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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.


Phosphorylated S751 is present at NMJs.Frozen sections of M. gastrocnemius were stained with MuSK specific antibodies (green) and with Alexa 594-conjugated α-BGT (red) to label AChRs. MuSK-EC recognizes an epitope of the MuSK extracellular domain, pY754/755 recognizes an epitope carrying phosphorylated Y754 and Y755 and pS751 is directed against an epitope containing phosphorylated S751. Note that S751 phosphorylation co-localizes with AChRs. Images were obtained by confocal microscopy and representative images are shown. Scale bar, 10 μm. EC, extracellular.
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f2: Phosphorylated S751 is present at NMJs.Frozen sections of M. gastrocnemius were stained with MuSK specific antibodies (green) and with Alexa 594-conjugated α-BGT (red) to label AChRs. MuSK-EC recognizes an epitope of the MuSK extracellular domain, pY754/755 recognizes an epitope carrying phosphorylated Y754 and Y755 and pS751 is directed against an epitope containing phosphorylated S751. Note that S751 phosphorylation co-localizes with AChRs. Images were obtained by confocal microscopy and representative images are shown. Scale bar, 10 μm. EC, extracellular.

Mentions: Next we asked whether S751 is also phosphorylated in MuSK in vivo. We stained muscle sections with antibodies against pS751. Parallel sections were stained with antibodies against MuSK or antibodies against pY754/Y755 of the MuSK activation loop. All samples were co-stained with α-BGT to label AChRs. Figure 2 shows that antibodies against pS751 specifically marked the postsynaptic region, which was identified as AChR-positive region. Antibodies against MuSK or pY754/Y755 presented a similar staining pattern as anti-pS751 antibodies. Pre-incubation of anti-pS751 antibodies with the peptide representing the pS751 epitope abolished the staining of NMJs, whereas pre-incubation with non-phoshorylated epitope did not interfere with antibody binding (Supplementary Fig. S2). In addition, localization and signal of pS751 was similar in different muscles (sternomastoid, gastrocnemius and intercostal, data not shown). We therefore conclude that S751 is phosphorylated in vivo and that MuSK carrying pS751 is present at NMJs.


Musk Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop
Phosphorylated S751 is present at NMJs.Frozen sections of M. gastrocnemius were stained with MuSK specific antibodies (green) and with Alexa 594-conjugated α-BGT (red) to label AChRs. MuSK-EC recognizes an epitope of the MuSK extracellular domain, pY754/755 recognizes an epitope carrying phosphorylated Y754 and Y755 and pS751 is directed against an epitope containing phosphorylated S751. Note that S751 phosphorylation co-localizes with AChRs. Images were obtained by confocal microscopy and representative images are shown. Scale bar, 10 μm. EC, extracellular.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5035991&req=5

f2: Phosphorylated S751 is present at NMJs.Frozen sections of M. gastrocnemius were stained with MuSK specific antibodies (green) and with Alexa 594-conjugated α-BGT (red) to label AChRs. MuSK-EC recognizes an epitope of the MuSK extracellular domain, pY754/755 recognizes an epitope carrying phosphorylated Y754 and Y755 and pS751 is directed against an epitope containing phosphorylated S751. Note that S751 phosphorylation co-localizes with AChRs. Images were obtained by confocal microscopy and representative images are shown. Scale bar, 10 μm. EC, extracellular.
Mentions: Next we asked whether S751 is also phosphorylated in MuSK in vivo. We stained muscle sections with antibodies against pS751. Parallel sections were stained with antibodies against MuSK or antibodies against pY754/Y755 of the MuSK activation loop. All samples were co-stained with α-BGT to label AChRs. Figure 2 shows that antibodies against pS751 specifically marked the postsynaptic region, which was identified as AChR-positive region. Antibodies against MuSK or pY754/Y755 presented a similar staining pattern as anti-pS751 antibodies. Pre-incubation of anti-pS751 antibodies with the peptide representing the pS751 epitope abolished the staining of NMJs, whereas pre-incubation with non-phoshorylated epitope did not interfere with antibody binding (Supplementary Fig. S2). In addition, localization and signal of pS751 was similar in different muscles (sternomastoid, gastrocnemius and intercostal, data not shown). We therefore conclude that S751 is phosphorylated in vivo and that MuSK carrying pS751 is present at NMJs.

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.