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Spatial and temporal regulation of cofilin activity by LIM kinase and Slingshot is critical for directional cell migration.

Nishita M, Tomizawa C, Yamamoto M, Horita Y, Ohashi K, Mizuno K - J. Cell Biol. (2005)

Bottom Line: Cofilin is inactivated by LIM kinase (LIMK)-1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L.In this study, we show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells.We propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.

ABSTRACT
Cofilin mediates lamellipodium extension and polarized cell migration by accelerating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by LIM kinase (LIMK)-1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L. In this study, we show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells. The knockdown of LIMK1 suppressed chemokine-induced lamellipodium formation and cell migration, whereas SSH1L knockdown produced and retained multiple lamellipodial protrusions around the cell after cell stimulation and impaired directional cell migration. Our results indicate that LIMK1 is required for cell migration by stimulating lamellipodium formation in the initial stages of cell response and that SSH1L is crucially involved in directional cell migration by restricting the membrane protrusion to one direction and locally stimulating cofilin activity in the lamellipodium in the front of the migrating cell. We propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement.

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Trp-458 is critical for the F-actin–mediated activation of SSH1L. (A) The role of amino acids 457–461 in F-actin–mediated SSH1L activation. Schematic structures of SSH1L and deletion mutants are shown. The conserved regions in the SSH family are indicated by the A, B, P (phosphatase), and S (Ser-rich) domains. Wild-type (WT) and deletion mutants of (myc + His)-tagged SSH1L were expressed in 293T cells, immunoprecipitated with an anti-myc antibody, and subjected to in vitro phosphatase assays using cofilin-(His)6 as a substrate in the presence or absence of F-actin. P-cofilin levels were measured by Pro-Q staining. Total cofilin and actin were measured by Coomassie blue staining. The expression of SSH1L mutants was analyzed by immunoblotting with the anti-myc antibody. *, Ig heavy chain. (B) Trp-458 is required for F-actin–mediated SSH1L activation. Point mutants of N461 and full-length (FL) SSH1L were subjected to in vitro cofilin phosphatase assays as described in A. Arrow indicates the replacement of Trp-458 with Ala.
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fig7: Trp-458 is critical for the F-actin–mediated activation of SSH1L. (A) The role of amino acids 457–461 in F-actin–mediated SSH1L activation. Schematic structures of SSH1L and deletion mutants are shown. The conserved regions in the SSH family are indicated by the A, B, P (phosphatase), and S (Ser-rich) domains. Wild-type (WT) and deletion mutants of (myc + His)-tagged SSH1L were expressed in 293T cells, immunoprecipitated with an anti-myc antibody, and subjected to in vitro phosphatase assays using cofilin-(His)6 as a substrate in the presence or absence of F-actin. P-cofilin levels were measured by Pro-Q staining. Total cofilin and actin were measured by Coomassie blue staining. The expression of SSH1L mutants was analyzed by immunoblotting with the anti-myc antibody. *, Ig heavy chain. (B) Trp-458 is required for F-actin–mediated SSH1L activation. Point mutants of N461 and full-length (FL) SSH1L were subjected to in vitro cofilin phosphatase assays as described in A. Arrow indicates the replacement of Trp-458 with Ala.

Mentions: As SSH1L is highly activated by its association with F-actin (Nagata-Ohashi et al., 2004), we hypothesized that the F-actin–mediated activation of SSH1L is critical for polarized cell migration. To identify the sites of SSH1L that are required for F-actin–mediated activation, a series of COOH-terminally deleted mutants of SSH1L were constructed. The truncated proteins were subjected to in vitro cofilin phosphatase assays in the absence or presence of F-actin by using P-cofilin as a substrate (Fig. 7 A). As previously reported (Nagata-Ohashi et al., 2004), wild-type (WT) SSH1L was activated by F-actin, but the COOH-terminally deleted mutant NP (composed of amino acid residues 1–456) was not. Unexpectedly, the other COOH-terminally deleted mutants (N960, N698, N486, and N461) were all activated by F-actin. This indicates that the region corresponding to amino acids 457–461 is required for the F-actin–mediated activation of SSH1L. To further define the residues that are essential for activation, various point mutants of N461 were constructed and subjected to cofilin phosphatase assays with or without F-actin (Fig. 7 B). The assays revealed that W458A (Trp-458 replaced by Ala) and 3A (Leu-457, Trp-458, and Arg-459 replaced by Ala) mutants of N461 were only weakly activated in the presence of F-actin, whereas other point mutants were remarkably activated by F-actin to a level similar to that of WT N461. Furthermore, the full-length SSH1L(W458A) mutant was poorly activated by F-actin (Fig. 7 B). Thus, Trp-458 is a critical residue for SSH1L activation by F-actin. Kinetic analysis revealed that although WT SSH1L was ∼10-fold activated by F-actin, SSH1L(W458A) was only approximately twofold activated by F-actin (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1). However, SSH1L(W458A) retained the basal cofilin phosphatase activity (activity in the absence of F-actin) to a level similar to that of WT SSH1L (Fig. S2 B), which indicates that Trp-458 is specifically involved in the process of F-actin–mediated activation.


Spatial and temporal regulation of cofilin activity by LIM kinase and Slingshot is critical for directional cell migration.

Nishita M, Tomizawa C, Yamamoto M, Horita Y, Ohashi K, Mizuno K - J. Cell Biol. (2005)

Trp-458 is critical for the F-actin–mediated activation of SSH1L. (A) The role of amino acids 457–461 in F-actin–mediated SSH1L activation. Schematic structures of SSH1L and deletion mutants are shown. The conserved regions in the SSH family are indicated by the A, B, P (phosphatase), and S (Ser-rich) domains. Wild-type (WT) and deletion mutants of (myc + His)-tagged SSH1L were expressed in 293T cells, immunoprecipitated with an anti-myc antibody, and subjected to in vitro phosphatase assays using cofilin-(His)6 as a substrate in the presence or absence of F-actin. P-cofilin levels were measured by Pro-Q staining. Total cofilin and actin were measured by Coomassie blue staining. The expression of SSH1L mutants was analyzed by immunoblotting with the anti-myc antibody. *, Ig heavy chain. (B) Trp-458 is required for F-actin–mediated SSH1L activation. Point mutants of N461 and full-length (FL) SSH1L were subjected to in vitro cofilin phosphatase assays as described in A. Arrow indicates the replacement of Trp-458 with Ala.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171197&req=5

fig7: Trp-458 is critical for the F-actin–mediated activation of SSH1L. (A) The role of amino acids 457–461 in F-actin–mediated SSH1L activation. Schematic structures of SSH1L and deletion mutants are shown. The conserved regions in the SSH family are indicated by the A, B, P (phosphatase), and S (Ser-rich) domains. Wild-type (WT) and deletion mutants of (myc + His)-tagged SSH1L were expressed in 293T cells, immunoprecipitated with an anti-myc antibody, and subjected to in vitro phosphatase assays using cofilin-(His)6 as a substrate in the presence or absence of F-actin. P-cofilin levels were measured by Pro-Q staining. Total cofilin and actin were measured by Coomassie blue staining. The expression of SSH1L mutants was analyzed by immunoblotting with the anti-myc antibody. *, Ig heavy chain. (B) Trp-458 is required for F-actin–mediated SSH1L activation. Point mutants of N461 and full-length (FL) SSH1L were subjected to in vitro cofilin phosphatase assays as described in A. Arrow indicates the replacement of Trp-458 with Ala.
Mentions: As SSH1L is highly activated by its association with F-actin (Nagata-Ohashi et al., 2004), we hypothesized that the F-actin–mediated activation of SSH1L is critical for polarized cell migration. To identify the sites of SSH1L that are required for F-actin–mediated activation, a series of COOH-terminally deleted mutants of SSH1L were constructed. The truncated proteins were subjected to in vitro cofilin phosphatase assays in the absence or presence of F-actin by using P-cofilin as a substrate (Fig. 7 A). As previously reported (Nagata-Ohashi et al., 2004), wild-type (WT) SSH1L was activated by F-actin, but the COOH-terminally deleted mutant NP (composed of amino acid residues 1–456) was not. Unexpectedly, the other COOH-terminally deleted mutants (N960, N698, N486, and N461) were all activated by F-actin. This indicates that the region corresponding to amino acids 457–461 is required for the F-actin–mediated activation of SSH1L. To further define the residues that are essential for activation, various point mutants of N461 were constructed and subjected to cofilin phosphatase assays with or without F-actin (Fig. 7 B). The assays revealed that W458A (Trp-458 replaced by Ala) and 3A (Leu-457, Trp-458, and Arg-459 replaced by Ala) mutants of N461 were only weakly activated in the presence of F-actin, whereas other point mutants were remarkably activated by F-actin to a level similar to that of WT N461. Furthermore, the full-length SSH1L(W458A) mutant was poorly activated by F-actin (Fig. 7 B). Thus, Trp-458 is a critical residue for SSH1L activation by F-actin. Kinetic analysis revealed that although WT SSH1L was ∼10-fold activated by F-actin, SSH1L(W458A) was only approximately twofold activated by F-actin (Fig. S2 A, available at http://www.jcb.org/cgi/content/full/jcb.200504029/DC1). However, SSH1L(W458A) retained the basal cofilin phosphatase activity (activity in the absence of F-actin) to a level similar to that of WT SSH1L (Fig. S2 B), which indicates that Trp-458 is specifically involved in the process of F-actin–mediated activation.

Bottom Line: Cofilin is inactivated by LIM kinase (LIMK)-1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L.In this study, we show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells.We propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomolecular Sciences, Graduate School of Life Sciences, Tohoku University, Sendai, Miyagi 980-8578, Japan.

ABSTRACT
Cofilin mediates lamellipodium extension and polarized cell migration by accelerating actin filament dynamics at the leading edge of migrating cells. Cofilin is inactivated by LIM kinase (LIMK)-1-mediated phosphorylation and is reactivated by cofilin phosphatase Slingshot (SSH)-1L. In this study, we show that cofilin activity is temporally and spatially regulated by LIMK1 and SSH1L in chemokine-stimulated Jurkat T cells. The knockdown of LIMK1 suppressed chemokine-induced lamellipodium formation and cell migration, whereas SSH1L knockdown produced and retained multiple lamellipodial protrusions around the cell after cell stimulation and impaired directional cell migration. Our results indicate that LIMK1 is required for cell migration by stimulating lamellipodium formation in the initial stages of cell response and that SSH1L is crucially involved in directional cell migration by restricting the membrane protrusion to one direction and locally stimulating cofilin activity in the lamellipodium in the front of the migrating cell. We propose that LIMK1- and SSH1L-mediated spatiotemporal regulation of cofilin activity is critical for chemokine-induced polarized lamellipodium formation and directional cell movement.

Show MeSH
Related in: MedlinePlus