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

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.


Related in: MedlinePlus

A phosphomimetic S751 mutation modulates MuSK phosphorylation and causes increased AChR phosphorylation.Myotubes expressing MuSK wild-type or S751 mutants were stimulated with agrin for different time periods and cell lysates were processed for further analysis. (A) MuSK was immunoprecipitated with anti-HA antibodies and assayed by immunoblotting using antibodies against phospho-tyrosine and HA, respectively. (B) Quantification of MuSK phosphorylation in the absence of agrin is shown. Mutation S751D increases basal MuSK phosphorylation compared to S751A (p = 0.00001) and WT (p = 0.00079). Values are presented as the median ± interquartile range (IQR). Outliers are plotted as individual points (n = 9). (C) The kinetics of MuSK phosphorylation in response to agrin is shown. MuSK phosphorylation after 1 h agrin stimulation is set to 1 and time points are quantified accordingly for each data set. Values are presented as the mean ± S.E.M. (n = 9). (D) AChRs were affinity-purified using biotin-α-BGT and assayed by immunoblotting using anti-phospho-tyrosine and anti-AChR β antibodies, respectively. (E) AChR β; phosphorylation in the absence of agrin was quantified. Phosphorylation increases with MuSK-S751D compared to MuSK-S751A (p = 0.0092) and MuSK-WT (p = 0.0200). Values are presented as the median ± IQR. Outliers are plotted as individual points (n = 6). (F) The kinetics of AChR β; phosphorylation upon agrin stimulation was quantified as in (C). Phosphorylation in cells expressing MuSK-S751D is increased after 15 min agrin stimulation compared to MuSK-S751A (p = 0.0228) and MuSK-WT (p = 0.0498). Values are presented as the mean ± S.E.M. (n = 6). (G) Cells were stimulated with non-saturating concentrations of agrin and processed as in (A). Total lysates were analysed with antibodies against HA, AChR α and Actin, respectively. (H) Quantification of MuSK phosphorylation is shown (MuSK-WT set to 1). Mutation S751D increases MuSK phosphorylation compared to S751A (15 min: p = 0.0036; 60 min: p = 0.005) and WT (15 min: p = 0.0035; 60 min: p = 0.004); n = 4. One-way ANOVA with Tukey’s multiple comparison test was used. Full-length blots are presented in Supplementary Fig. S8. IP, immunoprecipitation; pY, phospho-tyrosine; UT, untreated; WT, wild-type.
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f3: A phosphomimetic S751 mutation modulates MuSK phosphorylation and causes increased AChR phosphorylation.Myotubes expressing MuSK wild-type or S751 mutants were stimulated with agrin for different time periods and cell lysates were processed for further analysis. (A) MuSK was immunoprecipitated with anti-HA antibodies and assayed by immunoblotting using antibodies against phospho-tyrosine and HA, respectively. (B) Quantification of MuSK phosphorylation in the absence of agrin is shown. Mutation S751D increases basal MuSK phosphorylation compared to S751A (p = 0.00001) and WT (p = 0.00079). Values are presented as the median ± interquartile range (IQR). Outliers are plotted as individual points (n = 9). (C) The kinetics of MuSK phosphorylation in response to agrin is shown. MuSK phosphorylation after 1 h agrin stimulation is set to 1 and time points are quantified accordingly for each data set. Values are presented as the mean ± S.E.M. (n = 9). (D) AChRs were affinity-purified using biotin-α-BGT and assayed by immunoblotting using anti-phospho-tyrosine and anti-AChR β antibodies, respectively. (E) AChR β; phosphorylation in the absence of agrin was quantified. Phosphorylation increases with MuSK-S751D compared to MuSK-S751A (p = 0.0092) and MuSK-WT (p = 0.0200). Values are presented as the median ± IQR. Outliers are plotted as individual points (n = 6). (F) The kinetics of AChR β; phosphorylation upon agrin stimulation was quantified as in (C). Phosphorylation in cells expressing MuSK-S751D is increased after 15 min agrin stimulation compared to MuSK-S751A (p = 0.0228) and MuSK-WT (p = 0.0498). Values are presented as the mean ± S.E.M. (n = 6). (G) Cells were stimulated with non-saturating concentrations of agrin and processed as in (A). Total lysates were analysed with antibodies against HA, AChR α and Actin, respectively. (H) Quantification of MuSK phosphorylation is shown (MuSK-WT set to 1). Mutation S751D increases MuSK phosphorylation compared to S751A (15 min: p = 0.0036; 60 min: p = 0.005) and WT (15 min: p = 0.0035; 60 min: p = 0.004); n = 4. One-way ANOVA with Tukey’s multiple comparison test was used. Full-length blots are presented in Supplementary Fig. S8. IP, immunoprecipitation; pY, phospho-tyrosine; UT, untreated; WT, wild-type.

Mentions: Muscle cells lacking MuSK are unable to respond to agrin and fail to form AChR clusters211. We used MuSK−/− muscle cells to study S751 during MuSK activation and downstream signalling213. We mutated the serine (S) to alanine (A) to generate a phosphorylation-deficient mutant and to aspartate (D), which can mimic phosphorylation to generate a phosphorylation-active mutant. MuSK−/− muscle cell lines expressing these S751 mutants or MuSK wild-type were generated, differentiated into myotubes and stimulated with agrin (Fig. 3). To examine MuSK activation we isolated MuSK and to study MuSK downstream signalling we isolated AChRs from cell lysates. Phosphorylation and expression were analysed by immunoblotting. Cell lines expressed similar levels of MuSK and AChR (Supplementary Fig. S3). As shown in Fig. 3B, a phosphomimetic mutant, generated by a S751D mutation, increased the basal tyrosine phosphorylation level of MuSK in the absence of agrin compared to MuSK S751A and MuSK wild-type. The temporal activation profile of MuSK tyrosine phosphorylation in response to agrin was similar for MuSK wild-type, MuSK S751D and MuSK S751A (Fig. 3C). Consistent with these findings the basal phosphorylation of AChR β; was increased in muscle cells expressing MuSK S751D (Fig. 3D,E). Moreover, AChR β; phosphorylation after short agrin stimulation (15 minutes) was significantly increased in muscle cells expressing MuSK S751D compared to cells expressing MuSK S751A or MuSK wild-type (Fig. 3F).


Musk Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop
A phosphomimetic S751 mutation modulates MuSK phosphorylation and causes increased AChR phosphorylation.Myotubes expressing MuSK wild-type or S751 mutants were stimulated with agrin for different time periods and cell lysates were processed for further analysis. (A) MuSK was immunoprecipitated with anti-HA antibodies and assayed by immunoblotting using antibodies against phospho-tyrosine and HA, respectively. (B) Quantification of MuSK phosphorylation in the absence of agrin is shown. Mutation S751D increases basal MuSK phosphorylation compared to S751A (p = 0.00001) and WT (p = 0.00079). Values are presented as the median ± interquartile range (IQR). Outliers are plotted as individual points (n = 9). (C) The kinetics of MuSK phosphorylation in response to agrin is shown. MuSK phosphorylation after 1 h agrin stimulation is set to 1 and time points are quantified accordingly for each data set. Values are presented as the mean ± S.E.M. (n = 9). (D) AChRs were affinity-purified using biotin-α-BGT and assayed by immunoblotting using anti-phospho-tyrosine and anti-AChR β antibodies, respectively. (E) AChR β; phosphorylation in the absence of agrin was quantified. Phosphorylation increases with MuSK-S751D compared to MuSK-S751A (p = 0.0092) and MuSK-WT (p = 0.0200). Values are presented as the median ± IQR. Outliers are plotted as individual points (n = 6). (F) The kinetics of AChR β; phosphorylation upon agrin stimulation was quantified as in (C). Phosphorylation in cells expressing MuSK-S751D is increased after 15 min agrin stimulation compared to MuSK-S751A (p = 0.0228) and MuSK-WT (p = 0.0498). Values are presented as the mean ± S.E.M. (n = 6). (G) Cells were stimulated with non-saturating concentrations of agrin and processed as in (A). Total lysates were analysed with antibodies against HA, AChR α and Actin, respectively. (H) Quantification of MuSK phosphorylation is shown (MuSK-WT set to 1). Mutation S751D increases MuSK phosphorylation compared to S751A (15 min: p = 0.0036; 60 min: p = 0.005) and WT (15 min: p = 0.0035; 60 min: p = 0.004); n = 4. One-way ANOVA with Tukey’s multiple comparison test was used. Full-length blots are presented in Supplementary Fig. S8. IP, immunoprecipitation; pY, phospho-tyrosine; UT, untreated; WT, wild-type.
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f3: A phosphomimetic S751 mutation modulates MuSK phosphorylation and causes increased AChR phosphorylation.Myotubes expressing MuSK wild-type or S751 mutants were stimulated with agrin for different time periods and cell lysates were processed for further analysis. (A) MuSK was immunoprecipitated with anti-HA antibodies and assayed by immunoblotting using antibodies against phospho-tyrosine and HA, respectively. (B) Quantification of MuSK phosphorylation in the absence of agrin is shown. Mutation S751D increases basal MuSK phosphorylation compared to S751A (p = 0.00001) and WT (p = 0.00079). Values are presented as the median ± interquartile range (IQR). Outliers are plotted as individual points (n = 9). (C) The kinetics of MuSK phosphorylation in response to agrin is shown. MuSK phosphorylation after 1 h agrin stimulation is set to 1 and time points are quantified accordingly for each data set. Values are presented as the mean ± S.E.M. (n = 9). (D) AChRs were affinity-purified using biotin-α-BGT and assayed by immunoblotting using anti-phospho-tyrosine and anti-AChR β antibodies, respectively. (E) AChR β; phosphorylation in the absence of agrin was quantified. Phosphorylation increases with MuSK-S751D compared to MuSK-S751A (p = 0.0092) and MuSK-WT (p = 0.0200). Values are presented as the median ± IQR. Outliers are plotted as individual points (n = 6). (F) The kinetics of AChR β; phosphorylation upon agrin stimulation was quantified as in (C). Phosphorylation in cells expressing MuSK-S751D is increased after 15 min agrin stimulation compared to MuSK-S751A (p = 0.0228) and MuSK-WT (p = 0.0498). Values are presented as the mean ± S.E.M. (n = 6). (G) Cells were stimulated with non-saturating concentrations of agrin and processed as in (A). Total lysates were analysed with antibodies against HA, AChR α and Actin, respectively. (H) Quantification of MuSK phosphorylation is shown (MuSK-WT set to 1). Mutation S751D increases MuSK phosphorylation compared to S751A (15 min: p = 0.0036; 60 min: p = 0.005) and WT (15 min: p = 0.0035; 60 min: p = 0.004); n = 4. One-way ANOVA with Tukey’s multiple comparison test was used. Full-length blots are presented in Supplementary Fig. S8. IP, immunoprecipitation; pY, phospho-tyrosine; UT, untreated; WT, wild-type.
Mentions: Muscle cells lacking MuSK are unable to respond to agrin and fail to form AChR clusters211. We used MuSK−/− muscle cells to study S751 during MuSK activation and downstream signalling213. We mutated the serine (S) to alanine (A) to generate a phosphorylation-deficient mutant and to aspartate (D), which can mimic phosphorylation to generate a phosphorylation-active mutant. MuSK−/− muscle cell lines expressing these S751 mutants or MuSK wild-type were generated, differentiated into myotubes and stimulated with agrin (Fig. 3). To examine MuSK activation we isolated MuSK and to study MuSK downstream signalling we isolated AChRs from cell lysates. Phosphorylation and expression were analysed by immunoblotting. Cell lines expressed similar levels of MuSK and AChR (Supplementary Fig. S3). As shown in Fig. 3B, a phosphomimetic mutant, generated by a S751D mutation, increased the basal tyrosine phosphorylation level of MuSK in the absence of agrin compared to MuSK S751A and MuSK wild-type. The temporal activation profile of MuSK tyrosine phosphorylation in response to agrin was similar for MuSK wild-type, MuSK S751D and MuSK S751A (Fig. 3C). Consistent with these findings the basal phosphorylation of AChR β; was increased in muscle cells expressing MuSK S751D (Fig. 3D,E). Moreover, AChR β; phosphorylation after short agrin stimulation (15 minutes) was significantly increased in muscle cells expressing MuSK S751D compared to cells expressing MuSK S751A or MuSK wild-type (Fig. 3F).

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.


Related in: MedlinePlus