Data from high throughput siRNA screens (17
) and affinity purification-mass spectrometry experiments (17
) reveal complex regulation of the Wnt/β-catenin pathway by kinases and phosphatases. One observation that we made in this study is that phosphorylation changes catalyzed by stimulation of melanoma cells with the WNT3A ligand are distinct from those caused by inhibition of GSK3. We found that the subset of phosphopeptides that are decreased in abundance following GSK3 inhibition that contain a GSK3 consensus site (SpXXX
Sp) are largely unaffected by stimulus with the WNT3A ligand. These findings contrast with the model presented by Taelman et al.
) that proposes that the WNT3A ligand promotes trafficking of the majority of GSK3 to multivesicular bodies, thus sequestering GSK3 and preventing it from regulating cytosolic proteins. Instead, our data suggest that the WNT3A stimulus does not generally regulate proteins phosphorylated by GSK3 and are consistent with earlier studies that demonstrate that Wnt ligands can inhibit the ability of GSK3 proteins to phosphorylate CTNNB1 but not necessarily other substrates (39
). One possible explanation for the contrast with the earlier study is that the majority of GSK3 substrates may migrate with and remain accessible to GSK3 in multivesicular bodies upon cellular stimulation with WNT3A. Regardless, our data suggest that inhibiting GSK3 is not synonymous with activating the Wnt/β-catenin pathway, which is an important consideration when using GSK3 inhibition as a proxy for activation of β-catenin by Wnt ligands.
We found that WNT3A promotes phosphorylation changes in several proteins that have been previously reported to regulate RAS activation and phosphatidylinositol 3-kinase signaling (MTOR, RICTOR, PIK3CA, and RPS6KA4). Previous work has demonstrated that forced expression of a non-degradable phosphorylation mutant of CTNNB1 can increase the metastatic potential of melanoma cells harboring activating mutations in the NRAS/phosphatidylinositol 3-kinase pathways (40
). Because many of the WNT3A-dependent changes in phosphorylation that we observed occurred within 60 min of stimulus, our data suggest that the WNT3A ligand may also regulate progrowth pathways (e.g.
mitogen-activated protein kinase and phosphatidylinositol 3-kinase signaling) in melanoma cells that are possibly independent of CTNNB1.
An interesting finding in these studies is that PKN1
depletion increases LRP6 cell surface expression and phosphorylation at serine 1490, a site associated with active Wnt/β-catenin signaling. Similarly, additional studies have shown that manipulations that increase surface expression of FZD (42
) or LRP6 (44
) also increase expression of downstream markers of Wnt/β-catenin signaling. Collectively, these findings are inconsistent with the hypothesis that internalization of LRP6 is necessary for downstream Wnt/β-catenin signal transduction.
Two models have been proposed to explain how Wnt ligands inhibit GSK3, enabling stabilization of CTNNB1 (46
). The first is based on the idea that the WNT3A stimulus causes recruitment of GSK3 to the cell membrane where it is subsequently internalized into multivesicular bodies (38
), thus allowing cytosolic CTNNB1 to accumulate. In contrast, a second model proposes that phosphorylated LRP6 binds GSK3 and inhibits its catalytic activity (47
). Our data showing that PKN1 depletion increases LRP6 internalization while simultaneously decreasing GSK3-dependent phosphorylation of CTNNB1 are more consistent with the latter model.
PKN1 is an atypical PKC kinase that acts as a Rho effector in several contexts and can regulate AKT (51
) and MAPK8 (also known as Jnk) (55
) signaling (for a review, see Ref. 56
). PKN proteins localize to the endoplasmic reticulum and the endosome (57
) and are known to regulate vesicular traffic (60
). A protein array-based screen for substrates of the PKN1 kinase determined that it can phosphorylate several receptor proteins including EPHA5, EGF receptor, RET, and GRK4 (61
). Our finding that PKN1 is required for WNT3A-dependent internalization of LRP6 is consistent with data showing that PKN1 promotes RHOB-dependent endocytosis of the EGF receptor (58
). Also consistent with these findings, we observed that PKN1 co-purifies with several proteins known to regulate vesicle trafficking. Based on these findings, we hypothesize that PKN proteins might directly regulate surface expression of several receptor proteins including LRP6. However, because PKN1 is known to regulate the AKT (54
) and mitogen-activated protein kinase pathways (55
), its effects on WNT3A-dependent signaling could be mediated indirectly via its effects on those pathways. Further research to clarify how PKN1 regulates receptor dynamics may greatly enhance our understanding of the mechanisms governing LRP6 cell surface expression.
Finally, our data showing that depletion of PKN1 increases WNT3A-dependent apoptosis in melanoma cells bolster previous data that PKN1 may be relevant to cancer biology: 1) the depletion of PKN1 also promotes programmed cell death in models of multiple myeloma (64
), 2) PKN1 is overexpressed in prostate tumors (65
) and in certain cohorts of malignant melanoma (B
), and 3) PKN1 is a downstream effector of PDPK1, which is activated during phosphatidylinositol 3-kinase signaling (59
). Finally, our demonstration that depletion of PKN1
increases the number of cells undergoing programmed cell death upon treatment with BRAFi could contribute to improvements in therapies.