Mechanisms by which Wnt pathways integrate the organization of receptors, organelles, and cytoskeletal proteins to confer cell polarity and directional cell movement are incompletely understood. We show that acute responses to Wnt5a involve recruitment of actin, myosin IIB, Frizzled 3, and melanoma cell adhesion molecule into an intracellular structure in a melanoma cell line. In the presence of a chemokine gradient, this Wnt-mediated receptor-actin-myosin polarity (W-RAMP) structure accumulates asymmetrically at the cell periphery, where it triggers membrane contractility and nuclear movement in the direction of membrane retraction. The process requires endosome trafficking, is associated with multivesicular bodies, and is regulated by Wnt5a through the small guanosine triphosphatases Rab4 and RhoB. Thus, cell-autonomous mechanisms allow Wnt5a to control cell orientation, polarity, and directional movement in response to positional cues from chemokine gradients.Wnt signaling controls cell polarity and directional cell movement in developmental systems, as well as cell invasion in certain cancers. Features shared between noncanonical Wnt pathways include recruitment of Frizzled (Fz) receptors to the posterior end of cells, and asymmetric distribution of atypical cell adhesion molecules, often associated with Fz (1, 2). Thus, receptors redistribute, in response to Wnt, to define an axis of asymmetry. In developmental systems, these processes can be regulated by interactions with adjacent cells, which confer orientation with respect to surrounding tissues (1, 3). For example, during endoderm specification in Caenorhabditis elegans, the division plane in the four-cell blastomere is determined by a positional Wnt signal from a near-by P 2 cell (4). In contrast, Wnt pathway mutations in C. elegans that disrupt neuronal cell migration and polarity can be rescued by Wnt overexpression without requiring a localized source of ligand (5, 6). This suggests that directional presentation of Wnt to cells is not always needed for cell polarization, rather that Wnt ligands act permissively to allow cells to respond to environmental cues for longitudinal guidance and directional movement. In such cases, WM239A cells treated with Wnt5a were immunostained for melanoma cell adhesion molecule (MCAM, also known as MUC18 and CD146), an immunoglobulin G-family cell adhesion receptor implicated in melanoma tumorigenesis and metastasis (9). In untreated cells, MCAM was distributed uniformly. However, treatment of cells with Wnt5a for 30 min led to redistribution of MCAM into a polarized structure, which we call the Wnt5a-mediated receptor-actin-myosin polarity (W-RAMP) structure, for reasons described below (Fig. 1, A and B). The W-RAMP structure was also observed in some untreated cells, but this was suppressed by depleting Wnt5a with RNA interference (RNAi) (Fig. 1B), which indicated that the basal signal results from autocrine responses to endogeneous ligand. Similar W-RAMP structures were observed in WM1789 melanoma cells, as...

SUMMARY Melanoma and other cancers harbor oncogenic mutations in the protein kinase B-Raf, which leads to constitutive activation and dysregulation of MAP kinase signaling. In order to elucidate molecular determinants responsible for B-Raf control of cancer phenotypes, we present a method for phosphoprotein profiling, using negative ionization mass spectrometry to detect phosphopeptides based on their fragment ion signature caused by release of PO3−. The method provides an alternative strategy for phosphoproteomics, circumventing affinity enrichment of phosphopeptides and isotopic labeling of samples. Ninety phosphorylation events were regulated by oncogenic B-Raf signaling, based on their responses to treating melanoma cells with MKK1/2 inhibitor. Regulated phosphoproteins included known signaling effectors and cytoskeletal regulators. We investigated MINERVA/FAM129B, a target belonging to a protein family with unknown category and function, and established the importance of this protein and its MAP kinase-dependent phosphorylation in controlling melanoma cell invasion into 3-dimensional collagen matrix.

Human melanomas show oncogenic B-Raf mutations which activate the B-Raf/MKK/ERK cascade. We screened microarrays to identify cellular targets of this pathway, and found that genes upregulated by B-Raf/MKK/ERK showed highest association with cell cycle regulators, whereas genes downregulated were most highly associated with axon guidance genes, including plexin-semaphorin family members. Plexin B1 was strongly inhibited by MAP kinase signaling in melanoma cells and melanocytes. In primary melanoma cells, plexin B1 blocked tumorigenesis as measured by growth of colonies in soft agar, spheroids in extracellular matrix, and xenograft tumors. Tumor suppression depended on residues in the C-terminal domain of plexin B1 which mediate receptor GAP activity, and also correlated with AKT inhibition. Interestingly, the inhibitory response to plexin B1 was reduced or absent in cells from a matched metastatic tumor, suggesting that changes occur in metastatic cells which bypass the tumor suppressor mechanisms. Plexin B1 also inhibited cell migration, but this was seen in metastatic cells and not in matched primary cells. Thus, plexin B1 has tumor suppressor function in early-stage cells, while suppressing migration in late-stage cells. Our findings suggest that B-Raf/MKK/ERK provides a permissive environment for melanoma genesis by modulating plexin B1.