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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

S751 does not regulate autoactivation of MuSK in heterologous cells and phosphorylation of S751 is dependent on MuSK kinase activity.(A) HEK 293T cells were transiently transfected with wild-type and mutant MuSK constructs resulting in autoactivation of MuSK kinase. Cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK antibodies and immunoblotted with anti-phospho-tyrosine (pY) and anti-MuSK antibodies, respectively. Wild-type MuSK (WT) is strongly tyrosine phosphorylated. Kinase-dead MuSK (KD) lacks tyrosine phosphorylation whereas phosphorylation of kinase-active MuSK (KA) is further increased compared to wild-type. S751 mutants are similarly autoactivated as MuSK wild-type. Values are presented as the mean ± S.E.M. (Kruskal-Wallis with Dunn’s multiple comparison test, n = 5). (B) HEK 293T cells were transiently transfected with wild-type and mutant MuSK constructs resulting in autoactivation of MuSK kinase. Cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK antibodies and immunoblotted using antibodies against MuSK pY553, MuSK pY754/755 and MuSK, respectively. Total lysates were analysed with antibodies against MuSK and Actin, respectively. Phosphorylation of juxtamembrane Y553 and Y754, Y755 of the kinase loop occur at similar level in S751 mutant MuSK compared to MuSK-wild-type. Values are presented as the mean ± S.E.M. (Kruskal-Wallis with Dunn’s multiple comparison test, n = 5). (C) Muscle cells expressing MuSK kinase mutants were stimulated with agrin (+, A4B8; −, A0B0) and cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK or anti-Myc antibodies. Samples were analysed by immunoblotting using antibodies against phospho-tyrosine (PY), MuSK pS751 and MuSK, respectively. Kinase-dead MuSK (KD), MuSK Y750, 754, 755F (Y750F) and MuSK Y553F are unresponsive to agrin. Wild-type MuSK (WT) is phosphorylated in response to agrin and kinase-active MuSK (KA-2) is highly phosphorylated in the presence and absence of agrin. Note that phosphorylation of S751 depends on MuSK tyrosine phosphorylation. (D) HEK 293T were transiently transfected with wild-type and mutant MuSK constructs resulting in autoactivation of MuSK kinase. Cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK antibodies and immunoblotted with anti-MuSK pS751 and anti-MuSK antibodies, respectively. Phosphorylation of S751 is only detected upon autoactivation of the MuSK kinase. Full-length blots are presented in Supplementary Fig. S9.
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f4: S751 does not regulate autoactivation of MuSK in heterologous cells and phosphorylation of S751 is dependent on MuSK kinase activity.(A) HEK 293T cells were transiently transfected with wild-type and mutant MuSK constructs resulting in autoactivation of MuSK kinase. Cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK antibodies and immunoblotted with anti-phospho-tyrosine (pY) and anti-MuSK antibodies, respectively. Wild-type MuSK (WT) is strongly tyrosine phosphorylated. Kinase-dead MuSK (KD) lacks tyrosine phosphorylation whereas phosphorylation of kinase-active MuSK (KA) is further increased compared to wild-type. S751 mutants are similarly autoactivated as MuSK wild-type. Values are presented as the mean ± S.E.M. (Kruskal-Wallis with Dunn’s multiple comparison test, n = 5). (B) HEK 293T cells were transiently transfected with wild-type and mutant MuSK constructs resulting in autoactivation of MuSK kinase. Cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK antibodies and immunoblotted using antibodies against MuSK pY553, MuSK pY754/755 and MuSK, respectively. Total lysates were analysed with antibodies against MuSK and Actin, respectively. Phosphorylation of juxtamembrane Y553 and Y754, Y755 of the kinase loop occur at similar level in S751 mutant MuSK compared to MuSK-wild-type. Values are presented as the mean ± S.E.M. (Kruskal-Wallis with Dunn’s multiple comparison test, n = 5). (C) Muscle cells expressing MuSK kinase mutants were stimulated with agrin (+, A4B8; −, A0B0) and cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK or anti-Myc antibodies. Samples were analysed by immunoblotting using antibodies against phospho-tyrosine (PY), MuSK pS751 and MuSK, respectively. Kinase-dead MuSK (KD), MuSK Y750, 754, 755F (Y750F) and MuSK Y553F are unresponsive to agrin. Wild-type MuSK (WT) is phosphorylated in response to agrin and kinase-active MuSK (KA-2) is highly phosphorylated in the presence and absence of agrin. Note that phosphorylation of S751 depends on MuSK tyrosine phosphorylation. (D) HEK 293T were transiently transfected with wild-type and mutant MuSK constructs resulting in autoactivation of MuSK kinase. Cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK antibodies and immunoblotted with anti-MuSK pS751 and anti-MuSK antibodies, respectively. Phosphorylation of S751 is only detected upon autoactivation of the MuSK kinase. Full-length blots are presented in Supplementary Fig. S9.

Mentions: Next, we investigated the autoactivation of MuSK in heterologous cells by expressing wild-type and mutant MuSK in HEK 293T cells. Overexpression of MuSK induces dimerization of MuSK and its autophosphorylation. Figure 4A shows that MuSK wild-type was robustly tyrosine phosphorylated. Autophosphorylation was abolished in mutant MuSK protein that contained a K608A substitution in the autocatalytic loop161114. In contrast, autophosphorylation was further increased in a kinase-active mutant that carried the mutations LS746/747MT17. Autophoshorylation was not significantly altered in MuSK S751A and MuSK S751D compared to wild-type MuSK. Similarly, when we used phospho-specific antibodies against pY553 and pY754/755 we observed no significant change in phosphorylation in MuSK S751A and MuSK S751D compared to wild-type MuSK (Fig. 4B). Control experiments demonstrated that the expression level of the different constructs was similar (Supplementary Fig. S4). These results suggest that S751 modulates basal agrin-independent MuSK phosphorylation in muscle cells and subsequently promotes early AChR phosphorylation without affecting MuSK autoactivation.


Musk Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop
S751 does not regulate autoactivation of MuSK in heterologous cells and phosphorylation of S751 is dependent on MuSK kinase activity.(A) HEK 293T cells were transiently transfected with wild-type and mutant MuSK constructs resulting in autoactivation of MuSK kinase. Cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK antibodies and immunoblotted with anti-phospho-tyrosine (pY) and anti-MuSK antibodies, respectively. Wild-type MuSK (WT) is strongly tyrosine phosphorylated. Kinase-dead MuSK (KD) lacks tyrosine phosphorylation whereas phosphorylation of kinase-active MuSK (KA) is further increased compared to wild-type. S751 mutants are similarly autoactivated as MuSK wild-type. Values are presented as the mean ± S.E.M. (Kruskal-Wallis with Dunn’s multiple comparison test, n = 5). (B) HEK 293T cells were transiently transfected with wild-type and mutant MuSK constructs resulting in autoactivation of MuSK kinase. Cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK antibodies and immunoblotted using antibodies against MuSK pY553, MuSK pY754/755 and MuSK, respectively. Total lysates were analysed with antibodies against MuSK and Actin, respectively. Phosphorylation of juxtamembrane Y553 and Y754, Y755 of the kinase loop occur at similar level in S751 mutant MuSK compared to MuSK-wild-type. Values are presented as the mean ± S.E.M. (Kruskal-Wallis with Dunn’s multiple comparison test, n = 5). (C) Muscle cells expressing MuSK kinase mutants were stimulated with agrin (+, A4B8; −, A0B0) and cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK or anti-Myc antibodies. Samples were analysed by immunoblotting using antibodies against phospho-tyrosine (PY), MuSK pS751 and MuSK, respectively. Kinase-dead MuSK (KD), MuSK Y750, 754, 755F (Y750F) and MuSK Y553F are unresponsive to agrin. Wild-type MuSK (WT) is phosphorylated in response to agrin and kinase-active MuSK (KA-2) is highly phosphorylated in the presence and absence of agrin. Note that phosphorylation of S751 depends on MuSK tyrosine phosphorylation. (D) HEK 293T were transiently transfected with wild-type and mutant MuSK constructs resulting in autoactivation of MuSK kinase. Cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK antibodies and immunoblotted with anti-MuSK pS751 and anti-MuSK antibodies, respectively. Phosphorylation of S751 is only detected upon autoactivation of the MuSK kinase. Full-length blots are presented in Supplementary Fig. S9.
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f4: S751 does not regulate autoactivation of MuSK in heterologous cells and phosphorylation of S751 is dependent on MuSK kinase activity.(A) HEK 293T cells were transiently transfected with wild-type and mutant MuSK constructs resulting in autoactivation of MuSK kinase. Cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK antibodies and immunoblotted with anti-phospho-tyrosine (pY) and anti-MuSK antibodies, respectively. Wild-type MuSK (WT) is strongly tyrosine phosphorylated. Kinase-dead MuSK (KD) lacks tyrosine phosphorylation whereas phosphorylation of kinase-active MuSK (KA) is further increased compared to wild-type. S751 mutants are similarly autoactivated as MuSK wild-type. Values are presented as the mean ± S.E.M. (Kruskal-Wallis with Dunn’s multiple comparison test, n = 5). (B) HEK 293T cells were transiently transfected with wild-type and mutant MuSK constructs resulting in autoactivation of MuSK kinase. Cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK antibodies and immunoblotted using antibodies against MuSK pY553, MuSK pY754/755 and MuSK, respectively. Total lysates were analysed with antibodies against MuSK and Actin, respectively. Phosphorylation of juxtamembrane Y553 and Y754, Y755 of the kinase loop occur at similar level in S751 mutant MuSK compared to MuSK-wild-type. Values are presented as the mean ± S.E.M. (Kruskal-Wallis with Dunn’s multiple comparison test, n = 5). (C) Muscle cells expressing MuSK kinase mutants were stimulated with agrin (+, A4B8; −, A0B0) and cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK or anti-Myc antibodies. Samples were analysed by immunoblotting using antibodies against phospho-tyrosine (PY), MuSK pS751 and MuSK, respectively. Kinase-dead MuSK (KD), MuSK Y750, 754, 755F (Y750F) and MuSK Y553F are unresponsive to agrin. Wild-type MuSK (WT) is phosphorylated in response to agrin and kinase-active MuSK (KA-2) is highly phosphorylated in the presence and absence of agrin. Note that phosphorylation of S751 depends on MuSK tyrosine phosphorylation. (D) HEK 293T were transiently transfected with wild-type and mutant MuSK constructs resulting in autoactivation of MuSK kinase. Cell lysates were subjected to immunoprecipitation (IP) using anti-MuSK antibodies and immunoblotted with anti-MuSK pS751 and anti-MuSK antibodies, respectively. Phosphorylation of S751 is only detected upon autoactivation of the MuSK kinase. Full-length blots are presented in Supplementary Fig. S9.
Mentions: Next, we investigated the autoactivation of MuSK in heterologous cells by expressing wild-type and mutant MuSK in HEK 293T cells. Overexpression of MuSK induces dimerization of MuSK and its autophosphorylation. Figure 4A shows that MuSK wild-type was robustly tyrosine phosphorylated. Autophosphorylation was abolished in mutant MuSK protein that contained a K608A substitution in the autocatalytic loop161114. In contrast, autophosphorylation was further increased in a kinase-active mutant that carried the mutations LS746/747MT17. Autophoshorylation was not significantly altered in MuSK S751A and MuSK S751D compared to wild-type MuSK. Similarly, when we used phospho-specific antibodies against pY553 and pY754/755 we observed no significant change in phosphorylation in MuSK S751A and MuSK S751D compared to wild-type MuSK (Fig. 4B). Control experiments demonstrated that the expression level of the different constructs was similar (Supplementary Fig. S4). These results suggest that S751 modulates basal agrin-independent MuSK phosphorylation in muscle cells and subsequently promotes early AChR phosphorylation without affecting MuSK autoactivation.

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