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Musk Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop

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ABSTRACT

The neuromuscular junction (NMJ) forms when a motor neuron contacts a muscle fibre. A reciprocal exchange of signals initiates a cascade of signalling events that result in pre- and postsynaptic differentiation. At the centre of these signalling events stands muscle specific kinase (MuSK). MuSK activation, kinase activity and subsequent downstream signalling are crucial for NMJ formation as well as maintenance. Therefore MuSK kinase activity is tightly regulated to ensure proper NMJ development. We have identified a novel serine phosphorylation site at position 751 in MuSK that is increasingly phosphorylated upon agrin stimulation. S751 is also phosphorylated in muscle tissue and its phosphorylation depends on MuSK kinase activity. A phosphomimetic mutant of S751 increases MuSK kinase activity in response to non-saturating agrin concentrations . In addition, basal MuSK and AChR phosphorylation as well as AChR cluster size are increased. We believe that the phosphorylation of S751 provides a novel mechanism to relief the autoinhibition of the MuSK activation loop. Such a lower autoinhibition could foster or stabilize MuSK kinase activation, especially during stages when no or low level of agrin are present. Phosphorylation of S751 might therefore represent a novel mechanism to modulate MuSK kinase activity during prepatterning or NMJ maintenance.

No MeSH data available.


S751 is phosphorylated in response to agrin.(A) Phosphopeptides were identified by quantitative MS analysis. Quantification of these showed induction of pY553 (red) and pS751 (blue) upon agrin treatment. In addition, phosphorylation of MuSK S678 was detected but not significantly regulated (grey). Phosphosites of regulated peptides were localized with high confidence (phosphoRS site probabilities are indicated in brackets). (B) Cell lysates from agrin-stimulated muscle cells were subjected to immunoprecipitation with anti-MuSK antibodies. Samples were analysed by immunoblotting using antibodies against phospho-tyrosine, phospho-S751 and MuSK. (C) Quantification of immunoblots shows phosphorylation kinetics similar to the kinetics of MS analysis. Values are presented as the mean ± S.E.M. (two-way ANOVA with Sidak’s multiple comparison tests, n = 5, p = 0.004). Full-length blots are presented in Supplementary Fig. S7. IP, immunoprecipitation; pY, phospho-tyrosine; UT, untreated.
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f1: S751 is phosphorylated in response to agrin.(A) Phosphopeptides were identified by quantitative MS analysis. Quantification of these showed induction of pY553 (red) and pS751 (blue) upon agrin treatment. In addition, phosphorylation of MuSK S678 was detected but not significantly regulated (grey). Phosphosites of regulated peptides were localized with high confidence (phosphoRS site probabilities are indicated in brackets). (B) Cell lysates from agrin-stimulated muscle cells were subjected to immunoprecipitation with anti-MuSK antibodies. Samples were analysed by immunoblotting using antibodies against phospho-tyrosine, phospho-S751 and MuSK. (C) Quantification of immunoblots shows phosphorylation kinetics similar to the kinetics of MS analysis. Values are presented as the mean ± S.E.M. (two-way ANOVA with Sidak’s multiple comparison tests, n = 5, p = 0.004). Full-length blots are presented in Supplementary Fig. S7. IP, immunoprecipitation; pY, phospho-tyrosine; UT, untreated.

Mentions: In a previous study we have used a muscle C2C12 cell culture model system to identify and characterize the phosphoproteome during agrin-induced MuSK activation. We were able to show that MuSK induces the phosphorylation of a large set of proteins with a distinct temporal manner of regulation15. In total we identified 152 proteins whose phosphorylation was either up- or downregulated at least two-fold. As expected, MuSK itself was among these regulated phosphoproteins. Phosphopeptides carrying Y553, the major phosphorylation site in MuSK, were detected in this quantitative mass spectrometry (MS) analysis (Fig. 1A). Y553 was rapidly and transiently phosphorylated. Phosphorylation peaked at 15 minutes (17-fold induction) and was reduced close to basal level at 240 minutes. In addition to MuSK tyrosine phosphorylation, we identified a novel phosphorylation site on S751, which is upregulated during late agrin stimulation (Fig. 1A). The peptide carrying S751 showed high isolation interference but identification and phosphorylation site localization was achieved with high confidence (Supplementary Fig. S1). Since the majority of phosphopeptides (98%) in our analysis were unregulated, we argue that the interfering peptides were also unregulated. Under this assumption S751 phoshorylation peaked at 60 minutes (five-fold induction) and remained stably phosphorylated until 240 minutes. To confirm this observation we stimulated C2C12 myotubes with agrin for different time periods, isolated MuSK via immunoprecipitation from cell lysates and performed immunoblotting with antibodies directed against phosphorylated S751 and phospho-tyrosine, respectively (Fig. 1B). Quantification revealed phosphorylation kinetics similar to the kinetics identified in the MS analysis (Fig. 1C). S751 is located in the activation loop of the MuSK kinase domain between Y750 and Y754/Y755, which are critical for MuSK activation. The specific kinetics of S751 phosphorylation and its location suggest a role during MuSK activation and/or signalling.


Musk Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop
S751 is phosphorylated in response to agrin.(A) Phosphopeptides were identified by quantitative MS analysis. Quantification of these showed induction of pY553 (red) and pS751 (blue) upon agrin treatment. In addition, phosphorylation of MuSK S678 was detected but not significantly regulated (grey). Phosphosites of regulated peptides were localized with high confidence (phosphoRS site probabilities are indicated in brackets). (B) Cell lysates from agrin-stimulated muscle cells were subjected to immunoprecipitation with anti-MuSK antibodies. Samples were analysed by immunoblotting using antibodies against phospho-tyrosine, phospho-S751 and MuSK. (C) Quantification of immunoblots shows phosphorylation kinetics similar to the kinetics of MS analysis. Values are presented as the mean ± S.E.M. (two-way ANOVA with Sidak’s multiple comparison tests, n = 5, p = 0.004). Full-length blots are presented in Supplementary Fig. S7. IP, immunoprecipitation; pY, phospho-tyrosine; UT, untreated.
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f1: S751 is phosphorylated in response to agrin.(A) Phosphopeptides were identified by quantitative MS analysis. Quantification of these showed induction of pY553 (red) and pS751 (blue) upon agrin treatment. In addition, phosphorylation of MuSK S678 was detected but not significantly regulated (grey). Phosphosites of regulated peptides were localized with high confidence (phosphoRS site probabilities are indicated in brackets). (B) Cell lysates from agrin-stimulated muscle cells were subjected to immunoprecipitation with anti-MuSK antibodies. Samples were analysed by immunoblotting using antibodies against phospho-tyrosine, phospho-S751 and MuSK. (C) Quantification of immunoblots shows phosphorylation kinetics similar to the kinetics of MS analysis. Values are presented as the mean ± S.E.M. (two-way ANOVA with Sidak’s multiple comparison tests, n = 5, p = 0.004). Full-length blots are presented in Supplementary Fig. S7. IP, immunoprecipitation; pY, phospho-tyrosine; UT, untreated.
Mentions: In a previous study we have used a muscle C2C12 cell culture model system to identify and characterize the phosphoproteome during agrin-induced MuSK activation. We were able to show that MuSK induces the phosphorylation of a large set of proteins with a distinct temporal manner of regulation15. In total we identified 152 proteins whose phosphorylation was either up- or downregulated at least two-fold. As expected, MuSK itself was among these regulated phosphoproteins. Phosphopeptides carrying Y553, the major phosphorylation site in MuSK, were detected in this quantitative mass spectrometry (MS) analysis (Fig. 1A). Y553 was rapidly and transiently phosphorylated. Phosphorylation peaked at 15 minutes (17-fold induction) and was reduced close to basal level at 240 minutes. In addition to MuSK tyrosine phosphorylation, we identified a novel phosphorylation site on S751, which is upregulated during late agrin stimulation (Fig. 1A). The peptide carrying S751 showed high isolation interference but identification and phosphorylation site localization was achieved with high confidence (Supplementary Fig. S1). Since the majority of phosphopeptides (98%) in our analysis were unregulated, we argue that the interfering peptides were also unregulated. Under this assumption S751 phoshorylation peaked at 60 minutes (five-fold induction) and remained stably phosphorylated until 240 minutes. To confirm this observation we stimulated C2C12 myotubes with agrin for different time periods, isolated MuSK via immunoprecipitation from cell lysates and performed immunoblotting with antibodies directed against phosphorylated S751 and phospho-tyrosine, respectively (Fig. 1B). Quantification revealed phosphorylation kinetics similar to the kinetics identified in the MS analysis (Fig. 1C). S751 is located in the activation loop of the MuSK kinase domain between Y750 and Y754/Y755, which are critical for MuSK activation. The specific kinetics of S751 phosphorylation and its location suggest a role during MuSK activation and/or signalling.

View Article: PubMed Central - PubMed

ABSTRACT

The neuromuscular junction (NMJ) forms when a motor neuron contacts a muscle fibre. A reciprocal exchange of signals initiates a cascade of signalling events that result in pre- and postsynaptic differentiation. At the centre of these signalling events stands muscle specific kinase (MuSK). MuSK activation, kinase activity and subsequent downstream signalling are crucial for NMJ formation as well as maintenance. Therefore MuSK kinase activity is tightly regulated to ensure proper NMJ development. We have identified a novel serine phosphorylation site at position 751 in MuSK that is increasingly phosphorylated upon agrin stimulation. S751 is also phosphorylated in muscle tissue and its phosphorylation depends on MuSK kinase activity. A phosphomimetic mutant of S751 increases MuSK kinase activity in response to non-saturating agrin concentrations . In addition, basal MuSK and AChR phosphorylation as well as AChR cluster size are increased. We believe that the phosphorylation of S751 provides a novel mechanism to relief the autoinhibition of the MuSK activation loop. Such a lower autoinhibition could foster or stabilize MuSK kinase activation, especially during stages when no or low level of agrin are present. Phosphorylation of S751 might therefore represent a novel mechanism to modulate MuSK kinase activity during prepatterning or NMJ maintenance.

No MeSH data available.