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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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Related in: MedlinePlus

Model of chemotaxis leading edge structure/function by SSeCKS.In WT (SSeCKS+/+) cells (top), the ability of SSeCKS to scaffold Src at lipid raft sites allows for local activation of PI3K/AKT signaling, thereby producing locally enriched PIP2/3. SSeCKS’ additional ability to scaffold PIPs, including PIP2/3, at the chemotactic leading edge facilitates the local activation of Rac1, resulting in lamellipodial-dependent chemotaxis. In contrast, in the absence of SSeCKS’s scaffolding of PIPs, PIP3 enriches in budding filopodia, which attracts binding of Frabin through its PH and FYVE domains. This leads to local activation of Cdc42 and the subsequent domination of growing filopodia at the leading edge. Of note is that SSeCKS also affects actin cytoskeletal modeling not addressed directly in this paper, such as the generation of thickened, longitudinal stress fibers (SF) and the loss of arc SF in SSeCKS- cells.
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pone-0111534-g009: Model of chemotaxis leading edge structure/function by SSeCKS.In WT (SSeCKS+/+) cells (top), the ability of SSeCKS to scaffold Src at lipid raft sites allows for local activation of PI3K/AKT signaling, thereby producing locally enriched PIP2/3. SSeCKS’ additional ability to scaffold PIPs, including PIP2/3, at the chemotactic leading edge facilitates the local activation of Rac1, resulting in lamellipodial-dependent chemotaxis. In contrast, in the absence of SSeCKS’s scaffolding of PIPs, PIP3 enriches in budding filopodia, which attracts binding of Frabin through its PH and FYVE domains. This leads to local activation of Cdc42 and the subsequent domination of growing filopodia at the leading edge. Of note is that SSeCKS also affects actin cytoskeletal modeling not addressed directly in this paper, such as the generation of thickened, longitudinal stress fibers (SF) and the loss of arc SF in SSeCKS- cells.

Mentions: As mentioned above, SSeCKS encodes scaffolding domains for several possible mediators of cytoskeletal remodeling and leading edge formation, including domains for Src and PIP binding. Although the Src-scaffolding activity of SSeCKS was not necessary for the rescue of lamellipodia formation and Frabin translocation away from the leading edge (Fig. 7B), we noted that the leading edges of KO cells showed concentrations of FAK and active Src (SrcpoY416) at the tips of leading edge filopodia (Figs. 8A&B), although total Src protein and activation levels in MEF are not affected by SSeCKS [28]. Indeed, the FAK/Src configuration in KO MEF is consistent with increased enrichment of FAK and activated Src at the growing ends of so-called dorsal stress fibers, defined as being attached to focal adhesions at only one end [60]. Given that the enhanced chemotaxis of KO MEF requires PI3K activity (Fig. 3B), we endeavored a more comprehensive analysis of whether the enhanced chemotaxis and associated cytoskeletal and leading edge structures in KO cells is controlled locally by a Src-PI3K pathway that would directly influence production of local pools of PIP3 [12] and Frabin recruitment. Consistent with the Frabin localization and cytoskeletal remodeling result in Fig. 7B, FL and ΔSrc, but not ΔPBD-SSeCKS, could decrease the enhanced chemotaxis of KO MEF relative to levels in WT MEF (Fig. 8C). Inhibition of Src activity using the Src/Abl kinase inhibitor, SKI-606/bosutinib [61], decreased chemotaxis in WT and KO cells to statistically similar levels (Fig. 8D), yet induced lamellipodia formation and Frabin internalization from the leading edge in KO cells (Fig. 8E). However, although CA-PI3K did not increase chemotaxis (Fig. 8F), filopodia formation or Frabin enrichment at leading edge filopodia ends in KO cells (Fig. 8G), it did negate the ability of SKI-606 to inhibit chemotaxis (Fig. 8F) or to induce lamellipodia formation and Frabin internalization (Fig. 8G). This suggests that the enhanced chemotaxis of KO MEF is controlled by PI3K activity that is downstream of Src. Interestingly, whereas SKI-606 and ΔSrc-SSeCKS caused stress fibers to pull back from leading edge lamellipodia in KO cells (Figs. 8E&H), CA-PI3K seemed to induce fewer internal and more cell edge stress fibers (Fig. 8H), an effect that was not changed by SKI-606. Taken together with our earlier data, these findings suggest that in the absence of SSeCKS’ PIP scaffolding function, PIP2/3 concentrate at filopodial ends of the leading edge (Fig. 4). This leads to the enrichment of Frabin in these membrane sites via intrinsic PH and FYVE domains, and to the growing tips of F-actin fibers via an intrinsic FAB domain [62], resulting in increased chemotaxis through the local activation of Cdc42 (Fig. 9).


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)

Model of chemotaxis leading edge structure/function by SSeCKS.In WT (SSeCKS+/+) cells (top), the ability of SSeCKS to scaffold Src at lipid raft sites allows for local activation of PI3K/AKT signaling, thereby producing locally enriched PIP2/3. SSeCKS’ additional ability to scaffold PIPs, including PIP2/3, at the chemotactic leading edge facilitates the local activation of Rac1, resulting in lamellipodial-dependent chemotaxis. In contrast, in the absence of SSeCKS’s scaffolding of PIPs, PIP3 enriches in budding filopodia, which attracts binding of Frabin through its PH and FYVE domains. This leads to local activation of Cdc42 and the subsequent domination of growing filopodia at the leading edge. Of note is that SSeCKS also affects actin cytoskeletal modeling not addressed directly in this paper, such as the generation of thickened, longitudinal stress fibers (SF) and the loss of arc SF in SSeCKS- cells.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4214753&req=5

pone-0111534-g009: Model of chemotaxis leading edge structure/function by SSeCKS.In WT (SSeCKS+/+) cells (top), the ability of SSeCKS to scaffold Src at lipid raft sites allows for local activation of PI3K/AKT signaling, thereby producing locally enriched PIP2/3. SSeCKS’ additional ability to scaffold PIPs, including PIP2/3, at the chemotactic leading edge facilitates the local activation of Rac1, resulting in lamellipodial-dependent chemotaxis. In contrast, in the absence of SSeCKS’s scaffolding of PIPs, PIP3 enriches in budding filopodia, which attracts binding of Frabin through its PH and FYVE domains. This leads to local activation of Cdc42 and the subsequent domination of growing filopodia at the leading edge. Of note is that SSeCKS also affects actin cytoskeletal modeling not addressed directly in this paper, such as the generation of thickened, longitudinal stress fibers (SF) and the loss of arc SF in SSeCKS- cells.
Mentions: As mentioned above, SSeCKS encodes scaffolding domains for several possible mediators of cytoskeletal remodeling and leading edge formation, including domains for Src and PIP binding. Although the Src-scaffolding activity of SSeCKS was not necessary for the rescue of lamellipodia formation and Frabin translocation away from the leading edge (Fig. 7B), we noted that the leading edges of KO cells showed concentrations of FAK and active Src (SrcpoY416) at the tips of leading edge filopodia (Figs. 8A&B), although total Src protein and activation levels in MEF are not affected by SSeCKS [28]. Indeed, the FAK/Src configuration in KO MEF is consistent with increased enrichment of FAK and activated Src at the growing ends of so-called dorsal stress fibers, defined as being attached to focal adhesions at only one end [60]. Given that the enhanced chemotaxis of KO MEF requires PI3K activity (Fig. 3B), we endeavored a more comprehensive analysis of whether the enhanced chemotaxis and associated cytoskeletal and leading edge structures in KO cells is controlled locally by a Src-PI3K pathway that would directly influence production of local pools of PIP3 [12] and Frabin recruitment. Consistent with the Frabin localization and cytoskeletal remodeling result in Fig. 7B, FL and ΔSrc, but not ΔPBD-SSeCKS, could decrease the enhanced chemotaxis of KO MEF relative to levels in WT MEF (Fig. 8C). Inhibition of Src activity using the Src/Abl kinase inhibitor, SKI-606/bosutinib [61], decreased chemotaxis in WT and KO cells to statistically similar levels (Fig. 8D), yet induced lamellipodia formation and Frabin internalization from the leading edge in KO cells (Fig. 8E). However, although CA-PI3K did not increase chemotaxis (Fig. 8F), filopodia formation or Frabin enrichment at leading edge filopodia ends in KO cells (Fig. 8G), it did negate the ability of SKI-606 to inhibit chemotaxis (Fig. 8F) or to induce lamellipodia formation and Frabin internalization (Fig. 8G). This suggests that the enhanced chemotaxis of KO MEF is controlled by PI3K activity that is downstream of Src. Interestingly, whereas SKI-606 and ΔSrc-SSeCKS caused stress fibers to pull back from leading edge lamellipodia in KO cells (Figs. 8E&H), CA-PI3K seemed to induce fewer internal and more cell edge stress fibers (Fig. 8H), an effect that was not changed by SKI-606. Taken together with our earlier data, these findings suggest that in the absence of SSeCKS’ PIP scaffolding function, PIP2/3 concentrate at filopodial ends of the leading edge (Fig. 4). This leads to the enrichment of Frabin in these membrane sites via intrinsic PH and FYVE domains, and to the growing tips of F-actin fibers via an intrinsic FAB domain [62], resulting in increased chemotaxis through the local activation of Cdc42 (Fig. 9).

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