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Tyrosine phosphorylation of cofilin at Y68 by v-Src leads to its degradation through ubiquitin-proteasome pathway.

Yoo Y, Ho HJ, Wang C, Guan JL - Oncogene (2009)

Bottom Line: Cofilin phosphorylation at Y68 did not change its activity per se, but induced increased ubiquitination of cofilin and its degradation through the proteosome pathway.Furthermore, the negative effect of cofilin on cellular F-actin contents was inhibited by coexpression of v-Src, whereas that of cofilin mutant Y68F (Y68 mutated to F) was not affected, suggesting that v-Src-mediated cofilin phosphorylation at Y68 is required for the degradation of cofilin in vivo.Together, these results suggest a novel mechanism by which cofilin is regulated by v-Src through tyrosine phosphorylation at Y68 that triggers the degradation of cofilin through ubiquitination-proteosome pathway and consequently inhibits cofilin activity in reducing cellular F-actin contents and cell spreading.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine and Genetics, Departments of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.

ABSTRACT
Cofilin is a major regulator of actin dynamics involved in the regulation of cell spreading and migration through its actin depolymerizing and severing activities. v-Src is an activated Src tyrosine kinase and a potent oncogene known to phosphorylate a variety of cellular proteins in cell transformation process including altered cell adhesion, spreading and migration. Recently, it has been suggested that cofilin is a potential substrate of v-Src (Rush et al., 2005). Here, we show direct tyrosine phosphorylation of cofilin by v-Src and identify Y68 as the major phosphorylation site. Cofilin phosphorylation at Y68 did not change its activity per se, but induced increased ubiquitination of cofilin and its degradation through the proteosome pathway. Furthermore, the negative effect of cofilin on cellular F-actin contents was inhibited by coexpression of v-Src, whereas that of cofilin mutant Y68F (Y68 mutated to F) was not affected, suggesting that v-Src-mediated cofilin phosphorylation at Y68 is required for the degradation of cofilin in vivo. Lastly, inhibition of cell spreading by v-Src was rescued partially by coexpression of cofilin, and to a greater extent by the Y68F mutant, which is not subjected to v-Src-induced degradation through phosphorylation, suggesting that v-Src-mediated changes in cell spreading is, at least in part, through inhibiting the function of cofilin through phosphorylating it at Y68. Together, these results suggest a novel mechanism by which cofilin is regulated by v-Src through tyrosine phosphorylation at Y68 that triggers the degradation of cofilin through ubiquitination-proteosome pathway and consequently inhibits cofilin activity in reducing cellular F-actin contents and cell spreading.

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Phosphorylation of cofilin by v-Src reduces its activity in inducing stress fiber disassembly and actin depolymerization(A-F) HeLa cells were co-transfected with HA-tagged v-Src and Myc-tagged cofilin or Y68F mutant or control protein (GST) as indicated. Cells were fixed and subjected to immunofluorescence staining with anti-Myc or phalloidin to view filamentous actin. The transfected cells are marked by arrows. The percentage of transfected cells with positive phalloidin staining are indicated in the parentheses. (G) 293T cells were co-transfected with Myc-tagged cofilin, Y68F mutant or vector control and HA-tagged v-Src or vector control, as indicated. The cellular F- and G-actin contents were determined by western blotting after fractionation as described in the Experimental Procedures. Molecular weight markers are indicated on the right. (H) Relative ratio of F/G actin were obtained from three independent experiments and shown with the mean + S.E. after normalization to that of control cells (V+V, lane 1). *P<0.05; **P>0.05 in comparison with values from control cells. ***P<0.05 in comparison with values from cells transfected with v-Src and cofilin (v-Src + cofilin).
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Figure 3: Phosphorylation of cofilin by v-Src reduces its activity in inducing stress fiber disassembly and actin depolymerization(A-F) HeLa cells were co-transfected with HA-tagged v-Src and Myc-tagged cofilin or Y68F mutant or control protein (GST) as indicated. Cells were fixed and subjected to immunofluorescence staining with anti-Myc or phalloidin to view filamentous actin. The transfected cells are marked by arrows. The percentage of transfected cells with positive phalloidin staining are indicated in the parentheses. (G) 293T cells were co-transfected with Myc-tagged cofilin, Y68F mutant or vector control and HA-tagged v-Src or vector control, as indicated. The cellular F- and G-actin contents were determined by western blotting after fractionation as described in the Experimental Procedures. Molecular weight markers are indicated on the right. (H) Relative ratio of F/G actin were obtained from three independent experiments and shown with the mean + S.E. after normalization to that of control cells (V+V, lane 1). *P<0.05; **P>0.05 in comparison with values from control cells. ***P<0.05 in comparison with values from cells transfected with v-Src and cofilin (v-Src + cofilin).

Mentions: To study the physiological role of v-Src-mediated degradation of cofilin in vivo, we first analyzed the effect of co-expression of v-Src on the function of cofilin to reduce the cellular F-actin contents. HeLa cells were co-transfected with Myc-tagged cofilin or Y68F mutant and HA-tagged v-Src, and examined by immunofluorescence staining with Texas-Red conjugated phalloidin to detect cellular F-actin in stress fibers. Consistent with a previous report (Yap et al., 2005), over-expression of cofilin reduced the cellular F-actin contents compared with the control cells while transfection of the control vector did not affect it (Figs. 3A and 3B). Over-expression of the Y68F mutant also reduced F-actin contents to a similar extent (Fig. 3C), suggesting that mutation at this residue did not affect the cofilin activity per se in reducing F-actin contents. Interestingly, co-expression of v-Src with cofilin reversed its function to reduce the F-actin contents (Fig. 3E) whereas expression of v-Src alone did not affect stress fiber formation under the experimental conditions (Fig. 3D). In contrast, co-expression of v-Src with the Y68F mutant did not prevent the reduction of stress fibers by the Y68F mutant (Fig. 3F). We next measured the ratio of F-actin and G-actin (F/G actin) directly to quantify the effect of v-Src-mediated phosphorylation of cofilin on its activity to reduce F-actin contents. Consistent with data from the immunofluorescence, expression of cofilin reduced F/G actin significantly whereas expression of v-Src alone did not affect it under the experimental conditions (Figs. 3G and 3H). Furthermore, co-expression of v-Src with cofilin, but not the Y68F mutant, inhibited cofilin function to reduce F/G actin in cells. These results suggest that v-Src inhibits cofilin function to reduce F-actin contents through phosphorylation-mediated protein degradation in vivo.


Tyrosine phosphorylation of cofilin at Y68 by v-Src leads to its degradation through ubiquitin-proteasome pathway.

Yoo Y, Ho HJ, Wang C, Guan JL - Oncogene (2009)

Phosphorylation of cofilin by v-Src reduces its activity in inducing stress fiber disassembly and actin depolymerization(A-F) HeLa cells were co-transfected with HA-tagged v-Src and Myc-tagged cofilin or Y68F mutant or control protein (GST) as indicated. Cells were fixed and subjected to immunofluorescence staining with anti-Myc or phalloidin to view filamentous actin. The transfected cells are marked by arrows. The percentage of transfected cells with positive phalloidin staining are indicated in the parentheses. (G) 293T cells were co-transfected with Myc-tagged cofilin, Y68F mutant or vector control and HA-tagged v-Src or vector control, as indicated. The cellular F- and G-actin contents were determined by western blotting after fractionation as described in the Experimental Procedures. Molecular weight markers are indicated on the right. (H) Relative ratio of F/G actin were obtained from three independent experiments and shown with the mean + S.E. after normalization to that of control cells (V+V, lane 1). *P<0.05; **P>0.05 in comparison with values from control cells. ***P<0.05 in comparison with values from cells transfected with v-Src and cofilin (v-Src + cofilin).
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Figure 3: Phosphorylation of cofilin by v-Src reduces its activity in inducing stress fiber disassembly and actin depolymerization(A-F) HeLa cells were co-transfected with HA-tagged v-Src and Myc-tagged cofilin or Y68F mutant or control protein (GST) as indicated. Cells were fixed and subjected to immunofluorescence staining with anti-Myc or phalloidin to view filamentous actin. The transfected cells are marked by arrows. The percentage of transfected cells with positive phalloidin staining are indicated in the parentheses. (G) 293T cells were co-transfected with Myc-tagged cofilin, Y68F mutant or vector control and HA-tagged v-Src or vector control, as indicated. The cellular F- and G-actin contents were determined by western blotting after fractionation as described in the Experimental Procedures. Molecular weight markers are indicated on the right. (H) Relative ratio of F/G actin were obtained from three independent experiments and shown with the mean + S.E. after normalization to that of control cells (V+V, lane 1). *P<0.05; **P>0.05 in comparison with values from control cells. ***P<0.05 in comparison with values from cells transfected with v-Src and cofilin (v-Src + cofilin).
Mentions: To study the physiological role of v-Src-mediated degradation of cofilin in vivo, we first analyzed the effect of co-expression of v-Src on the function of cofilin to reduce the cellular F-actin contents. HeLa cells were co-transfected with Myc-tagged cofilin or Y68F mutant and HA-tagged v-Src, and examined by immunofluorescence staining with Texas-Red conjugated phalloidin to detect cellular F-actin in stress fibers. Consistent with a previous report (Yap et al., 2005), over-expression of cofilin reduced the cellular F-actin contents compared with the control cells while transfection of the control vector did not affect it (Figs. 3A and 3B). Over-expression of the Y68F mutant also reduced F-actin contents to a similar extent (Fig. 3C), suggesting that mutation at this residue did not affect the cofilin activity per se in reducing F-actin contents. Interestingly, co-expression of v-Src with cofilin reversed its function to reduce the F-actin contents (Fig. 3E) whereas expression of v-Src alone did not affect stress fiber formation under the experimental conditions (Fig. 3D). In contrast, co-expression of v-Src with the Y68F mutant did not prevent the reduction of stress fibers by the Y68F mutant (Fig. 3F). We next measured the ratio of F-actin and G-actin (F/G actin) directly to quantify the effect of v-Src-mediated phosphorylation of cofilin on its activity to reduce F-actin contents. Consistent with data from the immunofluorescence, expression of cofilin reduced F/G actin significantly whereas expression of v-Src alone did not affect it under the experimental conditions (Figs. 3G and 3H). Furthermore, co-expression of v-Src with cofilin, but not the Y68F mutant, inhibited cofilin function to reduce F/G actin in cells. These results suggest that v-Src inhibits cofilin function to reduce F-actin contents through phosphorylation-mediated protein degradation in vivo.

Bottom Line: Cofilin phosphorylation at Y68 did not change its activity per se, but induced increased ubiquitination of cofilin and its degradation through the proteosome pathway.Furthermore, the negative effect of cofilin on cellular F-actin contents was inhibited by coexpression of v-Src, whereas that of cofilin mutant Y68F (Y68 mutated to F) was not affected, suggesting that v-Src-mediated cofilin phosphorylation at Y68 is required for the degradation of cofilin in vivo.Together, these results suggest a novel mechanism by which cofilin is regulated by v-Src through tyrosine phosphorylation at Y68 that triggers the degradation of cofilin through ubiquitination-proteosome pathway and consequently inhibits cofilin activity in reducing cellular F-actin contents and cell spreading.

View Article: PubMed Central - PubMed

Affiliation: Division of Molecular Medicine and Genetics, Departments of Internal Medicine, University of Michigan Medical School, Ann Arbor, MI, USA.

ABSTRACT
Cofilin is a major regulator of actin dynamics involved in the regulation of cell spreading and migration through its actin depolymerizing and severing activities. v-Src is an activated Src tyrosine kinase and a potent oncogene known to phosphorylate a variety of cellular proteins in cell transformation process including altered cell adhesion, spreading and migration. Recently, it has been suggested that cofilin is a potential substrate of v-Src (Rush et al., 2005). Here, we show direct tyrosine phosphorylation of cofilin by v-Src and identify Y68 as the major phosphorylation site. Cofilin phosphorylation at Y68 did not change its activity per se, but induced increased ubiquitination of cofilin and its degradation through the proteosome pathway. Furthermore, the negative effect of cofilin on cellular F-actin contents was inhibited by coexpression of v-Src, whereas that of cofilin mutant Y68F (Y68 mutated to F) was not affected, suggesting that v-Src-mediated cofilin phosphorylation at Y68 is required for the degradation of cofilin in vivo. Lastly, inhibition of cell spreading by v-Src was rescued partially by coexpression of cofilin, and to a greater extent by the Y68F mutant, which is not subjected to v-Src-induced degradation through phosphorylation, suggesting that v-Src-mediated changes in cell spreading is, at least in part, through inhibiting the function of cofilin through phosphorylating it at Y68. Together, these results suggest a novel mechanism by which cofilin is regulated by v-Src through tyrosine phosphorylation at Y68 that triggers the degradation of cofilin through ubiquitination-proteosome pathway and consequently inhibits cofilin activity in reducing cellular F-actin contents and cell spreading.

Show MeSH
Related in: MedlinePlus