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Suppression of chemotaxis by SSeCKS via scaffolding of phosphoinositol phosphates and the recruitment of the Cdc42 GEF, Frabin, to the leading edge.

Ko HK, Guo LW, Su B, Gao L, Gelman IH - PLoS ONE (2014)

Bottom Line: Frabin knockdown in SSeCKS- MEF restores leading edge lamellipodia and chemotaxis inhibition.However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin.Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the actin cytoskeleton controlling directional movement.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York, United States of America.

ABSTRACT
Chemotaxis is controlled by interactions between receptors, Rho-family GTPases, phosphatidylinositol 3-kinases, and cytoskeleton remodeling proteins. We investigated how the metastasis suppressor, SSeCKS, attenuates chemotaxis. Chemotaxis activity inversely correlated with SSeCKS levels in mouse embryo fibroblasts (MEF), DU145 and MDA-MB-231 cancer cells. SSeCKS loss induced chemotactic velocity and linear directionality, correlating with replacement of leading edge lamellipodia with fascin-enriched filopodia-like extensions, the formation of thickened longitudinal F-actin stress fibers reaching to filopodial tips, relative enrichments at the leading edge of phosphatidylinositol (3,4,5)P3 (PIP3), Akt, PKC-ζ, Cdc42-GTP and active Src (SrcpoY416), and a loss of Rac1. Leading edge lamellipodia and chemotaxis inhibition in SSeCKS- MEF could be restored by full-length SSeCKS or SSeCKS deleted of its Src-binding domain (ΔSrc), but not by SSeCKS deleted of its three MARCKS (myristylated alanine-rich C kinase substrate) polybasic domains (ΔPBD), which bind PIP2 and PIP3. The enrichment of activated Cdc42 in SSeCKS- leading edge filopodia correlated with recruitment of the Cdc42-specific guanine nucleotide exchange factor, Frabin, likely recruited via multiple PIP2/3-binding domains. Frabin knockdown in SSeCKS- MEF restores leading edge lamellipodia and chemotaxis inhibition. However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin. Consistent with the notion that chemotaxis is controlled by SSeCKS-PIP (vs. -Src) scaffolding activity, constitutively-active phosphatidylinositol 3-kinase could override the ability of the Src inhibitor, SKI-606, to suppress chemotaxis and filopodial enrichment of Frabin in SSeCKS- MEF. Our data suggest a role for SSeCKS in controlling Rac1 vs. Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the actin cytoskeleton controlling directional movement.

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Enriched active Cdc42 in chemotactic leading edges in the absence of SSeCKS.A, Chemotactic WT or KO MEF were analyzed for localization of active Rac1 and Cdc42 by overlay assay as described in Experimental Procedures. Cells overlaid with GST PAK-PBD were probed by IFA for GST or F-actin, whereas cells overlaid with His-WASP-CBD were probed for His6 or F-actin. B, The leading edge staining enrichment for GST-PAK-PBD or His-WASP-CBD was quantified as described in Fig. 4C. **, p<0.02. C, KO-MEF transfected with CBD-GFP or mCBD-GFP, showing GFP fluorescence (upper panels) or F-actin staining (lower panels). D, Cdc42 activation in KO-MEF pseudopodia. Left panel- Pseudopodia isolated from WT- or KO-MEF as described in Materials and Methods were subjected to pulldown (PD) using CBD- or mCBD-beads followed by IB analysis for bound, active Cdc42. IB of direct lysates (“–”) are shown at right. Right panel- IB analysis of total MEF lysates (“–”) vs. PD using GST-PAK-PBD beads. Arrows, Cdc42 or Rac1 protein. E, Coomassie stained gel of the proteins used for PD in panel D. F, Lamellipodia or filopodia formation in KO MEF expressing DN-Cdc42 or CA-Rac1 (vs. vector). Scale bar (for whole figure), 10 µm. Long white arrows, chemotaxis direction.
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pone-0111534-g006: Enriched active Cdc42 in chemotactic leading edges in the absence of SSeCKS.A, Chemotactic WT or KO MEF were analyzed for localization of active Rac1 and Cdc42 by overlay assay as described in Experimental Procedures. Cells overlaid with GST PAK-PBD were probed by IFA for GST or F-actin, whereas cells overlaid with His-WASP-CBD were probed for His6 or F-actin. B, The leading edge staining enrichment for GST-PAK-PBD or His-WASP-CBD was quantified as described in Fig. 4C. **, p<0.02. C, KO-MEF transfected with CBD-GFP or mCBD-GFP, showing GFP fluorescence (upper panels) or F-actin staining (lower panels). D, Cdc42 activation in KO-MEF pseudopodia. Left panel- Pseudopodia isolated from WT- or KO-MEF as described in Materials and Methods were subjected to pulldown (PD) using CBD- or mCBD-beads followed by IB analysis for bound, active Cdc42. IB of direct lysates (“–”) are shown at right. Right panel- IB analysis of total MEF lysates (“–”) vs. PD using GST-PAK-PBD beads. Arrows, Cdc42 or Rac1 protein. E, Coomassie stained gel of the proteins used for PD in panel D. F, Lamellipodia or filopodia formation in KO MEF expressing DN-Cdc42 or CA-Rac1 (vs. vector). Scale bar (for whole figure), 10 µm. Long white arrows, chemotaxis direction.

Mentions: Based on the SSeCKS-regulated morphological differences and subcellular localization patterns of chemotaxis signaling mediators at the leading edge, we focused on the possibility that SSeCKS differentially regulates Rac and Cdc42 activation at leading edge membrane protrusions. The finding that the leading edge protrusions of KO MEF are predominantly filopodia- rather than lamellipodia-like (Fig. 1F) suggests a hyperactivation of Cdc42. To observe localized activated Rac and Cdc42, we overlaid fixed, chemotactic WT or KO MEF with either GST-tagged PAK-PBD, which binds active, GTP-bound forms of both Rac1 and Cdc42 [53], or His-tagged WASP-CBD, which preferentially binds GTP-Cdc42 [54], and the slides were then processed for epitope-tag-specific immunofluorescence staining. Enriched staining for the GST-PAK-PBD probe was found in lamellipodia and filopodia structures of WT and KO MEF (Fig. 6A). However, whereas the leading edge lamellipodia of WT MEF showed a mild enrichment of diffusely staining His-WASP-CBD probe, KO MEF showed strong punctate enrichments at the ends of their filopodial protrusions (Fig. 6A). Quantification of the GST-PAK-PBD and His-WASP-CBD staining within 0.5 µm of the leading edge confirmed a significant increase in His-WASP-CBD enrichment in KO vs. WT cells (Fig. 6B). The specificity of the enrichment of the CBD probe in leading edge filopodia in KO MEF was confirmed by showing that CBD-GFP, but not a mutant form lacking Cdc42 binding (CBDH246,249D; [55]), could enrich in filopodial tips (Fig. 6C). In order to further assess the relative protein and activation levels of Rac1 and Cdc42 in leading edge structures, we used the technique of Cho et al. [56] to isolate proteins from chemotactic cell projections found on the underside (i.e.- toward the chemoattractant) of 3 µm pores in Boyden chamber membranes. Fig. 6D shows that total Rac1 or Cdc42 protein levels did not differ in lysates of these projections. However, the level of activated Cdc42, based on binding to CBD-beads but not to mutantCBD-beads, was significantly higher in KO projection lysates than in those from WT cells. In contrast, GST-PAK-beads pulled down relatively equal levels of activated Rac1/Cdc42. Fig. 6E shows equal levels of purified CBD and PAK proteins used in overlay (Figs. 6A&C) and pulldown (Fig. 6D) assays.


Suppression of chemotaxis by SSeCKS via scaffolding of phosphoinositol phosphates and the recruitment of the Cdc42 GEF, Frabin, to the leading edge.

Ko HK, Guo LW, Su B, Gao L, Gelman IH - PLoS ONE (2014)

Enriched active Cdc42 in chemotactic leading edges in the absence of SSeCKS.A, Chemotactic WT or KO MEF were analyzed for localization of active Rac1 and Cdc42 by overlay assay as described in Experimental Procedures. Cells overlaid with GST PAK-PBD were probed by IFA for GST or F-actin, whereas cells overlaid with His-WASP-CBD were probed for His6 or F-actin. B, The leading edge staining enrichment for GST-PAK-PBD or His-WASP-CBD was quantified as described in Fig. 4C. **, p<0.02. C, KO-MEF transfected with CBD-GFP or mCBD-GFP, showing GFP fluorescence (upper panels) or F-actin staining (lower panels). D, Cdc42 activation in KO-MEF pseudopodia. Left panel- Pseudopodia isolated from WT- or KO-MEF as described in Materials and Methods were subjected to pulldown (PD) using CBD- or mCBD-beads followed by IB analysis for bound, active Cdc42. IB of direct lysates (“–”) are shown at right. Right panel- IB analysis of total MEF lysates (“–”) vs. PD using GST-PAK-PBD beads. Arrows, Cdc42 or Rac1 protein. E, Coomassie stained gel of the proteins used for PD in panel D. F, Lamellipodia or filopodia formation in KO MEF expressing DN-Cdc42 or CA-Rac1 (vs. vector). Scale bar (for whole figure), 10 µm. Long white arrows, chemotaxis direction.
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Related In: Results  -  Collection

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pone-0111534-g006: Enriched active Cdc42 in chemotactic leading edges in the absence of SSeCKS.A, Chemotactic WT or KO MEF were analyzed for localization of active Rac1 and Cdc42 by overlay assay as described in Experimental Procedures. Cells overlaid with GST PAK-PBD were probed by IFA for GST or F-actin, whereas cells overlaid with His-WASP-CBD were probed for His6 or F-actin. B, The leading edge staining enrichment for GST-PAK-PBD or His-WASP-CBD was quantified as described in Fig. 4C. **, p<0.02. C, KO-MEF transfected with CBD-GFP or mCBD-GFP, showing GFP fluorescence (upper panels) or F-actin staining (lower panels). D, Cdc42 activation in KO-MEF pseudopodia. Left panel- Pseudopodia isolated from WT- or KO-MEF as described in Materials and Methods were subjected to pulldown (PD) using CBD- or mCBD-beads followed by IB analysis for bound, active Cdc42. IB of direct lysates (“–”) are shown at right. Right panel- IB analysis of total MEF lysates (“–”) vs. PD using GST-PAK-PBD beads. Arrows, Cdc42 or Rac1 protein. E, Coomassie stained gel of the proteins used for PD in panel D. F, Lamellipodia or filopodia formation in KO MEF expressing DN-Cdc42 or CA-Rac1 (vs. vector). Scale bar (for whole figure), 10 µm. Long white arrows, chemotaxis direction.
Mentions: Based on the SSeCKS-regulated morphological differences and subcellular localization patterns of chemotaxis signaling mediators at the leading edge, we focused on the possibility that SSeCKS differentially regulates Rac and Cdc42 activation at leading edge membrane protrusions. The finding that the leading edge protrusions of KO MEF are predominantly filopodia- rather than lamellipodia-like (Fig. 1F) suggests a hyperactivation of Cdc42. To observe localized activated Rac and Cdc42, we overlaid fixed, chemotactic WT or KO MEF with either GST-tagged PAK-PBD, which binds active, GTP-bound forms of both Rac1 and Cdc42 [53], or His-tagged WASP-CBD, which preferentially binds GTP-Cdc42 [54], and the slides were then processed for epitope-tag-specific immunofluorescence staining. Enriched staining for the GST-PAK-PBD probe was found in lamellipodia and filopodia structures of WT and KO MEF (Fig. 6A). However, whereas the leading edge lamellipodia of WT MEF showed a mild enrichment of diffusely staining His-WASP-CBD probe, KO MEF showed strong punctate enrichments at the ends of their filopodial protrusions (Fig. 6A). Quantification of the GST-PAK-PBD and His-WASP-CBD staining within 0.5 µm of the leading edge confirmed a significant increase in His-WASP-CBD enrichment in KO vs. WT cells (Fig. 6B). The specificity of the enrichment of the CBD probe in leading edge filopodia in KO MEF was confirmed by showing that CBD-GFP, but not a mutant form lacking Cdc42 binding (CBDH246,249D; [55]), could enrich in filopodial tips (Fig. 6C). In order to further assess the relative protein and activation levels of Rac1 and Cdc42 in leading edge structures, we used the technique of Cho et al. [56] to isolate proteins from chemotactic cell projections found on the underside (i.e.- toward the chemoattractant) of 3 µm pores in Boyden chamber membranes. Fig. 6D shows that total Rac1 or Cdc42 protein levels did not differ in lysates of these projections. However, the level of activated Cdc42, based on binding to CBD-beads but not to mutantCBD-beads, was significantly higher in KO projection lysates than in those from WT cells. In contrast, GST-PAK-beads pulled down relatively equal levels of activated Rac1/Cdc42. Fig. 6E shows equal levels of purified CBD and PAK proteins used in overlay (Figs. 6A&C) and pulldown (Fig. 6D) assays.

Bottom Line: Frabin knockdown in SSeCKS- MEF restores leading edge lamellipodia and chemotaxis inhibition.However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin.Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the actin cytoskeleton controlling directional movement.

View Article: PubMed Central - PubMed

Affiliation: Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, New York, United States of America.

ABSTRACT
Chemotaxis is controlled by interactions between receptors, Rho-family GTPases, phosphatidylinositol 3-kinases, and cytoskeleton remodeling proteins. We investigated how the metastasis suppressor, SSeCKS, attenuates chemotaxis. Chemotaxis activity inversely correlated with SSeCKS levels in mouse embryo fibroblasts (MEF), DU145 and MDA-MB-231 cancer cells. SSeCKS loss induced chemotactic velocity and linear directionality, correlating with replacement of leading edge lamellipodia with fascin-enriched filopodia-like extensions, the formation of thickened longitudinal F-actin stress fibers reaching to filopodial tips, relative enrichments at the leading edge of phosphatidylinositol (3,4,5)P3 (PIP3), Akt, PKC-ζ, Cdc42-GTP and active Src (SrcpoY416), and a loss of Rac1. Leading edge lamellipodia and chemotaxis inhibition in SSeCKS- MEF could be restored by full-length SSeCKS or SSeCKS deleted of its Src-binding domain (ΔSrc), but not by SSeCKS deleted of its three MARCKS (myristylated alanine-rich C kinase substrate) polybasic domains (ΔPBD), which bind PIP2 and PIP3. The enrichment of activated Cdc42 in SSeCKS- leading edge filopodia correlated with recruitment of the Cdc42-specific guanine nucleotide exchange factor, Frabin, likely recruited via multiple PIP2/3-binding domains. Frabin knockdown in SSeCKS- MEF restores leading edge lamellipodia and chemotaxis inhibition. However, SSeCKS failed to co-immunoprecipitate with Rac1, Cdc42 or Frabin. Consistent with the notion that chemotaxis is controlled by SSeCKS-PIP (vs. -Src) scaffolding activity, constitutively-active phosphatidylinositol 3-kinase could override the ability of the Src inhibitor, SKI-606, to suppress chemotaxis and filopodial enrichment of Frabin in SSeCKS- MEF. Our data suggest a role for SSeCKS in controlling Rac1 vs. Cdc42-induced cellular dynamics at the leading chemotactic edge through the scaffolding of phospholipids and signal mediators, and through the reorganization of the actin cytoskeleton controlling directional movement.

Show MeSH
Related in: MedlinePlus