M-Ras/Shoc2 signaling modulates E-cadherin turnover and cell-cell adhesion during collective cell migration

Abstract

Collective cell migration is required for normal embryonic development and contributes to various biological processes, including wound healing and cancer cell invasion. The M-Ras GTPase and its effector, the Shoc2 scaffold, are proteins mutated in the developmental RASopathy Noonan syndrome, and, here, we report that activated M-Ras recruits Shoc2 to cell surface junctions where M-Ras/Shoc2 signaling contributes to the dynamic regulation of cell-cell junction turnover required for collective cell migration. MCF10A cells expressing the dominant-inhibitory M-Ras^S27N variant or those lacking Shoc2 exhibited reduced junction turnover and were unable to migrate effectively as a group. Through further depletion/reconstitution studies, we found that M-Ras/Shoc2 signaling contributes to junction turnover by modulating the E-cadherin/p120-catenin interaction and, in turn, the junctional expression of E-cadherin. The regulatory effect of the M-Ras/Shoc2 complex was mediated at least in part through the phosphoregulation of p120-catenin and required downstream ERK cascade activation. Strikingly, cells rescued with the Noonan-associated, myristoylated-Shoc2 mutant (Myr-Shoc2) displayed a gain-of-function (GOF) phenotype, with the cells exhibiting increased junction turnover and reduced E-cadherin/p120-catenin binding and migrating as a faster but less cohesive group. Consistent with these results, Noonan-associated C-Raf mutants that bypass the need for M-Ras/Shoc2 signaling exhibited a similar GOF phenotype when expressed in Shoc2-depleted MCF10A cells. Finally, expression of the Noonan-associated Myr-Shoc2 or C-Raf mutants, but not their WT counterparts, induced gastrulation defects indicative of aberrant cell migration in zebrafish embryos, further demonstrating the function of the M-Ras/Shoc2/ERK cascade signaling axis in the dynamic control of coordinated cell movement.

Keywords: C-Raf; M-Ras; Noonan syndrome; Shoc2; collective cell migration.

Fig. 1.

Activated M-Ras recruits Shoc2 to cell–cell junctions. (A) Shown are BRET saturation curves examining the interaction of Shoc2-Rluc8 or C-Raf-R-luc8 and Venus-M-Ras^Q71L, K-Ras^Q61L, N-Ras^Q61L, or H-Ras^Q61L. BRET₅₀ values, indicative of binding affinity, are also listed. (B) Endogenous Shoc2 complexes were immunoprecipitated (IP) from MCF10A cells stably expressing HA-tagged M-Ras^Q71L, HA-K-Ras^Q61L, or the indicated M-Ras^Q71L variants, and the complexes examined for HA-Ras binding and Shoc2 levels. (C) MCF10A cells stably expressing HA-M-Ras^Q71L, HA-K-Ras^Q61L, or vector alone were fractionated into cytosol and membrane-rich fractions, following which the fractions were probed for Shoc2, HA-Ras, E-cadherin (E-Cad), RhoGDI (cytosolic), and the transferrin receptor (TfRc, membrane). (D) Live cell imaging of cells expressing GFP-WT-Shoc2 and mCherry, GFP and mCherry-M-Ras^Q71L, or GFP-WT-Shoc2 and mCherry-M-Ras^Q71L, showing that M-Ras^Q71L can recruit Shoc2 to cell–cell junctions. (E) Immunofluorescent staining of endogenous Shoc2 and M-Ras proteins at cell–cell junctions in serum-stimulated MCF10A cells. Magenta lines indicate free cell edges, and white arrows indicate cell–cell junctions.

Fig. 2.

Shoc2 is required for M-Ras–driven collective migration of MCF10A cells. (A) Confluent monolayers of MCF10A cells stably expressing the indicated HA-M-Ras proteins were wounded, and images taken at 0 and 18 h after wounding. HA-M-Ras and tubulin levels are shown. (B) M-Ras^Q71L–expressing MCF10A cells were transfected with control (siNeg) or Shoc2 siRNAs before wounding. Images were taken at 0 and 18 h after wounding. Lysates were also examined for Shoc2, HA-M-Ras^Q71L, and tubulin levels. (C) M-Ras^Q71L–expressing MCF10A cells transfected with control or Shoc2 siRNAs were plated at low density on collagen-coated surfaces, and isolated cells were tracked for their direction and velocity of movement over a 12-h period. (D) Serum-starved clusters of MCF10A cells stably expressing the indicated proteins were induced to scatter by the addition of growth media. Shown are images taken at 0 and 16 h after scatter induction. Red lines indicate free cell edges in A, B, and D.

Fig. 3.

Analysis of Shoc2 mutant proteins: protein interactions, localization, and migratory properties. (A) Pyo-Shoc2 proteins were immunoprecipitated from lysates of MCF10A cells stably expressing HA-M-Ras^Q71L together with the indicated Pyo-Shoc2 proteins. Pyo-Shoc2 complexes were probed for the presence of HA-M-Ras, PP1c, Scribble (Scrib), and Pyo-Shoc2. (B) BRET analysis of binding interactions between the indicated Shoc2-Rluc8 proteins and Venus-M-Ras^Q71L. (C) Live cell imaging of cells expressing the indicated GFP-Shoc2 proteins and mCherry-M-Ras^Q71L. (D and E) Control MCF10A cells or lines stably expressing siShoc2-resistant GFP-Shoc2 proteins were transfected with control or Shoc2 siRNAs before analysis in wound-healing (D) and cell-scattering assays (E). GFP-Shoc2 and tubulin levels are also shown. Error bars represent mean ± SD, ****P > 0.0001. Red lines indicate free cell edges.

Fig. 4.

Computational analysis of Shoc2 function in collective cell migration. (A–F) Wound-healing and cell-scattering assays were conducted using cells stably expressing shNeg, shShoc2, or shShoc2 reexpressing either Pyo-WT-Shoc2 or Pyo-Myr-Shoc2. (A) Percent normalized wound coverage and nearest neighbor scatter distances were determined from images taken at 1-h and 30-min intervals, respectively. (B) PIV analysis was conducted to determine the velocity fields underlying cell motions captured by time-lapse movies (Top) of wound healing. Speed of cell migration (Middle) and persistence of cell movement in a given direction (Bottom) are depicted with a heat map, and directionality is indicated with white arrows. A black arrow points to a Myr-Shoc2–expressing cell that has escaped from the leading edge. Red lines indicate the leading edge border. Rose plots are also shown depicting the aggregate directionality distributions compiled over all times and space for each cell condition. (C) Speed distributions were determined over all times and space of wound healing, and shown is the mean average speed for each cell condition. (D) Variation in the directionality of motion was quantified and represented as the mean coefficient of directional variation. (E) Spatial velocity correlation analysis was used to determine the length of coordinated cell movement. (F) Biorthogonal decomposition (BOD) analysis was used to assess cohesiveness of the monolayer during sheet movement. Analysis reveals the number of connected components (“modes”) needed to cover the monolayer.

Fig. 5.

M-Ras/Shoc2 signaling modulates cell–cell adhesion through effects on E-cadherin turnover. (A) MCF10A cells stably expressing GFP-α-catenin alone or coexpressing GFP-α-catenin and the indicated Shoc2 proteins were transfected with control or Shoc2 siRNAs before junction analysis. Circular regions of interest (ROIs, indicated by red circles) at the cell–cell junctions were photobleached, and the recovery of GFP-α-catenin into the bleached areas was quantified and compared using exponential functions representing the fast and slow phases of recovery. (Error bars represent mean ± SD, ****P > 0.0001). (B) For each of the lines, mean fluorescence recovery over time is indicated as a solid line, with SEM depicted as dotted lines. (C) MCF10A cells stably expressing control shNeg or shShoc2 vectors were analyzed for E-cadherin localization during cell scattering. Shown are images taken at 0 and 9 h after scatter induction. (D) Confluent monolayers of MCF10A cells stably expressing shNeg or shShoc2 vectors were analyzed for E-cadherin and p120-catenin localization. Also analyzed were shShoc2-cells reexpressing WT- or D175N-Shoc2 proteins. Yellow arrows in C and D indicate cell–cell junctions. (E) Proteins on the surface of MCF10A cells stably expressing shNeg or shShoc2 vectors were biotinylated before cell lysis. The biotinylated surface proteins were then isolated using avidin-coupled beads, following which the beads were analyzed for the presence of EGFR and E-cadherin. Lysates were also monitored for EGFR, E-cadherin, and Shoc2 levels. (F) Endogenous p120-catenin or E-cadherin was immunoprecipitated from lysates of MCF10A cells stably expressing shNeg or shShoc2 vectors. p120-catenin immunoprecipitates were probed for E-cadherin, phosphotyrosine (pY), or pT310 levels, and E-cadherin immunoprecipitates were probed for pY levels. Total Shoc2, E-cadherin, and p120-catenin levels are also shown. (G) MCF10A cells treated for 18 h with DMSO or U0126 were analyzed as in F. (H) Purified p120-catenin was incubated with purified active ERK or GSK3 in the presence of γ[³²P]-ATP, following which the p120-catenin protein was isolated and digested with trypsin. The resulting tryptic phosphopeptides were separated by reversed phase HPLC (Top), and the phosphopeptide isolated in fraction 4 was subjected to phosphoamino acid analysis (PAA), Edman degradation (Edman), and sequencing to determine the residue phosphorylated (indicated in red).

Fig. 6.

GOF activity of Noonan-associated Myr-Shoc2 and C-Raf mutants. (A) The indicated Flag-C-Raf proteins were isolated from MCF10A cells and probed for the phosphorylation state of S259 using pS259-C-Raf antibodies. (B and C) Wound-healing (B) and cell-scattering assays (C) were conducted using cells stably expressing shNeg, shShoc2, or shShoc2 and either Flag-tagged WT-, S257L- or P261S-C-Raf. Red arrows indicate cells dissociating from the leading edge of monolayers expressing S257L- and P261S-C-Raf in B. Red lines indicate free cell edges in C. (D and E) MCF10A cells stably expressing shShoc2 or shShoc2 and the indicated Shoc2 or C-Raf proteins were generated. The cell lines were then examined for binding between p120-catenin and E-cadherin in immunoprecipitation assays (D) and for E-cadherin and p120-catenin localization in live cell imaging studies (E). Arrows indicate the “stretched” appearance of the cell–cell junctions in lines expressing the Noonan-associated mutants. (F) mRNA encoding the indicated Shoc2 and C-Raf proteins were injected into one-cell stage zebrafish embryos, and the embryos were measured at 11 h postfertilization (hpf) to determine the major to minor axis ratio. Shown are representative images of the embryos, expression levels of the indicated proteins, and the average axis ratio of embryos analyzed in three independent experiments. (G) mRNA-injected embryos were treated with DMSO or 7 μM PD0325901 (MEK In) from 4.5 to 5.5 hpf, and embryos were measured at 11 hpf. The average axis ratio of embryos analyzed in three independent experiments is shown. Error bars represent mean ± SD, ****P > 0.0001.