser1490. HEK293T cells were transfected with siRNAs, as indicated, for 48 hours and then treated with Wnt3a (50 ng/ml) for 30 min. Phosphorylated proteins were assayed by Western blotting. The experiments were repeated at least three times. Representative images are shown. The asterisk in (A) indicates nonspecific bands. (C and D) Control MO oligos (Ctr MO, 10 nM) or MO (10 nM) targeting (C) Xenopus PI4KII{alpha} or (D) PIP5KI{alpha} or PIP5KIβ was injected with XWnt8 (2 pg) or Xβ-catenin (10 pg) mRNA into four-cell stage embryos. n > 40 for all of the Xenopus embryo studies (where n is the number of examined embryos). Open bars, no double axis; light gray bars, incomplete double axis; black bars, complete double axis. (E) Four-cell stage embryos were injected with XPIP5KIβ MO (40 ng), XPI4KII{alpha} MO (40 ng), or XPIP5KI{alpha} MO (40 ng) with or without XPIP5KIβ (10 pg) or XPI4KII{alpha} (5 pg) RNA in the dorsal region and cultured to tailbud stages. XPIP5KI{alpha} MO, n = 30; XPIP5KIβ MO, n = 45; XPIP5KIβ MO+XPIP5KIβ, n = 29; XPI4KII{alpha} MO, n = 55; and XPI4KII{alpha} MO+XPI4KII{alpha}, n = 30. [View Larger Version of this Image (30K GIF file)]
 

Figure 2 Fig. 2.. Effect of PtdIns (4,5)P2 on Wnt3a signaling. (A) Effect of exogenous PtdIns (4,5)P2 on Wnt3a-induced phosphorylation of LRP 6 at Ser1490. HEK293T cells were treated with various PtdIns lipids in a lipid carrier for 10 min and incubated with Wnt3a (20 ng/ml) for an additional 20 min before being assayed by immunoblotting. (B and C) Rescuing the effects of PI kinase siRNAs by direct delivery of PtdIns lipids. The asterisk in (B) indicates nonspecific bands. (D and E) Reduction in PtdIns (4,5)P2 levels decreases LRP6 Ser1490 phosphorylation. HEK293T cells transfected with FRB (PM-FRB-CFP), FKBP (mRFP-FKBP12), or FK-IP (mRFP-FKBP12-5-ptase-dom) were treated with Wnt3a (20 ng/ml) in the presence or absence of rapamycin (100 nM) for 30 min before they were collected for the lipid assay (D) and immunoblotting analysis (E). *P < 0.01 compared with the same transfection in the absence of rapamycin (Student's t test). Error bars indicate SDs. [View Larger Version of this Image (60K GIF file)]
 

Figure 3 Fig. 3.. Stimulation of PtdIns (4,5)P2 formation by Wnt3a through Fz and Dvl. (A) Effect of Wnt3a treatment on PtdIns (4,5)P2 content. HEK293T Cells were stimulated with Wnt3a protein (50 ng/ml) before lipid extraction. PtdIns (4,5)P2 content was determined by HPLC. *P < 0.01 compared with time 0 (Student's t test). (B) Requirement of PI4KII{alpha} and PIP5KIβ for Wnt3a-induced formation of PtdIns (4,5)P2. Cells were transfected with siRNAs, as indicated, for 48 hours and then treated with Wnt3a (50 ng/ml) for 30 min. PtdIns (4,5)P2 were detected by ELISA. (C) Effect of Fz siRNAs on Wnt3a-induced formation of PtdIns (4,5)P2. Cells were transfected with control siRNA or a combination of Fz2, Fz4, and Fz5 siRNAs for 48 hours and then treated with Wnt3a (50 ng/ml) for 30 min before assays. *P < 0.01 compared with control siRNA transfection in the absence of Wnt3a (Student's t test). (D) Effect of Fz siRNAs on Wnt3a-induced phosphorylation of LRP6 at Ser1490. Cells were transfected as in (C) for 48 hours and then treated with Wnt3a (50 ng/ml) for 30 min. (E) Effect of Fz overexpression on accumulation of PtdIns (4,5)P2. HEK293T cells were transfected with the LacZ, Fz5, or LRP6 expression plasmids for 18 hours, and PtdIns (4,5)P2 levels were determined by ELISA. *P < 0.01 compared with the sample expressing LacZ (Student's t test). (F) Effect of Fz5 expression on phosphorylation of LRP6 at Ser1490. Cells were transfected with Fz5 expression plasmid for 18 hours and then treated with Wnt3a (20 ng/ml) for 20 min. (G) Effect of Dvl expression on the PtdIns (4,5)P2 levels. HEK293T cells were transfected with the mouse Dvl1, 2, or 3 expression plasmid for 18 hours before the PtdIns (4,5)P2 ELISA assay. *P < 0.01 compared with the sample expressing LacZ (Student's t test). (H and I) Effect of Dvl siRNAs on formation of PtdIns (4,5)P2 and phosphorylation of LRP6 at Ser1490. HEK293T cells were transfected with control siRNA or Dvl siRNA mixture targeting Dvl1, 2, and 3 for 48 hours and then treated with Wnt3a (50 ng/ml) for 30 min. *P < 0.01 compared with control siRNA transfection in the absence of Wnt3a (Student's t test). (J) Interaction of Dvl3 with endogenous PIP5KIβ. HEK293T cells (Dvl) stably expressing Dvl3-HA7 were used in immunoprecipitation by an antibody against HA (anti-HA). The parent HEK293T cells (HEK) were used as a control. Immunocomplexes were detected by the anti-Dvl3 and anti-PIP5KIβ antibodies. (K) Effect of purified recombinant Dvl3 protein on kinase activity of purified recombinant PIP5KIβ protein. PIP5KIβ (50 nM) was incubated with the GST (glutathione S-transferase)or Dvl3 proteins for 2 hours at room temperature. One-tenth of the samples was taken for Western blotting, and the rest was subjected to in vitro kinase assay with PtdIns (4)P as a substrate. The product PtdIns (4,5)P2 is separated by TLC, detected, and quantified by a phosphoimager. Error bars indicate SDs in all panels. [View Larger Version of this Image (34K GIF file)]
 

Figure 4 Fig. 4.. Requirement of PtdIns (4,5)P2 for formation of LRP6 aggregate and membrane translocation of axin and GSK3. (A) Requirement of PtdIns (4,5)P2 for Wnt3a-induced LRP6 aggregation. HEK293T cells were transfected and treated with Wnt3a and rapamycin as indicated. Cell lysates were subjected to sucrose density-gradient ultracentrifugation, and fractions were analyzed by Western analysis. (B) PtdIns (4,5)P2 content in two sucrose density-gradient ultracentrifugation fraction pools. Fractions 8 to 11 and 1 to 4 from (A) were pooled, and PtdIns (4,5)P2 amounts were measured by ELISA. Open bars correspond to the samples from the top panel in (A); black bars to the second panel; striped bars to the third panel; and dotted bars to the last panel. The PtdIns (4,5)P2 amounts are presented relative to those in untreated cells. Error bars indicate SDs. (C) Requirement of PtdIns (4,5)P2 for phosphorylation of LRP6 at Thr1479. HEK293T cells were transfected with plasmids, as indicated, for 20 hours and then treated with Wnt3a (20 ng/ml) for 30 min in the presence or absence of rapamycin (100 nM) before they were collected for immunoblotting analysis. The asterisk indicates nonspecific bands. (D) Requirement of PtdIns (4,5)P2 for Wnt3a-induced membrane recruitment of axin1. HEK293T cells were transfected and treated as indicated. The membrane fractions were prepared and analyzed by Western analysis. (E) Model for Wnt3a cross-membrane signaling. [View Larger Version of this Image (29K GIF file)]
 


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