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ALDH1L1 inhibits cell motility via dephosphorylation of cofilin by PP1 and PP2A.

Oleinik NV, Krupenko NI, Krupenko SA - Oncogene (2010)

Bottom Line: Inhibition of FDH-induced apoptosis by the Jun N-terminal kinase inhibitor SP600125 or the pan-caspase inhibitor zVAD-fmk did not restore motility or levels of phosphor-cofilin, indicating that the observed effects are independent of FDH function in apoptosis.Interestingly, cofilin small interfering RNA or expression of phosphorylation-deficient S3A cofilin mutant resulted in a decrease of G-actin and the actin stress fiber formation, the effects seen upon FDH expression.Our experiments showed that these effects were folate specific and not a general response to nutrient starvation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA.

ABSTRACT
Here we report that ALDH1L1 (FDH, a folate enzyme with tumor suppressor-like properties) inhibits cell motility. The underlying mechanism involves F-actin stabilization, re-distribution of cytoplasmic actin toward strong preponderance of filamentous actin and formation of actin stress fibers. A549 cells expressing FDH showed a much slower recovery of green fluorescent protein-actin fluorescence in a fluorescence recovery after photobleaching assay, as well as an increase in G-actin polymerization and a decrease in F-actin depolymerization rates in pyren-actin fluorescence assays indicating the inhibition of actin dynamics. These effects were associated with robust dephosphorylation of the actin depolymerizing factor cofilin by PP1 and PP2A serine/threonine protein phosphatases, but not the cofilin-specific phosphatases slingshot and chronophin. In fact, the PP1/PP2A inhibitor calyculin prevented cofilin dephosphorylation and restored motility. Inhibition of FDH-induced apoptosis by the Jun N-terminal kinase inhibitor SP600125 or the pan-caspase inhibitor zVAD-fmk did not restore motility or levels of phosphor-cofilin, indicating that the observed effects are independent of FDH function in apoptosis. Interestingly, cofilin small interfering RNA or expression of phosphorylation-deficient S3A cofilin mutant resulted in a decrease of G-actin and the actin stress fiber formation, the effects seen upon FDH expression. In contrast, the expression of S3D mutant, mimicking constitutive phosphorylation, prevented these effects further supporting the cofilin-dependent mechanism. Dephosphorylation of cofilin and inhibition of motility in response to FDH can also be prevented by the increased folate in media. Furthermore, folate depletion itself, in the absence of FDH, resulted in cofilin dephosphorylation and inhibition of motility in several cell lines. Our experiments showed that these effects were folate specific and not a general response to nutrient starvation. Overall, this study shows the presence of distinct intracellular signaling pathways regulating motility in response to folate status and points toward mechanisms involving folates in promoting a malignant phenotype.

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FDH activates dephosphorylation of cofilin by PP1 and PP2A. (a) Time dependent in vitro dephosphorylation of p-cofilin by lysate from FDH-expressing cells (top panel); the same assay in the presence of 1.0 μM of PP1/PP2A inhibitor calyculin (bottom panel); bar graph, levels of p-cofilin quantified from the panels (-Inh, in the absence of calyculin; +Inh, in the presence calyculin) using Quantity One software (Bio-Rad). Average (±SD) of three independent experiments is shown. (b) Western blot analysis of phosphatases pulled down with cofilin antibody from FDH expressing and FDH-deficient cells. (c) Motility of FDH-expressing A549 cells (wound healing assays) is restored by the addition of calyculin. (d) Calyculin prevents FDH-induced cofilin dephosphorylation in vivo. Time (h) after addition of calyculin to the cell culture is indicated. FDH expression was induced 24 h before addition of calyculin.
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Figure 6: FDH activates dephosphorylation of cofilin by PP1 and PP2A. (a) Time dependent in vitro dephosphorylation of p-cofilin by lysate from FDH-expressing cells (top panel); the same assay in the presence of 1.0 μM of PP1/PP2A inhibitor calyculin (bottom panel); bar graph, levels of p-cofilin quantified from the panels (-Inh, in the absence of calyculin; +Inh, in the presence calyculin) using Quantity One software (Bio-Rad). Average (±SD) of three independent experiments is shown. (b) Western blot analysis of phosphatases pulled down with cofilin antibody from FDH expressing and FDH-deficient cells. (c) Motility of FDH-expressing A549 cells (wound healing assays) is restored by the addition of calyculin. (d) Calyculin prevents FDH-induced cofilin dephosphorylation in vivo. Time (h) after addition of calyculin to the cell culture is indicated. FDH expression was induced 24 h before addition of calyculin.

Mentions: To study whether the decrease of phospho-cofilin is a result of activated dephosphorylation and not the lack of kinase activity, we have monitored phospho-cofilin levels in A549 cell lysates after mixing them with the lysates from FDH expressing cells. We observed rapid time-dependent dephosphorylation of cofilin upon addition of the FDH-containing lysate (with presumably activated cofilin phosphatases) (Fig. 6a). To identify the phosphatase responsible for cofilin dephosphorylation in response to FDH, we used pull down assays with a cofilin-specific antibody. Immunoblot analysis revealed PP1 and PP2A in the pull-down preparation, while slingshot or chronophin, two cofilin-specific phosphatases (Huang et al., 2006), were not detected (Fig. 6b). To confirm that PP1 and PP2A were responsible for cofilin dephosphorylation, we monitored phospho-cofilin dephosphorylation by the lysate from FDH-expressing cells in the presence of the specific PP1/PP2A inhibitor, calyculin, which does not inhibit slingshot or chronophin (Huang et al., 2006; Niwa et al., 2002). In agreement with the results of the pull-down experiments, calyculin blocked dephosphorylation of cofilin (Fig. 6a).


ALDH1L1 inhibits cell motility via dephosphorylation of cofilin by PP1 and PP2A.

Oleinik NV, Krupenko NI, Krupenko SA - Oncogene (2010)

FDH activates dephosphorylation of cofilin by PP1 and PP2A. (a) Time dependent in vitro dephosphorylation of p-cofilin by lysate from FDH-expressing cells (top panel); the same assay in the presence of 1.0 μM of PP1/PP2A inhibitor calyculin (bottom panel); bar graph, levels of p-cofilin quantified from the panels (-Inh, in the absence of calyculin; +Inh, in the presence calyculin) using Quantity One software (Bio-Rad). Average (±SD) of three independent experiments is shown. (b) Western blot analysis of phosphatases pulled down with cofilin antibody from FDH expressing and FDH-deficient cells. (c) Motility of FDH-expressing A549 cells (wound healing assays) is restored by the addition of calyculin. (d) Calyculin prevents FDH-induced cofilin dephosphorylation in vivo. Time (h) after addition of calyculin to the cell culture is indicated. FDH expression was induced 24 h before addition of calyculin.
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Related In: Results  -  Collection

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Figure 6: FDH activates dephosphorylation of cofilin by PP1 and PP2A. (a) Time dependent in vitro dephosphorylation of p-cofilin by lysate from FDH-expressing cells (top panel); the same assay in the presence of 1.0 μM of PP1/PP2A inhibitor calyculin (bottom panel); bar graph, levels of p-cofilin quantified from the panels (-Inh, in the absence of calyculin; +Inh, in the presence calyculin) using Quantity One software (Bio-Rad). Average (±SD) of three independent experiments is shown. (b) Western blot analysis of phosphatases pulled down with cofilin antibody from FDH expressing and FDH-deficient cells. (c) Motility of FDH-expressing A549 cells (wound healing assays) is restored by the addition of calyculin. (d) Calyculin prevents FDH-induced cofilin dephosphorylation in vivo. Time (h) after addition of calyculin to the cell culture is indicated. FDH expression was induced 24 h before addition of calyculin.
Mentions: To study whether the decrease of phospho-cofilin is a result of activated dephosphorylation and not the lack of kinase activity, we have monitored phospho-cofilin levels in A549 cell lysates after mixing them with the lysates from FDH expressing cells. We observed rapid time-dependent dephosphorylation of cofilin upon addition of the FDH-containing lysate (with presumably activated cofilin phosphatases) (Fig. 6a). To identify the phosphatase responsible for cofilin dephosphorylation in response to FDH, we used pull down assays with a cofilin-specific antibody. Immunoblot analysis revealed PP1 and PP2A in the pull-down preparation, while slingshot or chronophin, two cofilin-specific phosphatases (Huang et al., 2006), were not detected (Fig. 6b). To confirm that PP1 and PP2A were responsible for cofilin dephosphorylation, we monitored phospho-cofilin dephosphorylation by the lysate from FDH-expressing cells in the presence of the specific PP1/PP2A inhibitor, calyculin, which does not inhibit slingshot or chronophin (Huang et al., 2006; Niwa et al., 2002). In agreement with the results of the pull-down experiments, calyculin blocked dephosphorylation of cofilin (Fig. 6a).

Bottom Line: Inhibition of FDH-induced apoptosis by the Jun N-terminal kinase inhibitor SP600125 or the pan-caspase inhibitor zVAD-fmk did not restore motility or levels of phosphor-cofilin, indicating that the observed effects are independent of FDH function in apoptosis.Interestingly, cofilin small interfering RNA or expression of phosphorylation-deficient S3A cofilin mutant resulted in a decrease of G-actin and the actin stress fiber formation, the effects seen upon FDH expression.Our experiments showed that these effects were folate specific and not a general response to nutrient starvation.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Medical University of South Carolina, Charleston, SC, USA.

ABSTRACT
Here we report that ALDH1L1 (FDH, a folate enzyme with tumor suppressor-like properties) inhibits cell motility. The underlying mechanism involves F-actin stabilization, re-distribution of cytoplasmic actin toward strong preponderance of filamentous actin and formation of actin stress fibers. A549 cells expressing FDH showed a much slower recovery of green fluorescent protein-actin fluorescence in a fluorescence recovery after photobleaching assay, as well as an increase in G-actin polymerization and a decrease in F-actin depolymerization rates in pyren-actin fluorescence assays indicating the inhibition of actin dynamics. These effects were associated with robust dephosphorylation of the actin depolymerizing factor cofilin by PP1 and PP2A serine/threonine protein phosphatases, but not the cofilin-specific phosphatases slingshot and chronophin. In fact, the PP1/PP2A inhibitor calyculin prevented cofilin dephosphorylation and restored motility. Inhibition of FDH-induced apoptosis by the Jun N-terminal kinase inhibitor SP600125 or the pan-caspase inhibitor zVAD-fmk did not restore motility or levels of phosphor-cofilin, indicating that the observed effects are independent of FDH function in apoptosis. Interestingly, cofilin small interfering RNA or expression of phosphorylation-deficient S3A cofilin mutant resulted in a decrease of G-actin and the actin stress fiber formation, the effects seen upon FDH expression. In contrast, the expression of S3D mutant, mimicking constitutive phosphorylation, prevented these effects further supporting the cofilin-dependent mechanism. Dephosphorylation of cofilin and inhibition of motility in response to FDH can also be prevented by the increased folate in media. Furthermore, folate depletion itself, in the absence of FDH, resulted in cofilin dephosphorylation and inhibition of motility in several cell lines. Our experiments showed that these effects were folate specific and not a general response to nutrient starvation. Overall, this study shows the presence of distinct intracellular signaling pathways regulating motility in response to folate status and points toward mechanisms involving folates in promoting a malignant phenotype.

Show MeSH
Related in: MedlinePlus