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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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v-Src phosphorylation of cofilin at Y68(A) 293T cells were co-transfected with His- and Myc-tagged cofilin or the mutants and HA-tagged v-Src or vector control, as indicated. Lysates were precipitated with Ni-beads, followed by western blotting with anti-phosphotyrosine, 4G10 (top) or anti-Myc (middle) antibody. Whole cell lysates (WCL) were also immunoblotted with anti-HA (bottom). (B) Recombinant GST-cofilin or GST-Y68F purified from bacteria were incubated in a kinase buffer (ATP is omitted in lane 2, as indicated by labels above the upper panel) with HA-v-Src or HA-FAK immunoprecipitated from 293T cells transfected with corresponding expression vectors, as indicated. In vitro kinase assays were performed as described in the Experimental Procedures and the reaction mixtures were subjected to immunoblotting with 4G10 antibody (upper) or anti-GST (lower). (C) 293T cells were co-transfected with Flag-tagged v-Src and HA-tagged cofilin or Y68F mutant or vector control as indicated. Lysates were immunoprecipitated with anti-HA antibody, followed by western blotting with anti-Flag antibody (upper). WCL were also immunoblotted with anti-Flag (lower). Molecular weight markers are indicated on the right.
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Figure 1: v-Src phosphorylation of cofilin at Y68(A) 293T cells were co-transfected with His- and Myc-tagged cofilin or the mutants and HA-tagged v-Src or vector control, as indicated. Lysates were precipitated with Ni-beads, followed by western blotting with anti-phosphotyrosine, 4G10 (top) or anti-Myc (middle) antibody. Whole cell lysates (WCL) were also immunoblotted with anti-HA (bottom). (B) Recombinant GST-cofilin or GST-Y68F purified from bacteria were incubated in a kinase buffer (ATP is omitted in lane 2, as indicated by labels above the upper panel) with HA-v-Src or HA-FAK immunoprecipitated from 293T cells transfected with corresponding expression vectors, as indicated. In vitro kinase assays were performed as described in the Experimental Procedures and the reaction mixtures were subjected to immunoblotting with 4G10 antibody (upper) or anti-GST (lower). (C) 293T cells were co-transfected with Flag-tagged v-Src and HA-tagged cofilin or Y68F mutant or vector control as indicated. Lysates were immunoprecipitated with anti-HA antibody, followed by western blotting with anti-Flag antibody (upper). WCL were also immunoblotted with anti-Flag (lower). Molecular weight markers are indicated on the right.

Mentions: To study the potential regulatory mechanism of cofilin by v-Src, we first determined whether v-Src can mediate tyrosine phosphorylation of cofilin. 293T cells were transfected with plasmids encoding HA-tagged v-Src and His-Myc-tagged cofilin. The overexpressed cofilins were then pulldown with Ni-beads, followed by immunoblotting with anti-phosphotyrosine antibody, 4G10. Fig. 1A shows that cofilin is specifically phosphorylated on tyrosine in cells co-transfected with v-Src (compare lane 2 with lane 1). To identify the site of phosphorylation by v-Src, each of the 6 tyrosine residues in cofilin was mutated to phenylalanine and the mutants were examined for their phosphorylation by v-Src. As shown in Fig. 1A, the Y68F mutant exhibited the greatest reduction in phosphorylation by v-Src (compare lane 3 with lanes 2 and 4–8), suggesting that Y68 is the major phosphorylation site of v-Src. Mutation of all six sites completely abolished cofilin phosphorylation by v-Src, as expected (lane 9). Co-transfection of plasmids encoding HA-FAK and Myc-cofilin or its mutants in similar experiments did not result in tyrosine phosphorylation of cofilin (data not shown), providing further support for the specificity of cofilin phosphorylation by v-Src. We then examined whether v-Src could directly phosphorylate cofilin at Y68. In vitro phosphorylation assay was performed using purified GST fusion proteins GST-cofilin or GST-Y68F, and recombinant HA-v-Src or HA-FAK immobilized on agarose beads by immunoprecipitation of lysates from 293T cells expressing the kinases, as described in the Materials and Methods. Fig. 1B shows that GST-cofilin was phosphorylated by v-Src in an ATP-dependent manner in vitro (lanes 1–4). Interestingly, mutation of Y68 to F significantly decreased cofilin phosphorylation by v-Src in vitro (compare lane 5 and lane 4), suggesting that Y68 is a major phosphorylation site by v-Src. The weak signal in lane 5 could be due to phosphorylation of GST-Y68F mutant at other sites by v-Src. We also observed a lower band in both lanes 4 and 5, which is probably a degradation product of GST-cofilin with the GST part degraded at least partially as this lower band was not recognized by anti-GST (lower panel). Consistent with the co-transfection studies, recombinant FAK did not phosphorylate either GST-cofilin or GST-Y68F in vitro (lanes 6 and 7). Lastly, we examined whether v-Src could associate with its substrate cofilin by co-immunoprecipitation of lysates from 293T cells that had been transfected with plasmids encoding Flag-tagged v-Src and HA-tagged cofilin, Y68F mutant, or vector alone. Fig. 1C shows that v-Src associated with both its substrate cofilin and the Y68F mutant, suggesting that the lack of phosphorylation of the Y68F mutant was not caused by a disruption of v-Src association with the mutant. Taken together, these results demonstrate that v-Src binds to cofilin and phosphorylates it at Y68.


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)

v-Src phosphorylation of cofilin at Y68(A) 293T cells were co-transfected with His- and Myc-tagged cofilin or the mutants and HA-tagged v-Src or vector control, as indicated. Lysates were precipitated with Ni-beads, followed by western blotting with anti-phosphotyrosine, 4G10 (top) or anti-Myc (middle) antibody. Whole cell lysates (WCL) were also immunoblotted with anti-HA (bottom). (B) Recombinant GST-cofilin or GST-Y68F purified from bacteria were incubated in a kinase buffer (ATP is omitted in lane 2, as indicated by labels above the upper panel) with HA-v-Src or HA-FAK immunoprecipitated from 293T cells transfected with corresponding expression vectors, as indicated. In vitro kinase assays were performed as described in the Experimental Procedures and the reaction mixtures were subjected to immunoblotting with 4G10 antibody (upper) or anti-GST (lower). (C) 293T cells were co-transfected with Flag-tagged v-Src and HA-tagged cofilin or Y68F mutant or vector control as indicated. Lysates were immunoprecipitated with anti-HA antibody, followed by western blotting with anti-Flag antibody (upper). WCL were also immunoblotted with anti-Flag (lower). Molecular weight markers are indicated on the right.
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Related In: Results  -  Collection

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Figure 1: v-Src phosphorylation of cofilin at Y68(A) 293T cells were co-transfected with His- and Myc-tagged cofilin or the mutants and HA-tagged v-Src or vector control, as indicated. Lysates were precipitated with Ni-beads, followed by western blotting with anti-phosphotyrosine, 4G10 (top) or anti-Myc (middle) antibody. Whole cell lysates (WCL) were also immunoblotted with anti-HA (bottom). (B) Recombinant GST-cofilin or GST-Y68F purified from bacteria were incubated in a kinase buffer (ATP is omitted in lane 2, as indicated by labels above the upper panel) with HA-v-Src or HA-FAK immunoprecipitated from 293T cells transfected with corresponding expression vectors, as indicated. In vitro kinase assays were performed as described in the Experimental Procedures and the reaction mixtures were subjected to immunoblotting with 4G10 antibody (upper) or anti-GST (lower). (C) 293T cells were co-transfected with Flag-tagged v-Src and HA-tagged cofilin or Y68F mutant or vector control as indicated. Lysates were immunoprecipitated with anti-HA antibody, followed by western blotting with anti-Flag antibody (upper). WCL were also immunoblotted with anti-Flag (lower). Molecular weight markers are indicated on the right.
Mentions: To study the potential regulatory mechanism of cofilin by v-Src, we first determined whether v-Src can mediate tyrosine phosphorylation of cofilin. 293T cells were transfected with plasmids encoding HA-tagged v-Src and His-Myc-tagged cofilin. The overexpressed cofilins were then pulldown with Ni-beads, followed by immunoblotting with anti-phosphotyrosine antibody, 4G10. Fig. 1A shows that cofilin is specifically phosphorylated on tyrosine in cells co-transfected with v-Src (compare lane 2 with lane 1). To identify the site of phosphorylation by v-Src, each of the 6 tyrosine residues in cofilin was mutated to phenylalanine and the mutants were examined for their phosphorylation by v-Src. As shown in Fig. 1A, the Y68F mutant exhibited the greatest reduction in phosphorylation by v-Src (compare lane 3 with lanes 2 and 4–8), suggesting that Y68 is the major phosphorylation site of v-Src. Mutation of all six sites completely abolished cofilin phosphorylation by v-Src, as expected (lane 9). Co-transfection of plasmids encoding HA-FAK and Myc-cofilin or its mutants in similar experiments did not result in tyrosine phosphorylation of cofilin (data not shown), providing further support for the specificity of cofilin phosphorylation by v-Src. We then examined whether v-Src could directly phosphorylate cofilin at Y68. In vitro phosphorylation assay was performed using purified GST fusion proteins GST-cofilin or GST-Y68F, and recombinant HA-v-Src or HA-FAK immobilized on agarose beads by immunoprecipitation of lysates from 293T cells expressing the kinases, as described in the Materials and Methods. Fig. 1B shows that GST-cofilin was phosphorylated by v-Src in an ATP-dependent manner in vitro (lanes 1–4). Interestingly, mutation of Y68 to F significantly decreased cofilin phosphorylation by v-Src in vitro (compare lane 5 and lane 4), suggesting that Y68 is a major phosphorylation site by v-Src. The weak signal in lane 5 could be due to phosphorylation of GST-Y68F mutant at other sites by v-Src. We also observed a lower band in both lanes 4 and 5, which is probably a degradation product of GST-cofilin with the GST part degraded at least partially as this lower band was not recognized by anti-GST (lower panel). Consistent with the co-transfection studies, recombinant FAK did not phosphorylate either GST-cofilin or GST-Y68F in vitro (lanes 6 and 7). Lastly, we examined whether v-Src could associate with its substrate cofilin by co-immunoprecipitation of lysates from 293T cells that had been transfected with plasmids encoding Flag-tagged v-Src and HA-tagged cofilin, Y68F mutant, or vector alone. Fig. 1C shows that v-Src associated with both its substrate cofilin and the Y68F mutant, suggesting that the lack of phosphorylation of the Y68F mutant was not caused by a disruption of v-Src association with the mutant. Taken together, these results demonstrate that v-Src binds to cofilin and phosphorylates it at Y68.

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