(d) Injection of Merlin mRNA into the dorsal side of embryos clogged formation of the embryonic axis (ventralization). antibody and immunoblotted with the indicated antibodies. All western blotting and immunoprecipitation experiments were performed more than three occasions, and these data are representative Wnt3a activates PAK and PAK phosphorylates Merlin inside a PIP2-dependent manner As the connection between Merlin and LRP6 was shown to be inhibited by Wnt3a-CM treatment and the phospho-mimetic form of Merlin (Merlin-SD) did not bind to LRP6 (Number 1), we reasoned the phosphorylation status of Ser518 in Merlin might be enhanced by treatment with Wnt3a-CM. Phosphorylation of Ser518 in Merlin was enhanced in a dose- and time-dependent manner in HEK293T cells incubated with Wnt3a-CM (Number 5a and Supplementary Number S5B). As phosphorylation of LRP6 is also enhanced by Wnt3a,37 we tested whether or not the phosphorylation status of LRP6 influences the connection with Merlin. Mutated LRP6, in which all five serines in the PPPSP motif are changed to alanine (VSVG-LRP6-M5),12 interacted with Merlin in the absence of Wnt3a. This connection was inhibited by treatment with Wnt3a-CM, which suggests that Wnt3a-mediated disruption of the connection between LRP6 and Merlin could be attributed to phosphorylation of Merlin and not LRP6 phosphorylation (Number 5b). Open in a separate window Number 5 Phosphorylation of Merlin upon Wnt3a treatment is definitely mediated by PAK inside a PIP2-dependent manner. (a) Treatment with Wnt3a-CM induced phosphorylation of Merlin. HEK293T cells were treated with L-CM or Wnt3a-CM (+, 2-fold diluted CM; ++, initial CM) over night and harvested, followed by western blotting with indicated antibodies. (b) Reduced connection between LRP6 and Merlin upon Wnt3a-CM treatment is definitely irrelevant to the phosphorylation status of LRP6. HEK293T cells transfected with VSVG-LRP6, VSVG-LRP6-M5, or Flag-Merlin were treated with Wnt3a-CM over night and subjected to immunoprecipitation with anti-VSVG antibody. (c) Phosphorylation of PAK improved upon Wnt3a-CM treatment. HEK293T cells were treated with Wnt3a-CM in the indicated time and harvested, followed by western blotting with indicated antibodies. (d) Ectopic manifestation of the dominant-negative form of PAK1 (dnPAK1) inhibited Wnt3a-mediated phosphorylation of LRP6 and Merlin. Rabbit polyclonal to AFG3L1 After vacant vector or dnPAK1 was transfected, cells were treated with L-CM or Wnt3a-CM for 2?h before being harvested, followed by western blotting with indicated antibodies. (e) Inhibition of PAK activity clogged Wnt3a-mediated phosphorylation of LRP6. Cells were pretreated with DMSO or IPA-3 (30?and embryos To examine the inhibitory part of Merlin in Wnt signaling in a more physiological context, we employed embryos, which are a well-known magic size system to evaluate the part of candidate proteins in XRP44X Wnt XRP44X signaling.44 As shown in Number 6a, co-injection of Merlin with xWnt8 or LRP6N in animal cap explants significantly blocked xWnt8 or LRP6N-mediated induced expression of Wnt target genes such as and and induced by Wnt8 or LRP6N (Number 6e). However, co-injection of Merlin-SA or Merlin-SD into animal cap explants did not inhibit enhanced manifestation of and induced by ectopic manifestation of embryos strongly support the idea the unphosphorylated active form of Merlin inhibits Wnt/mRNA. Manifestation levels of Wnt target genes (mRNA was injected at one cell stage, and total RNA was extracted from your dorsal marginal zone (DMZ) at stage 10.5 to synthesize cDNA for RT-PCR analysis. (c) Formation of secondary embryonic axis by injection of 1 1?pg of LRP6N mRNA into the ventral part of two blastomeres from four-cell stage embryos was severely reduced by co-injection of 200?pg of mRNA. (d) Injection of Merlin mRNA into the dorsal part of embryos clogged formation of the embryonic axis (ventralization). (e and f) Animal caps isolated from embryos were injected with 2?pg of Wnt8, 10?pg of LRP6N, or 100?pg of (was examined by RT-PCR at stage 10.5 (left panel). Ectopic manifestation of Flag-Merlin was examined by western blotting (ideal panel). (g) morpholino (Nf2 MO) was injected into the dorsal or ventral part of two blastomeres from four-cell stage embryos (ideal panel), and total RNAs were extracted from your DMZ XRP44X (remaining panel) or VMZ (middle panel) at indicated phases for RT-PCR. Ct (Control), DMZ, or VMZ samples were from non-injected embryos; XRP44X AC, animal cap like a.
