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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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Degradation and ubiquitination of cofilin induced by v-Src phosphorylation at Y68 residue(A, B) 293T cells were co-transfected with Myc-tagged cofilin, Y68F, or S3A mutant, and HA-tagged v-Src or vector control as indicated. Lysates were then prepared and analyzed by western blotting with anti-Myc, anti-HA, or anti-vinculin, as indicated. In panel B, cells were treated with cycloheximide and MG132 or DMSO as control for 4 hrs before lysis as described in Experimental Procedures. (C) NIH3T3 and v-Src-transformed-NIH3T3 (v-Src-3T3) cells were treated with nothing (lanes 1 and 2), cycloheximide with DMSO (lanes 3 and 4) or with MG132 (lanes 5 and 6) for 4 hrs. Lysates were then prepared and analyzed by western blotting with anti-cofilin (upper) and anti-vinculin (lower). Molecular weight markers are indicated on the right. (D) 293T cells were co-transfected with HA-tagged cofilin or Y68F mutant, and Myc-tagged ubiquitin and Flag-tagged v-Src or vector control as indicated. Cells were treated with MG132 for 4 hrs and then lysates were prepared and immunoprecipitated with anti-HA antibody, followed by western blotting with anti-Myc (top) or anti-HA (middle). The Ubiquitinated cofilin bands (marked on the right) were quantified by densitometer from 3 independent experiments and relative intensity was shown with the mean + S.E (bottom). (E) NIH3T3 and v-Src-3T3 cells were treated with MG132 for 4 hr and lysates were prepared and immunoprecipitated with anti-cofilin antibody, followed by western blotting with anti-ubiquitin (upper) or anti-cofilin (lower). The ubiquitinated cofilin bands are marked on the right. Molecular weight markers are indicated on the right.
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Figure 2: Degradation and ubiquitination of cofilin induced by v-Src phosphorylation at Y68 residue(A, B) 293T cells were co-transfected with Myc-tagged cofilin, Y68F, or S3A mutant, and HA-tagged v-Src or vector control as indicated. Lysates were then prepared and analyzed by western blotting with anti-Myc, anti-HA, or anti-vinculin, as indicated. In panel B, cells were treated with cycloheximide and MG132 or DMSO as control for 4 hrs before lysis as described in Experimental Procedures. (C) NIH3T3 and v-Src-transformed-NIH3T3 (v-Src-3T3) cells were treated with nothing (lanes 1 and 2), cycloheximide with DMSO (lanes 3 and 4) or with MG132 (lanes 5 and 6) for 4 hrs. Lysates were then prepared and analyzed by western blotting with anti-cofilin (upper) and anti-vinculin (lower). Molecular weight markers are indicated on the right. (D) 293T cells were co-transfected with HA-tagged cofilin or Y68F mutant, and Myc-tagged ubiquitin and Flag-tagged v-Src or vector control as indicated. Cells were treated with MG132 for 4 hrs and then lysates were prepared and immunoprecipitated with anti-HA antibody, followed by western blotting with anti-Myc (top) or anti-HA (middle). The Ubiquitinated cofilin bands (marked on the right) were quantified by densitometer from 3 independent experiments and relative intensity was shown with the mean + S.E (bottom). (E) NIH3T3 and v-Src-3T3 cells were treated with MG132 for 4 hr and lysates were prepared and immunoprecipitated with anti-cofilin antibody, followed by western blotting with anti-ubiquitin (upper) or anti-cofilin (lower). The ubiquitinated cofilin bands are marked on the right. Molecular weight markers are indicated on the right.

Mentions: During the course of studies on cofilin phosphorylation by v-Src, we noticed that the expression level of cofilin and the S3A mutant, but not the Y68F mutant, was reduced specifically in the presence of co-expressed v-Src (Fig. 2A). Because the ectopically expressed cofilin is under the control of an exogenous promoter, these results suggest that v-Src likely affects the expression level of cofilin through posttranscriptional mechanisms. This possibility is supported by the observation that treatment of the cells with an inhibitor of protein synthesis cycloheximide did not affect the decreased protein level of cofilin induced by v-Src (Fig. 2B, lanes 1–3). We then examined the effects of v-Src on the protein levels of endogenous cofilin using a NIH 3T3 cell line stably expressing v-Src. Fig. 2C shows that expression of cofilin in these cells is also decreased compared to the control NIH 3T3 cells both in the presence and absence of cycloheximide (lanes 1–4). The expression level of endogenous cofilin is also reduced by expression of v-Src with or without treatment of cycloheximide in 293T and CHO cells (data not shown). Together, these data suggest that phosphorylation of cofilin by v-Src at Y68 may regulate the expression of cofilin by stimulation of its degradation.


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)

Degradation and ubiquitination of cofilin induced by v-Src phosphorylation at Y68 residue(A, B) 293T cells were co-transfected with Myc-tagged cofilin, Y68F, or S3A mutant, and HA-tagged v-Src or vector control as indicated. Lysates were then prepared and analyzed by western blotting with anti-Myc, anti-HA, or anti-vinculin, as indicated. In panel B, cells were treated with cycloheximide and MG132 or DMSO as control for 4 hrs before lysis as described in Experimental Procedures. (C) NIH3T3 and v-Src-transformed-NIH3T3 (v-Src-3T3) cells were treated with nothing (lanes 1 and 2), cycloheximide with DMSO (lanes 3 and 4) or with MG132 (lanes 5 and 6) for 4 hrs. Lysates were then prepared and analyzed by western blotting with anti-cofilin (upper) and anti-vinculin (lower). Molecular weight markers are indicated on the right. (D) 293T cells were co-transfected with HA-tagged cofilin or Y68F mutant, and Myc-tagged ubiquitin and Flag-tagged v-Src or vector control as indicated. Cells were treated with MG132 for 4 hrs and then lysates were prepared and immunoprecipitated with anti-HA antibody, followed by western blotting with anti-Myc (top) or anti-HA (middle). The Ubiquitinated cofilin bands (marked on the right) were quantified by densitometer from 3 independent experiments and relative intensity was shown with the mean + S.E (bottom). (E) NIH3T3 and v-Src-3T3 cells were treated with MG132 for 4 hr and lysates were prepared and immunoprecipitated with anti-cofilin antibody, followed by western blotting with anti-ubiquitin (upper) or anti-cofilin (lower). The ubiquitinated cofilin bands are marked on the right. Molecular weight markers are indicated on the right.
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Related In: Results  -  Collection

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Figure 2: Degradation and ubiquitination of cofilin induced by v-Src phosphorylation at Y68 residue(A, B) 293T cells were co-transfected with Myc-tagged cofilin, Y68F, or S3A mutant, and HA-tagged v-Src or vector control as indicated. Lysates were then prepared and analyzed by western blotting with anti-Myc, anti-HA, or anti-vinculin, as indicated. In panel B, cells were treated with cycloheximide and MG132 or DMSO as control for 4 hrs before lysis as described in Experimental Procedures. (C) NIH3T3 and v-Src-transformed-NIH3T3 (v-Src-3T3) cells were treated with nothing (lanes 1 and 2), cycloheximide with DMSO (lanes 3 and 4) or with MG132 (lanes 5 and 6) for 4 hrs. Lysates were then prepared and analyzed by western blotting with anti-cofilin (upper) and anti-vinculin (lower). Molecular weight markers are indicated on the right. (D) 293T cells were co-transfected with HA-tagged cofilin or Y68F mutant, and Myc-tagged ubiquitin and Flag-tagged v-Src or vector control as indicated. Cells were treated with MG132 for 4 hrs and then lysates were prepared and immunoprecipitated with anti-HA antibody, followed by western blotting with anti-Myc (top) or anti-HA (middle). The Ubiquitinated cofilin bands (marked on the right) were quantified by densitometer from 3 independent experiments and relative intensity was shown with the mean + S.E (bottom). (E) NIH3T3 and v-Src-3T3 cells were treated with MG132 for 4 hr and lysates were prepared and immunoprecipitated with anti-cofilin antibody, followed by western blotting with anti-ubiquitin (upper) or anti-cofilin (lower). The ubiquitinated cofilin bands are marked on the right. Molecular weight markers are indicated on the right.
Mentions: During the course of studies on cofilin phosphorylation by v-Src, we noticed that the expression level of cofilin and the S3A mutant, but not the Y68F mutant, was reduced specifically in the presence of co-expressed v-Src (Fig. 2A). Because the ectopically expressed cofilin is under the control of an exogenous promoter, these results suggest that v-Src likely affects the expression level of cofilin through posttranscriptional mechanisms. This possibility is supported by the observation that treatment of the cells with an inhibitor of protein synthesis cycloheximide did not affect the decreased protein level of cofilin induced by v-Src (Fig. 2B, lanes 1–3). We then examined the effects of v-Src on the protein levels of endogenous cofilin using a NIH 3T3 cell line stably expressing v-Src. Fig. 2C shows that expression of cofilin in these cells is also decreased compared to the control NIH 3T3 cells both in the presence and absence of cycloheximide (lanes 1–4). The expression level of endogenous cofilin is also reduced by expression of v-Src with or without treatment of cycloheximide in 293T and CHO cells (data not shown). Together, these data suggest that phosphorylation of cofilin by v-Src at Y68 may regulate the expression of cofilin by stimulation of its degradation.

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