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In studies of developmental signaling pathways stimulated by the Wnt proteins and their receptors, Xenopus Wnt-5A (Xwnt-5A) and a prospective Wnt receptor, rat Frizzled 2 (Rfz2), have been shown to stimulate inositol signaling and Ca2+ fluxes in zebrafish   . As protein kinase C (PKC) isoforms can respond to Ca2+ signals , we asked whether expression of different Wnt and Frizzled homologs modulates PKC. Expression of Rfz2 and Xwnt-5A resulted in translocation of PKC to the plasma membrane, whereas expression of rat Frizzled 1 (Rfz1), which activates a Wnt pathway using beta-catenin but not Ca2+ fluxes , did not. Rfz2 and Xwnt-5A were also able to stimulate PKC activity in an in vitro kinase assay. Agents that inhibit Rfz2-induced signaling through G-protein subunits blocked Rfz2-induced translocation of PKC. To determine if other Frizzled homologs differentially stimulate PKC, we tested mouse Frizzled (Mfz) homologs for their ability to induce PKC translocation relative to their ability to induce the expression of two target genes of beta-catenin, siamois and Xnr3. Mfz7 and Mfz8 stimulated siamois and Xnr3 expression but not PKC activation, whereas Mfz3, Mfz4 and Mfz6 reciprocally stimulated PKC activation but not expression of siamois or Xnr3. These results demonstrate that some but not all Wnt and Frizzled signals modulate PKC localization and stimulate PKC activity via a G-protein-dependent mechanism. In agreement with other studies   .   these data support the existence of multiple Wnt and Frizzled signaling pathways in vertebrates.
Xwnt-5A and Rfz2, but not Xwnt-8 nor Rfz1, elicit PKC translocation. (a) Following co-injection of Xwnt-5A RNA, Myc-tagged PKC is localized at the plasma membrane. (b) Co-injection of Xwnt-8 RNA has a negligible effect upon PKC localization. (c) Following co-injection of Rfz2 RNA, PKC is localized at the plasma membrane. (d) Co-injection of Rfz1 RNA has no apparent effect upon PKC localization. (e) Injection of control (prolactin) RNA shows PKC is present throughout the cytoplasm in the absence of exogenous Wnt signals. (f) Treatment for 30 min with PMA, a potent PKC activator, is sufficient to relocalize PKC to the plasma membrane.
RNAs that induce PKC translocation also increase PKC activity in an in vitro kinase assay. (a) Relative phosphorylation of a PKC substrate peptide by immunoprecipitated XPKCα–Myc from embryos co-injected with RNA encoding either Xwnt-8 (n = 3 experiments), Xwnt-5A (n = 3), Rfz1 (n = 3) or Rfz2 (n = 6). As we wanted to measure relative kinase activities, uninjected embryos (n = 8) were measured for basal kinase activity, and this level was set to 1. PMA was added to the reaction tubes as a positive control. (b) Embryo lysates from panel (a) were immunoblotted with an anti-Myc monoclonal antibody, 9E10, to confirm equal expression of PKC after Frizzled injection.
Agents that block Gi or Go-βγ subunit signaling block Rfz2-induced PKC translocation. Two-cell Xenopus embryos were injected with RNAs encoding XPKCα–GFP, Rfz2, and either pertussis toxin, α-transducin, Gi2α, Gsα or prolactin. (a,c) Examples of confocal images used. (b,d) Images (17 different cells from 4 different experiments for each condition in (e); see also Supplementary material published with this paper on the internet) were analyzed using NIH Image software to plot the pixel intensity profile (y-axis: 0 = black, 255 = white) over a linear section of the cell (dashed lines in (a,c) as representative cells). (e) The average pixel intensity of membrane regions divided by the average pixel intensity of intracellular regions was used as a relative measure of PKC membrane localization. (f) Confirming the results in (e), injection of PTX was also able to reduce PKC activity in Rfz2-injected embryos, as measured directly by the independent in vitro kinase assay.
Frizzled homologs display a differential ability to increase PKC activity versus siamois and Xnr3 expression. (a) Both cells of two-cell Xenopus embryos were injected with XPKCα–Myc RNA along with RNA encoding Rfz1, Rfz2, Mfz3, Mfz4, Mfz6, Mfz7 or Mfz8 as indicated. The subcellular localization of ectopic PKC is shown. (b) PKC localization in animal cap cells from experiments as in (a) was imaged by confocal microscopy and quantified as in Figure 3. Frizzled homologs which showed less than 1.1-fold relative membrane versus cytoplasmic staining are denoted ‘−’, while Frizzled homologs which showed fivefold or greater membrane versus cytoplasmic staining are denoted ‘+’. (c) Frizzled homologs were assayed for the ability to increase siamois and Xnr3 expression in Xenopus embryos by RT–PCR amplification. EF1-α expression was measured as a loading control and −RT denotes this reaction without reverse transcriptase.