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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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Reversion of v-Src inhibition of cell spreading by cofilin and Y68F mutant(A and B) CHO cells were transfected with Myc-tagged cofilin or Y68F mutant, and HA-tagged v-Src or vector control along with a plasmid encoding GFP as a transfection marker, as indicated. Cell spreading assays were performed as described in the Experimental Procedures. Representative micrographs are shown in A. Panel B shows the mean + S.E. of percentage of spread cells (among transfected cells as identified by GFP expression) from three independent experiments are shown as relative spreading after normalization to that in mock transfected cells. *P<0.05 in comparison with value from mock cells. **P<0.05 in comparison with value from cells transfected with v-Src and vector (v-Src + V). ***P<0.05 in comparison with value from cells transfected with v-Src and cofilin (v-Src + cofilin). (C) Aliquots of WCL were analyzed by western blotting with anti-Myc (upper) or anti-HA (lower). Molecular weight markers are indicated on the right.
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Figure 4: Reversion of v-Src inhibition of cell spreading by cofilin and Y68F mutant(A and B) CHO cells were transfected with Myc-tagged cofilin or Y68F mutant, and HA-tagged v-Src or vector control along with a plasmid encoding GFP as a transfection marker, as indicated. Cell spreading assays were performed as described in the Experimental Procedures. Representative micrographs are shown in A. Panel B shows the mean + S.E. of percentage of spread cells (among transfected cells as identified by GFP expression) from three independent experiments are shown as relative spreading after normalization to that in mock transfected cells. *P<0.05 in comparison with value from mock cells. **P<0.05 in comparison with value from cells transfected with v-Src and vector (v-Src + V). ***P<0.05 in comparison with value from cells transfected with v-Src and cofilin (v-Src + cofilin). (C) Aliquots of WCL were analyzed by western blotting with anti-Myc (upper) or anti-HA (lower). Molecular weight markers are indicated on the right.

Mentions: Cofilin has been shown to promote cell spreading by stimulating actin cytoskeleton dynamics and membrane protrusion (Bamburg, 1999; Bamburg et al., 1999; Carlier et al., 1999; Huang et al., 2006; Ichetovkin et al., 2002). V-Src is also known to reduce cell spreading in many cell types through phosphorylation of target cellular proteins, even though the precise mechanisms are still not well understood (Brown & Cooper, 1996; Frame, 2002; Lin et al., 2006). Therefore, we investigated whether v-Src could regulate cell spreading through its phosphorylation of cofilin at Y68 by examining the effects of co-expression of v-Src with cofilin or its Y68F mutant on cell spreading. CHO cells were co-transfected with Myc-tagged cofilin or the Y68F mutant, and HA-tagged v-Src or control vector, along with a plasmid encoding GFP as a transfection marker. Cell spreading was assessed for the transfected cells after plating on fibronectin (FN), as described previously (Yoo et al., 2006). As shown in Figs. 4A and 4B, expression of v-Src in CHO cells reduced its spreading as expected. Consistent with the possibility that v-Src inhibits cell spreading through its induction of degradation of cofilin which plays a positive role in cell spreading, co-expression of cofilin with v-Src partially reversed the decrease in cell spreading induced by v-Src. Interestingly, co-expression of the Y68F mutant, which showed reduced phosphorylation by v-Src and consequent degradation (see Figs. 1 and 2), almost completely abolished the inhibitory effects by v-Src. Western blotting analysis of aliquots of the transfected cells confirmed the higher expression of the Y68F mutant (due to less degradation) compared to the wild type, when co-expressed with v-Src (Fig. 4C). Together, these results suggest that inhibition of cell spreading by v-Src, at least in part, is through its phosphorylation of cofilin at Y68 and subsequence induction of cofilin degradation via ubiquitination-proteosome pathway.


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)

Reversion of v-Src inhibition of cell spreading by cofilin and Y68F mutant(A and B) CHO cells were transfected with Myc-tagged cofilin or Y68F mutant, and HA-tagged v-Src or vector control along with a plasmid encoding GFP as a transfection marker, as indicated. Cell spreading assays were performed as described in the Experimental Procedures. Representative micrographs are shown in A. Panel B shows the mean + S.E. of percentage of spread cells (among transfected cells as identified by GFP expression) from three independent experiments are shown as relative spreading after normalization to that in mock transfected cells. *P<0.05 in comparison with value from mock cells. **P<0.05 in comparison with value from cells transfected with v-Src and vector (v-Src + V). ***P<0.05 in comparison with value from cells transfected with v-Src and cofilin (v-Src + cofilin). (C) Aliquots of WCL were analyzed by western blotting with anti-Myc (upper) or anti-HA (lower). Molecular weight markers are indicated on the right.
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Figure 4: Reversion of v-Src inhibition of cell spreading by cofilin and Y68F mutant(A and B) CHO cells were transfected with Myc-tagged cofilin or Y68F mutant, and HA-tagged v-Src or vector control along with a plasmid encoding GFP as a transfection marker, as indicated. Cell spreading assays were performed as described in the Experimental Procedures. Representative micrographs are shown in A. Panel B shows the mean + S.E. of percentage of spread cells (among transfected cells as identified by GFP expression) from three independent experiments are shown as relative spreading after normalization to that in mock transfected cells. *P<0.05 in comparison with value from mock cells. **P<0.05 in comparison with value from cells transfected with v-Src and vector (v-Src + V). ***P<0.05 in comparison with value from cells transfected with v-Src and cofilin (v-Src + cofilin). (C) Aliquots of WCL were analyzed by western blotting with anti-Myc (upper) or anti-HA (lower). Molecular weight markers are indicated on the right.
Mentions: Cofilin has been shown to promote cell spreading by stimulating actin cytoskeleton dynamics and membrane protrusion (Bamburg, 1999; Bamburg et al., 1999; Carlier et al., 1999; Huang et al., 2006; Ichetovkin et al., 2002). V-Src is also known to reduce cell spreading in many cell types through phosphorylation of target cellular proteins, even though the precise mechanisms are still not well understood (Brown & Cooper, 1996; Frame, 2002; Lin et al., 2006). Therefore, we investigated whether v-Src could regulate cell spreading through its phosphorylation of cofilin at Y68 by examining the effects of co-expression of v-Src with cofilin or its Y68F mutant on cell spreading. CHO cells were co-transfected with Myc-tagged cofilin or the Y68F mutant, and HA-tagged v-Src or control vector, along with a plasmid encoding GFP as a transfection marker. Cell spreading was assessed for the transfected cells after plating on fibronectin (FN), as described previously (Yoo et al., 2006). As shown in Figs. 4A and 4B, expression of v-Src in CHO cells reduced its spreading as expected. Consistent with the possibility that v-Src inhibits cell spreading through its induction of degradation of cofilin which plays a positive role in cell spreading, co-expression of cofilin with v-Src partially reversed the decrease in cell spreading induced by v-Src. Interestingly, co-expression of the Y68F mutant, which showed reduced phosphorylation by v-Src and consequent degradation (see Figs. 1 and 2), almost completely abolished the inhibitory effects by v-Src. Western blotting analysis of aliquots of the transfected cells confirmed the higher expression of the Y68F mutant (due to less degradation) compared to the wild type, when co-expressed with v-Src (Fig. 4C). Together, these results suggest that inhibition of cell spreading by v-Src, at least in part, is through its phosphorylation of cofilin at Y68 and subsequence induction of cofilin degradation via ubiquitination-proteosome pathway.

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