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Dystroglycan, Tks5 and Src mediated assembly of podosomes in myoblasts.

Thompson O, Kleino I, Crimaldi L, Gimona M, Saksela K, Winder SJ - PLoS ONE (2008)

Bottom Line: Dystroglycan overexpression inhibited podosome formation by sequestering Tks5 and Src.Mutation of dystroglycan tyrosine 890, previously identified as a Src substrate, restored podosome formation.We propose therefore, that Src-dependent phosphorylation of beta-dystroglycan results in the formation of a Src/dystroglycan complex that drives the SH3-mediated association between dystroglycan and Tks5 which together regulate podosome formation in myoblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, UK.

ABSTRACT

Background: Dystroglycan is a ubiquitously expressed cell adhesion receptor best understood in its role as part of the dystrophin glycoprotein complex of mature skeletal muscle. Less is known of the role of dystroglycan in more fundamental aspects of cell adhesion in other cell types, nor of its role in myoblast cell adhesion.

Principal findings: We have examined the role of dystroglycan in the early stages of myoblast adhesion and spreading and found that dystroglycan initially associates with other adhesion proteins in large puncta morphologically similar to podosomes. Using a human SH3 domain phage display library we identified Tks5, a key regulator of podosomes, as interacting with beta-dystroglycan. We verified the interaction by immunoprecipitation, GST-pulldown and immunfluorescence localisation. Both proteins localise to puncta during early phases of spreading, but importantly following stimulation with phorbol ester, also localise to structures indistinguishable from podosomes. Dystroglycan overexpression inhibited podosome formation by sequestering Tks5 and Src. Mutation of dystroglycan tyrosine 890, previously identified as a Src substrate, restored podosome formation.

Conclusions: We propose therefore, that Src-dependent phosphorylation of beta-dystroglycan results in the formation of a Src/dystroglycan complex that drives the SH3-mediated association between dystroglycan and Tks5 which together regulate podosome formation in myoblasts.

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DG and Tks5 localise to podosomes in myoblasts.Myoblast cells, untreated (−) and following stimulation with PDBu for 30 min (+) were stained for endogenous β-DG (red), Tks5 (green) and F-actin (blue). Unstimulated cells exhibit only faint β-DG and Tks5 localisation toward the periphery in cellular protrusions, potentially in podosome cluster precursors [43]. Following stimulation, β-DG and Tks5 were clearly localised along with F-actin to podosome structures following stimulation. The actin staining can be seen clearly to form a more central core to the podosome with Tks5 and dystroglycan around the periphery (inset). Scale bar 20 µm.
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pone-0003638-g004: DG and Tks5 localise to podosomes in myoblasts.Myoblast cells, untreated (−) and following stimulation with PDBu for 30 min (+) were stained for endogenous β-DG (red), Tks5 (green) and F-actin (blue). Unstimulated cells exhibit only faint β-DG and Tks5 localisation toward the periphery in cellular protrusions, potentially in podosome cluster precursors [43]. Following stimulation, β-DG and Tks5 were clearly localised along with F-actin to podosome structures following stimulation. The actin staining can be seen clearly to form a more central core to the podosome with Tks5 and dystroglycan around the periphery (inset). Scale bar 20 µm.

Mentions: We verified an interaction between Tks5 and β-DG by reciprocal immunoprecipitation from myoblast lysates. As shown in figure 3A, antibodies against either β-DG or Tks5 were able to immunoprecipitate the other protein. To further substantiate a role for the 3rd SH3 domain we used a GST-SH3 pulldown to recover β-DG from myoblast lysates. DG was recovered specifically with the 3rd but not the 5th SH3 domain of Tks5 fused to GST. Significantly the β-DG bound to the Tks5 was only recognised by an anti-phospho β-DG antibody that is able to recognise DG that is phosphorylated on tyrosine 890 (Y890). A monoclonal antibody that recognises a similar epitope, but that can't bind to β-DG that is phosphorylated on Y890 did not detect any β-DG in either pulldown (Figure 3B). This result suggests that not only is Src activation necessary for podosome formation, and for the recruitment of Tks5 to podosomes, but that when β-DG is associated with Tks5 in cells, it too is phosphorylated on tyrosine at Y890. In order to demonstrate an interaction between DG and Tks5 in a cellular context, we employed a GFP-tagged Tks5 construct fused with a mitochondrial targeting sequence at its amino terminus. As can be seen in Figure 3C, the mito-Tks5-GFP construct was efficiently targeted to mitochondria and localised extensively with mito-tracker red staining in the myoblast cells. We then co-expressed the mito-Tks5-GFP construct with a construct comprising the cytoplasmic domain of β-DG only with an amino-terminal Myc tag. As we have demonstrated previously this construct is entirely cytosolic [8], [12], and therefore its localisation should respond to that of a binding partner that was localised to a discrete domain such as the mitochondrion. As can be seen, this is indeed the case; the cytoplasmic Myc-cβ-DG construct is colocalised with the mito-Tks5-GFP (Figure 3D) rather than appearing diffusely in the cytoplasm (Figure 3E) substantiating an interaction between the two proteins. We further verified the association between Tks5 and DG by immunfluorescence localisation of the two proteins with F-actin in myoblasts before and after stimulation with PDBu. As can be seen in Figure 4, whilst there is a faint and indistinct area of colocalisation between Tks5 and β-DG towards the leading extremities of unstimulated myoblasts, which may correspond to the podosome cluster precursors described by Matsudaira and colleagues [43], following PDBu stimulation all three proteins colocalise robustly to distinct podosome structures behind the leading edge of the cell. And as in typical podosome structures, there is a clear actin core with a more diffuse and wider distribution of dystroglycan and Tks5 (Figure 4 inset).


Dystroglycan, Tks5 and Src mediated assembly of podosomes in myoblasts.

Thompson O, Kleino I, Crimaldi L, Gimona M, Saksela K, Winder SJ - PLoS ONE (2008)

DG and Tks5 localise to podosomes in myoblasts.Myoblast cells, untreated (−) and following stimulation with PDBu for 30 min (+) were stained for endogenous β-DG (red), Tks5 (green) and F-actin (blue). Unstimulated cells exhibit only faint β-DG and Tks5 localisation toward the periphery in cellular protrusions, potentially in podosome cluster precursors [43]. Following stimulation, β-DG and Tks5 were clearly localised along with F-actin to podosome structures following stimulation. The actin staining can be seen clearly to form a more central core to the podosome with Tks5 and dystroglycan around the periphery (inset). Scale bar 20 µm.
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pone-0003638-g004: DG and Tks5 localise to podosomes in myoblasts.Myoblast cells, untreated (−) and following stimulation with PDBu for 30 min (+) were stained for endogenous β-DG (red), Tks5 (green) and F-actin (blue). Unstimulated cells exhibit only faint β-DG and Tks5 localisation toward the periphery in cellular protrusions, potentially in podosome cluster precursors [43]. Following stimulation, β-DG and Tks5 were clearly localised along with F-actin to podosome structures following stimulation. The actin staining can be seen clearly to form a more central core to the podosome with Tks5 and dystroglycan around the periphery (inset). Scale bar 20 µm.
Mentions: We verified an interaction between Tks5 and β-DG by reciprocal immunoprecipitation from myoblast lysates. As shown in figure 3A, antibodies against either β-DG or Tks5 were able to immunoprecipitate the other protein. To further substantiate a role for the 3rd SH3 domain we used a GST-SH3 pulldown to recover β-DG from myoblast lysates. DG was recovered specifically with the 3rd but not the 5th SH3 domain of Tks5 fused to GST. Significantly the β-DG bound to the Tks5 was only recognised by an anti-phospho β-DG antibody that is able to recognise DG that is phosphorylated on tyrosine 890 (Y890). A monoclonal antibody that recognises a similar epitope, but that can't bind to β-DG that is phosphorylated on Y890 did not detect any β-DG in either pulldown (Figure 3B). This result suggests that not only is Src activation necessary for podosome formation, and for the recruitment of Tks5 to podosomes, but that when β-DG is associated with Tks5 in cells, it too is phosphorylated on tyrosine at Y890. In order to demonstrate an interaction between DG and Tks5 in a cellular context, we employed a GFP-tagged Tks5 construct fused with a mitochondrial targeting sequence at its amino terminus. As can be seen in Figure 3C, the mito-Tks5-GFP construct was efficiently targeted to mitochondria and localised extensively with mito-tracker red staining in the myoblast cells. We then co-expressed the mito-Tks5-GFP construct with a construct comprising the cytoplasmic domain of β-DG only with an amino-terminal Myc tag. As we have demonstrated previously this construct is entirely cytosolic [8], [12], and therefore its localisation should respond to that of a binding partner that was localised to a discrete domain such as the mitochondrion. As can be seen, this is indeed the case; the cytoplasmic Myc-cβ-DG construct is colocalised with the mito-Tks5-GFP (Figure 3D) rather than appearing diffusely in the cytoplasm (Figure 3E) substantiating an interaction between the two proteins. We further verified the association between Tks5 and DG by immunfluorescence localisation of the two proteins with F-actin in myoblasts before and after stimulation with PDBu. As can be seen in Figure 4, whilst there is a faint and indistinct area of colocalisation between Tks5 and β-DG towards the leading extremities of unstimulated myoblasts, which may correspond to the podosome cluster precursors described by Matsudaira and colleagues [43], following PDBu stimulation all three proteins colocalise robustly to distinct podosome structures behind the leading edge of the cell. And as in typical podosome structures, there is a clear actin core with a more diffuse and wider distribution of dystroglycan and Tks5 (Figure 4 inset).

Bottom Line: Dystroglycan overexpression inhibited podosome formation by sequestering Tks5 and Src.Mutation of dystroglycan tyrosine 890, previously identified as a Src substrate, restored podosome formation.We propose therefore, that Src-dependent phosphorylation of beta-dystroglycan results in the formation of a Src/dystroglycan complex that drives the SH3-mediated association between dystroglycan and Tks5 which together regulate podosome formation in myoblasts.

View Article: PubMed Central - PubMed

Affiliation: Department of Biomedical Science, University of Sheffield, Western Bank, Sheffield, UK.

ABSTRACT

Background: Dystroglycan is a ubiquitously expressed cell adhesion receptor best understood in its role as part of the dystrophin glycoprotein complex of mature skeletal muscle. Less is known of the role of dystroglycan in more fundamental aspects of cell adhesion in other cell types, nor of its role in myoblast cell adhesion.

Principal findings: We have examined the role of dystroglycan in the early stages of myoblast adhesion and spreading and found that dystroglycan initially associates with other adhesion proteins in large puncta morphologically similar to podosomes. Using a human SH3 domain phage display library we identified Tks5, a key regulator of podosomes, as interacting with beta-dystroglycan. We verified the interaction by immunoprecipitation, GST-pulldown and immunfluorescence localisation. Both proteins localise to puncta during early phases of spreading, but importantly following stimulation with phorbol ester, also localise to structures indistinguishable from podosomes. Dystroglycan overexpression inhibited podosome formation by sequestering Tks5 and Src. Mutation of dystroglycan tyrosine 890, previously identified as a Src substrate, restored podosome formation.

Conclusions: We propose therefore, that Src-dependent phosphorylation of beta-dystroglycan results in the formation of a Src/dystroglycan complex that drives the SH3-mediated association between dystroglycan and Tks5 which together regulate podosome formation in myoblasts.

Show MeSH
Related in: MedlinePlus