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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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SSeCKS controls the localization of signaling mediators that regulate leading edge protrusion formation.A, SSeCKS scaffolds PKCζ. PKCζ IB analysis of HEK293T cell lysates transfected with PKCζ-GFP or pEGFP (vect) after pull down using GST- or GST-SSeCKS-beads. Aliquots of lysates (5 µg, representing 1% input) are shown on the right. Note that PKCζ (arrow) binds one of the two PKC-binding domains identified previously [29], and that deletion of the minimal binding domain (a.a.- 745–753) abrogates this binding. B, Chemotactic WT or KO MEF were stained by IFA for Akt, Rac1, Cdc42, PKC-ζ or Par6, and the leading edge staining was quantified in Panel C as in Fig. 4C. *, p<0.05, **, p<0.01. Scale bar, 10 µm. D, IB analysis for the proteins stained in panel A, plus GAPDH controls. MWt markers are shown at right.
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pone-0111534-g005: SSeCKS controls the localization of signaling mediators that regulate leading edge protrusion formation.A, SSeCKS scaffolds PKCζ. PKCζ IB analysis of HEK293T cell lysates transfected with PKCζ-GFP or pEGFP (vect) after pull down using GST- or GST-SSeCKS-beads. Aliquots of lysates (5 µg, representing 1% input) are shown on the right. Note that PKCζ (arrow) binds one of the two PKC-binding domains identified previously [29], and that deletion of the minimal binding domain (a.a.- 745–753) abrogates this binding. B, Chemotactic WT or KO MEF were stained by IFA for Akt, Rac1, Cdc42, PKC-ζ or Par6, and the leading edge staining was quantified in Panel C as in Fig. 4C. *, p<0.05, **, p<0.01. Scale bar, 10 µm. D, IB analysis for the proteins stained in panel A, plus GAPDH controls. MWt markers are shown at right.

Mentions: The localized enrichment of PIP2 and PIP3 at the leading edge is known to recruit and activate several signaling mediators of chemotaxis, such as Rac1, Cdc42 and AKT [2]. Additionally, an important mechanism that influences chemotaxis is the ability of activated Cdc42 to recruit Par6 and atypical PKC isoforms to the polarized leading edge [51], [52]. We speculated that SSeCKS might alter chemotaxis by affecting the recruitment of one or more of these factors at the leading edge. Indeed, in the case of PKC, SSeCKS can directly scaffold conventional, novel [29], and atypical (Fig. 5A) isoforms. KO MEF exhibited increased enrichment levels of Akt, PKC-ζ and Cdc42 at leading edge filopodial protrusions, whereas WT MEF exhibited higher enrichment levels of Rac1 in lamellipodia (Figs. 5B & C). In contrast, SSeCKS did not seem to affect Par6 enrichment at the leading edge. Significantly, WT and KO MEF showed no major differences in the total cellular levels of these proteins (Fig. 5D).


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)

SSeCKS controls the localization of signaling mediators that regulate leading edge protrusion formation.A, SSeCKS scaffolds PKCζ. PKCζ IB analysis of HEK293T cell lysates transfected with PKCζ-GFP or pEGFP (vect) after pull down using GST- or GST-SSeCKS-beads. Aliquots of lysates (5 µg, representing 1% input) are shown on the right. Note that PKCζ (arrow) binds one of the two PKC-binding domains identified previously [29], and that deletion of the minimal binding domain (a.a.- 745–753) abrogates this binding. B, Chemotactic WT or KO MEF were stained by IFA for Akt, Rac1, Cdc42, PKC-ζ or Par6, and the leading edge staining was quantified in Panel C as in Fig. 4C. *, p<0.05, **, p<0.01. Scale bar, 10 µm. D, IB analysis for the proteins stained in panel A, plus GAPDH controls. MWt markers are shown at right.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4214753&req=5

pone-0111534-g005: SSeCKS controls the localization of signaling mediators that regulate leading edge protrusion formation.A, SSeCKS scaffolds PKCζ. PKCζ IB analysis of HEK293T cell lysates transfected with PKCζ-GFP or pEGFP (vect) after pull down using GST- or GST-SSeCKS-beads. Aliquots of lysates (5 µg, representing 1% input) are shown on the right. Note that PKCζ (arrow) binds one of the two PKC-binding domains identified previously [29], and that deletion of the minimal binding domain (a.a.- 745–753) abrogates this binding. B, Chemotactic WT or KO MEF were stained by IFA for Akt, Rac1, Cdc42, PKC-ζ or Par6, and the leading edge staining was quantified in Panel C as in Fig. 4C. *, p<0.05, **, p<0.01. Scale bar, 10 µm. D, IB analysis for the proteins stained in panel A, plus GAPDH controls. MWt markers are shown at right.
Mentions: The localized enrichment of PIP2 and PIP3 at the leading edge is known to recruit and activate several signaling mediators of chemotaxis, such as Rac1, Cdc42 and AKT [2]. Additionally, an important mechanism that influences chemotaxis is the ability of activated Cdc42 to recruit Par6 and atypical PKC isoforms to the polarized leading edge [51], [52]. We speculated that SSeCKS might alter chemotaxis by affecting the recruitment of one or more of these factors at the leading edge. Indeed, in the case of PKC, SSeCKS can directly scaffold conventional, novel [29], and atypical (Fig. 5A) isoforms. KO MEF exhibited increased enrichment levels of Akt, PKC-ζ and Cdc42 at leading edge filopodial protrusions, whereas WT MEF exhibited higher enrichment levels of Rac1 in lamellipodia (Figs. 5B & C). In contrast, SSeCKS did not seem to affect Par6 enrichment at the leading edge. Significantly, WT and KO MEF showed no major differences in the total cellular levels of these proteins (Fig. 5D).

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