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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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Src inhibition prevents dystroglycan localisation/podosome formation.Myoblast cells were seeded onto tissue culture plastic and allowed to spread for one hour before fixation and staining for β-dystroglycan, F-actin and the indicated markers of cell adhesion. In A cells were untreated and formed dystroglycan-containing puncta as before. Cells in B, were treated with the Src Inhibitor PP2 (10 µM) for 1 hour before and during re-seeding. No dystroglycan-containing puncta were formed in these cells. Focal adhesions appeared to form as normal, as judged by staining for paxillin, talin, vinculin and phospho-tyrosine (pTyr). Dystroglycan staining in green in all merged images. Scale bars 20 µm.
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pone-0003638-g008: Src inhibition prevents dystroglycan localisation/podosome formation.Myoblast cells were seeded onto tissue culture plastic and allowed to spread for one hour before fixation and staining for β-dystroglycan, F-actin and the indicated markers of cell adhesion. In A cells were untreated and formed dystroglycan-containing puncta as before. Cells in B, were treated with the Src Inhibitor PP2 (10 µM) for 1 hour before and during re-seeding. No dystroglycan-containing puncta were formed in these cells. Focal adhesions appeared to form as normal, as judged by staining for paxillin, talin, vinculin and phospho-tyrosine (pTyr). Dystroglycan staining in green in all merged images. Scale bars 20 µm.

Mentions: The dominant-negative effect of excess DG on podosome formation provided a useful tool to reveal functionally important regions of dystroglycan involved in podosome formation. By mutating potential binding sites or phosphorylation sites within the cytoplasmic domain of β-DG we could also potentially restore podosome formation by inhibiting the dominant-negative effect of DG overexpression. We therefore mutagenised potential tyrosine phosphorylation sites and proline residues in the SH3 domain binding consensus PxxP motifs within the β-DG cytoplasmic domain that might mediate SH3 domains interactions with Tks5. As can be seen in Figure 7, most mutations did not significantly reduce the inhibition of podosome formation compared to control, as dystroglycan-GFP carrying these mutations still had a dominant-negative effect. This was not due to the constructs failing to express as can be seen from the inset, the levels of DG-GFP, despite being a transient expression, are relatively similar. The fact these mutants did not behave exactly like wild-type and completely inhibit podosome formation, may reflect short range conformational effects induced by the mutations that perturbed interactions in neighbouring regions of the β-dystroglycan cytoplasmic domain. However mutations in Y890 restored podosome formation in myoblasts suppressing the dominant-negative effect of over-expressing dystroglycan-GFP (Figure 7). This result suggests that the phosphorylation of Y890 by Src is an important event in podosome formation. Overexpression of Y890A DG significantly restored podosomes to 70% of control levels, whereas mutation of other tyrosine residues or mutation of potential SH3 binding sites did not significantly affect podosome formation (Figure 7). These results highlight the importance of phosphorylation of β-DG Y890 in podosome assembly and corroborate the finding that DG associated with Tks5 is tyrosine phosphorylated (Figure 3B). Earlier work has shown dystroglycan Y890 to be a substrate for Src family kinases. We therefore treated early spreading myoblast cells with the Src inhibitor PP2 to observe the effect on podosome assembly. Compared to untreated cells, PP2 completely prevented the formation of the dystroglycan-rich puncta or podosomes instead DG was localised to the cell periphery along with actin and cortactin (Figure 8AB). As we have shown previously inhibition of Src does not affect focal adhesion formation per se [44], accordingly paxillin, talin and vinculin are all visible in peripheral focal adhesion-like structures (Figure 8AB), rather Src activity appears to be essential for focal adhesion turnover or disassembly [45]. By contrast in podosomes, Src activity appears to be required for the phosphorylation of Tks5 and β-DG in podosome assembly. Phosphorylation of DG Y890 would form a potential SH2 domain interaction site and DG phosphorylated at this site has been demonstrated previously to interact with Src itself [46]. We therefore carried out cell fractionation experiments in cells expressing different levels of dystroglycan and examined the distribution of dystroglycan and Src between cytoplasmic, membrane and cytoskeletal fractions. Due to its association with the actin cytoskeleton, DG is not readily extracted from membranes by Triton, but rather partitions with the insoluble cytoskeletal fraction [47]. We therefore utilised a protocol first extracting in CHAPS to release DG from the membrane, followed by a triton extraction step to precipitate the cytoskeleton and associated proteins in the triton insoluble fraction. This method has been employed with previous success to extract ERM family proteins and dystroglycan [48], [49]. Overexpression of DG results in increased levels of free DG in the CHAPS soluble fraction ([8]; Figure 9A–D). Furthermore, compared with the cytoskeleton associated (triton insoluble) dystroglycan a greater proportion of the CHAPS soluble DG is phosphorylated on Y890. In control cells the distribution of Src is almost exclusively in the triton insoluble fraction, but in cells overexpressing DG-GFP, Src localisation switches dramatically to the CHAPS soluble fraction. It would therefore appear that Y890 phosphorylation results in the creation of an SH2 domain binding site which also recruits Src to the CHAPS soluble fraction (Figure 9A,). This is supported by the finding that Src can be co-immunoprecipitated with both Tks5 and dystroglycan, suggesting the existence of a ternary complex between Tks5, Src and dystroglycan (Figure 9E). DG overexpression can therefore redirect the regulatory functions of both Tks5 and Src, the combined effects of which result in the inhibition of podosome formation. This adds further support to the idea that levels of dystroglycan must be finely balanced to maintain an appropriate level of scaffolding and signalling to regulate podosome formation.


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)

Src inhibition prevents dystroglycan localisation/podosome formation.Myoblast cells were seeded onto tissue culture plastic and allowed to spread for one hour before fixation and staining for β-dystroglycan, F-actin and the indicated markers of cell adhesion. In A cells were untreated and formed dystroglycan-containing puncta as before. Cells in B, were treated with the Src Inhibitor PP2 (10 µM) for 1 hour before and during re-seeding. No dystroglycan-containing puncta were formed in these cells. Focal adhesions appeared to form as normal, as judged by staining for paxillin, talin, vinculin and phospho-tyrosine (pTyr). Dystroglycan staining in green in all merged images. Scale bars 20 µm.
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Related In: Results  -  Collection

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pone-0003638-g008: Src inhibition prevents dystroglycan localisation/podosome formation.Myoblast cells were seeded onto tissue culture plastic and allowed to spread for one hour before fixation and staining for β-dystroglycan, F-actin and the indicated markers of cell adhesion. In A cells were untreated and formed dystroglycan-containing puncta as before. Cells in B, were treated with the Src Inhibitor PP2 (10 µM) for 1 hour before and during re-seeding. No dystroglycan-containing puncta were formed in these cells. Focal adhesions appeared to form as normal, as judged by staining for paxillin, talin, vinculin and phospho-tyrosine (pTyr). Dystroglycan staining in green in all merged images. Scale bars 20 µm.
Mentions: The dominant-negative effect of excess DG on podosome formation provided a useful tool to reveal functionally important regions of dystroglycan involved in podosome formation. By mutating potential binding sites or phosphorylation sites within the cytoplasmic domain of β-DG we could also potentially restore podosome formation by inhibiting the dominant-negative effect of DG overexpression. We therefore mutagenised potential tyrosine phosphorylation sites and proline residues in the SH3 domain binding consensus PxxP motifs within the β-DG cytoplasmic domain that might mediate SH3 domains interactions with Tks5. As can be seen in Figure 7, most mutations did not significantly reduce the inhibition of podosome formation compared to control, as dystroglycan-GFP carrying these mutations still had a dominant-negative effect. This was not due to the constructs failing to express as can be seen from the inset, the levels of DG-GFP, despite being a transient expression, are relatively similar. The fact these mutants did not behave exactly like wild-type and completely inhibit podosome formation, may reflect short range conformational effects induced by the mutations that perturbed interactions in neighbouring regions of the β-dystroglycan cytoplasmic domain. However mutations in Y890 restored podosome formation in myoblasts suppressing the dominant-negative effect of over-expressing dystroglycan-GFP (Figure 7). This result suggests that the phosphorylation of Y890 by Src is an important event in podosome formation. Overexpression of Y890A DG significantly restored podosomes to 70% of control levels, whereas mutation of other tyrosine residues or mutation of potential SH3 binding sites did not significantly affect podosome formation (Figure 7). These results highlight the importance of phosphorylation of β-DG Y890 in podosome assembly and corroborate the finding that DG associated with Tks5 is tyrosine phosphorylated (Figure 3B). Earlier work has shown dystroglycan Y890 to be a substrate for Src family kinases. We therefore treated early spreading myoblast cells with the Src inhibitor PP2 to observe the effect on podosome assembly. Compared to untreated cells, PP2 completely prevented the formation of the dystroglycan-rich puncta or podosomes instead DG was localised to the cell periphery along with actin and cortactin (Figure 8AB). As we have shown previously inhibition of Src does not affect focal adhesion formation per se [44], accordingly paxillin, talin and vinculin are all visible in peripheral focal adhesion-like structures (Figure 8AB), rather Src activity appears to be essential for focal adhesion turnover or disassembly [45]. By contrast in podosomes, Src activity appears to be required for the phosphorylation of Tks5 and β-DG in podosome assembly. Phosphorylation of DG Y890 would form a potential SH2 domain interaction site and DG phosphorylated at this site has been demonstrated previously to interact with Src itself [46]. We therefore carried out cell fractionation experiments in cells expressing different levels of dystroglycan and examined the distribution of dystroglycan and Src between cytoplasmic, membrane and cytoskeletal fractions. Due to its association with the actin cytoskeleton, DG is not readily extracted from membranes by Triton, but rather partitions with the insoluble cytoskeletal fraction [47]. We therefore utilised a protocol first extracting in CHAPS to release DG from the membrane, followed by a triton extraction step to precipitate the cytoskeleton and associated proteins in the triton insoluble fraction. This method has been employed with previous success to extract ERM family proteins and dystroglycan [48], [49]. Overexpression of DG results in increased levels of free DG in the CHAPS soluble fraction ([8]; Figure 9A–D). Furthermore, compared with the cytoskeleton associated (triton insoluble) dystroglycan a greater proportion of the CHAPS soluble DG is phosphorylated on Y890. In control cells the distribution of Src is almost exclusively in the triton insoluble fraction, but in cells overexpressing DG-GFP, Src localisation switches dramatically to the CHAPS soluble fraction. It would therefore appear that Y890 phosphorylation results in the creation of an SH2 domain binding site which also recruits Src to the CHAPS soluble fraction (Figure 9A,). This is supported by the finding that Src can be co-immunoprecipitated with both Tks5 and dystroglycan, suggesting the existence of a ternary complex between Tks5, Src and dystroglycan (Figure 9E). DG overexpression can therefore redirect the regulatory functions of both Tks5 and Src, the combined effects of which result in the inhibition of podosome formation. This adds further support to the idea that levels of dystroglycan must be finely balanced to maintain an appropriate level of scaffolding and signalling to regulate podosome formation.

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