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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 MuSK S751 phosphomimetic mutant increases the size of AChR clusters but does not increase the number of AChRs.(A) MuSK−/− muscle cells expressing MuSK wild-type and S751 mutant proteins were differentiated and stimulated with agrin for 16 h (overnight) to induce AChR cluster formation. To measure AChR cluster stability, agrin was removed and the cells were maintained in agrin-free medium for indicated times. Representative images of clusters stained with Alexa 594-conjugated α-BGT are shown. Scale bar, 10 μm. (B) The number of AChR clusters normalized to myotube area (Kruskal-Wallis with Dunn’s multiple comparison test, mean ± S.E.M., n = 4 with > 75 myotubes in total for each treatment) and the mean feret of clusters (one-way ANOVA with Tukey’s multiple comparison test, mean ± S.E.M., *p < 0.05, **p < 0.01, n ≥ 100 from four different experiments) were quantified using ImageJ. Note that AChR clusters are significantly bigger in cells expressing MuSK-S751D after agrin stimulation and after 6 h of agrin withdrawal. (C) Graph showing the percentage of microclusters per myotube. MuSK-S751D expressing cells form more microclusters compared to cells expressing MuSK-S751 A (p = 0.0270) and MuSK-WT (p = 0.0382). Values are presented as the mean ± S.E.M. (one-way ANOVA with Tukey’s multiple comparison test, n = 3 with > 100 myotubes in total for each cell line). WD, withdrawal; o/n, overnight; WT, wild-type.
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f6: A MuSK S751 phosphomimetic mutant increases the size of AChR clusters but does not increase the number of AChRs.(A) MuSK−/− muscle cells expressing MuSK wild-type and S751 mutant proteins were differentiated and stimulated with agrin for 16 h (overnight) to induce AChR cluster formation. To measure AChR cluster stability, agrin was removed and the cells were maintained in agrin-free medium for indicated times. Representative images of clusters stained with Alexa 594-conjugated α-BGT are shown. Scale bar, 10 μm. (B) The number of AChR clusters normalized to myotube area (Kruskal-Wallis with Dunn’s multiple comparison test, mean ± S.E.M., n = 4 with > 75 myotubes in total for each treatment) and the mean feret of clusters (one-way ANOVA with Tukey’s multiple comparison test, mean ± S.E.M., *p < 0.05, **p < 0.01, n ≥ 100 from four different experiments) were quantified using ImageJ. Note that AChR clusters are significantly bigger in cells expressing MuSK-S751D after agrin stimulation and after 6 h of agrin withdrawal. (C) Graph showing the percentage of microclusters per myotube. MuSK-S751D expressing cells form more microclusters compared to cells expressing MuSK-S751 A (p = 0.0270) and MuSK-WT (p = 0.0382). Values are presented as the mean ± S.E.M. (one-way ANOVA with Tukey’s multiple comparison test, n = 3 with > 100 myotubes in total for each cell line). WD, withdrawal; o/n, overnight; WT, wild-type.

Mentions: The hallmark of MuSK signalling is the clustering of AChRs to discrete patches on the muscle membrane. Changes in MuSK kinase activity or alterations in the downstream signalling pathway greatly influence AChR clustering11131416. Defects in AChR clustering usually result in increased or decreased number of clusters or increased or decreased size of clusters. We stimulated myotubes expressing MuSK wild-type, S751A or S751D with neural agrin A4B8 overnight followed by staining of AChRs with α-BGT. Figure 6A shows that AChR clusters were formed in all cell lines. To examine AChR cluster stability we removed agrin and cultured the cells for an additional 6 or 12 hours in agrin-free medium. Again we stained AChR clusters with α-BGT. AChR clusters decreased in number and size upon agrin withdrawal (Fig. 6A). As control we treated myotubes overnight with muscle agrin A0B0, which does not activate MuSK and therefore fails to induce AChR clusters1321. Number and size of AChR clusters were subsequently analysed (Fig. 6B). Mutation of S751 either to alanine or aspartate did not affect the ability of myotubes to form AChR clusters. The number of clusters was similar in all cell lines after overnight agrin stimulation as well as after agrin withdrawal. However, when we measured AChR cluster size we found that myotubes expressing MuSK S751D formed bigger clusters upon agrin treatment compared to myotubes expressing MuSK wild-type or S751A. Moreover, clusters in MuSK S751D expressing myotubes remained big after 6 hours agrin removal compared to MuSK wild-type and MuSK S751A expressing cells. In addition, more MuSK S751D expressing myotubes formed microclusters compared to myotubes expressing MuSK wild-type or MuSK S751A (Fig. 6C). These data suggest that the increased basal kinase activity of MuSK S751D positively modulates AChR clustering.


Musk Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop
A MuSK S751 phosphomimetic mutant increases the size of AChR clusters but does not increase the number of AChRs.(A) MuSK−/− muscle cells expressing MuSK wild-type and S751 mutant proteins were differentiated and stimulated with agrin for 16 h (overnight) to induce AChR cluster formation. To measure AChR cluster stability, agrin was removed and the cells were maintained in agrin-free medium for indicated times. Representative images of clusters stained with Alexa 594-conjugated α-BGT are shown. Scale bar, 10 μm. (B) The number of AChR clusters normalized to myotube area (Kruskal-Wallis with Dunn’s multiple comparison test, mean ± S.E.M., n = 4 with > 75 myotubes in total for each treatment) and the mean feret of clusters (one-way ANOVA with Tukey’s multiple comparison test, mean ± S.E.M., *p < 0.05, **p < 0.01, n ≥ 100 from four different experiments) were quantified using ImageJ. Note that AChR clusters are significantly bigger in cells expressing MuSK-S751D after agrin stimulation and after 6 h of agrin withdrawal. (C) Graph showing the percentage of microclusters per myotube. MuSK-S751D expressing cells form more microclusters compared to cells expressing MuSK-S751 A (p = 0.0270) and MuSK-WT (p = 0.0382). Values are presented as the mean ± S.E.M. (one-way ANOVA with Tukey’s multiple comparison test, n = 3 with > 100 myotubes in total for each cell line). WD, withdrawal; o/n, overnight; WT, wild-type.
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f6: A MuSK S751 phosphomimetic mutant increases the size of AChR clusters but does not increase the number of AChRs.(A) MuSK−/− muscle cells expressing MuSK wild-type and S751 mutant proteins were differentiated and stimulated with agrin for 16 h (overnight) to induce AChR cluster formation. To measure AChR cluster stability, agrin was removed and the cells were maintained in agrin-free medium for indicated times. Representative images of clusters stained with Alexa 594-conjugated α-BGT are shown. Scale bar, 10 μm. (B) The number of AChR clusters normalized to myotube area (Kruskal-Wallis with Dunn’s multiple comparison test, mean ± S.E.M., n = 4 with > 75 myotubes in total for each treatment) and the mean feret of clusters (one-way ANOVA with Tukey’s multiple comparison test, mean ± S.E.M., *p < 0.05, **p < 0.01, n ≥ 100 from four different experiments) were quantified using ImageJ. Note that AChR clusters are significantly bigger in cells expressing MuSK-S751D after agrin stimulation and after 6 h of agrin withdrawal. (C) Graph showing the percentage of microclusters per myotube. MuSK-S751D expressing cells form more microclusters compared to cells expressing MuSK-S751 A (p = 0.0270) and MuSK-WT (p = 0.0382). Values are presented as the mean ± S.E.M. (one-way ANOVA with Tukey’s multiple comparison test, n = 3 with > 100 myotubes in total for each cell line). WD, withdrawal; o/n, overnight; WT, wild-type.
Mentions: The hallmark of MuSK signalling is the clustering of AChRs to discrete patches on the muscle membrane. Changes in MuSK kinase activity or alterations in the downstream signalling pathway greatly influence AChR clustering11131416. Defects in AChR clustering usually result in increased or decreased number of clusters or increased or decreased size of clusters. We stimulated myotubes expressing MuSK wild-type, S751A or S751D with neural agrin A4B8 overnight followed by staining of AChRs with α-BGT. Figure 6A shows that AChR clusters were formed in all cell lines. To examine AChR cluster stability we removed agrin and cultured the cells for an additional 6 or 12 hours in agrin-free medium. Again we stained AChR clusters with α-BGT. AChR clusters decreased in number and size upon agrin withdrawal (Fig. 6A). As control we treated myotubes overnight with muscle agrin A0B0, which does not activate MuSK and therefore fails to induce AChR clusters1321. Number and size of AChR clusters were subsequently analysed (Fig. 6B). Mutation of S751 either to alanine or aspartate did not affect the ability of myotubes to form AChR clusters. The number of clusters was similar in all cell lines after overnight agrin stimulation as well as after agrin withdrawal. However, when we measured AChR cluster size we found that myotubes expressing MuSK S751D formed bigger clusters upon agrin treatment compared to myotubes expressing MuSK wild-type or S751A. Moreover, clusters in MuSK S751D expressing myotubes remained big after 6 hours agrin removal compared to MuSK wild-type and MuSK S751A expressing cells. In addition, more MuSK S751D expressing myotubes formed microclusters compared to myotubes expressing MuSK wild-type or MuSK S751A (Fig. 6C). These data suggest that the increased basal kinase activity of MuSK S751D positively modulates AChR clustering.

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