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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

Mutation of S751 does not alter MuSK surface expression and MuSK protein stability.(A) MuSK wild-type (WT), S751D, S751A constructs with a N-terminal HA-Tag and a C-terminal Myc-Tag were expressed in C2C12 muscle cells. Differentiated myotubes were stimulated with agrin for 30 min (+, A4B8; −, A0B0) and incubated with anti-HA antibodies to label surface MuSK. Cells were lysed and HA-bound MuSK protein was purified by immunoprecipitation (IP). Samples were analysed by immunoblotting using anti-Myc antibodies. Total lysates were used as loading controls and immunoblotted with antibodies against Myc and Tubulin, respectively. (B) Quantification of MuSK surface expression normalized to total MuSK protein is shown. Cell lines express similar levels of surface MuSK. MuSK wild-type and MuSK S751D show slightly increased MuSK surface expression upon agrin treatment compared to MuSK S751A. Values are presented as the mean ± S.E.M. (one-way ANOVA with Tukey’s multiple comparison test, n = 4) (C) C2C12 myotubes expressing MuSK wild-type (WT), S751D or S751A with a N-terminal HA-Tag and a C-terminal Myc-Tag were incubated with cycloheximide (CHX) for different time periods. Cells were lysed and total lysates were analysed by immunoblotting using antibodies against anti-Myc and anti-Tubulin antibodies, respectively. Note that MuSK expression is highly decreased after 6 h of cycloheximide incubation whereas Tubulin is stable. (D) Quantification of MuSK expression upon cycloheximide treatment is shown (normalized against Tubulin). MuSK-S751A is significantly faster degraded within the first hour of cycloheximide treatment compared to MuSK–WT (p = 0.044) and MuSK-S751D (p = 0.017). In contrast, expression level is similar for all tested MuSK proteins at later time points. Values are presented as the mean ± S.E.M. (two-way ANOVA with Tukey’s multiple comparison test, n = 3 in duplicates). Full-length blots are presented in Supplementary Fig. S10.
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f5: Mutation of S751 does not alter MuSK surface expression and MuSK protein stability.(A) MuSK wild-type (WT), S751D, S751A constructs with a N-terminal HA-Tag and a C-terminal Myc-Tag were expressed in C2C12 muscle cells. Differentiated myotubes were stimulated with agrin for 30 min (+, A4B8; −, A0B0) and incubated with anti-HA antibodies to label surface MuSK. Cells were lysed and HA-bound MuSK protein was purified by immunoprecipitation (IP). Samples were analysed by immunoblotting using anti-Myc antibodies. Total lysates were used as loading controls and immunoblotted with antibodies against Myc and Tubulin, respectively. (B) Quantification of MuSK surface expression normalized to total MuSK protein is shown. Cell lines express similar levels of surface MuSK. MuSK wild-type and MuSK S751D show slightly increased MuSK surface expression upon agrin treatment compared to MuSK S751A. Values are presented as the mean ± S.E.M. (one-way ANOVA with Tukey’s multiple comparison test, n = 4) (C) C2C12 myotubes expressing MuSK wild-type (WT), S751D or S751A with a N-terminal HA-Tag and a C-terminal Myc-Tag were incubated with cycloheximide (CHX) for different time periods. Cells were lysed and total lysates were analysed by immunoblotting using antibodies against anti-Myc and anti-Tubulin antibodies, respectively. Note that MuSK expression is highly decreased after 6 h of cycloheximide incubation whereas Tubulin is stable. (D) Quantification of MuSK expression upon cycloheximide treatment is shown (normalized against Tubulin). MuSK-S751A is significantly faster degraded within the first hour of cycloheximide treatment compared to MuSK–WT (p = 0.044) and MuSK-S751D (p = 0.017). In contrast, expression level is similar for all tested MuSK proteins at later time points. Values are presented as the mean ± S.E.M. (two-way ANOVA with Tukey’s multiple comparison test, n = 3 in duplicates). Full-length blots are presented in Supplementary Fig. S10.

Mentions: Serine phosphorylation of RTKs not only regulates downstream signalling but also endocytosis and internalization of RTKs22232425. To examine whether S751 phosphorylation influences MuSK surface expression we expressed MuSK wild-type, MuSK S751A and MuSK S751D with a N-terminal HA-Tag in C2C12 muscle cells. Surface MuSK was isolated from myotubes untreated or treated with agrin via the HA-Tag. Subsequent analysis of protein expression by immunoblotting revealed that similar amounts of MuSK wild-type, MuSK S751A and MuSK S751D were present on the cell surface (Fig. 5A,B). This observation is further supported by determining MuSK surface expression using biotinylation of surface proteins (Supplementary Fig. S5). Establishing that mutations in S751 did not affect surface expression, we next tested whether MuSK dimerization is affected. Here we took advantage of the fact that our cell system expressed endogenous wild-type MuSK and exogenous MuSK either wild-type, S751A or S751D, which carry, in addition to the N-terminal HA-Tag, a C-terminal Myc-Tag. We used an antibody that recognizes only endogenous MuSK for immunoprecipitation. Samples were subsequently analysed by immunoblotting against endogenous MuSK using anti-MuSK antibodies and exogenous MuSK using antibodies against the Myc-Tag. Quantification showed that similar amounts of wild-type and mutant MuSK were isolated together with endogenous MuSK suggesting that MuSK dimerization is not affected by S751 (Supplementary Fig. S6).


Musk Kinase Activity is Modulated By A Serine Phosphorylation Site in The Kinase Loop
Mutation of S751 does not alter MuSK surface expression and MuSK protein stability.(A) MuSK wild-type (WT), S751D, S751A constructs with a N-terminal HA-Tag and a C-terminal Myc-Tag were expressed in C2C12 muscle cells. Differentiated myotubes were stimulated with agrin for 30 min (+, A4B8; −, A0B0) and incubated with anti-HA antibodies to label surface MuSK. Cells were lysed and HA-bound MuSK protein was purified by immunoprecipitation (IP). Samples were analysed by immunoblotting using anti-Myc antibodies. Total lysates were used as loading controls and immunoblotted with antibodies against Myc and Tubulin, respectively. (B) Quantification of MuSK surface expression normalized to total MuSK protein is shown. Cell lines express similar levels of surface MuSK. MuSK wild-type and MuSK S751D show slightly increased MuSK surface expression upon agrin treatment compared to MuSK S751A. Values are presented as the mean ± S.E.M. (one-way ANOVA with Tukey’s multiple comparison test, n = 4) (C) C2C12 myotubes expressing MuSK wild-type (WT), S751D or S751A with a N-terminal HA-Tag and a C-terminal Myc-Tag were incubated with cycloheximide (CHX) for different time periods. Cells were lysed and total lysates were analysed by immunoblotting using antibodies against anti-Myc and anti-Tubulin antibodies, respectively. Note that MuSK expression is highly decreased after 6 h of cycloheximide incubation whereas Tubulin is stable. (D) Quantification of MuSK expression upon cycloheximide treatment is shown (normalized against Tubulin). MuSK-S751A is significantly faster degraded within the first hour of cycloheximide treatment compared to MuSK–WT (p = 0.044) and MuSK-S751D (p = 0.017). In contrast, expression level is similar for all tested MuSK proteins at later time points. Values are presented as the mean ± S.E.M. (two-way ANOVA with Tukey’s multiple comparison test, n = 3 in duplicates). Full-length blots are presented in Supplementary Fig. S10.
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f5: Mutation of S751 does not alter MuSK surface expression and MuSK protein stability.(A) MuSK wild-type (WT), S751D, S751A constructs with a N-terminal HA-Tag and a C-terminal Myc-Tag were expressed in C2C12 muscle cells. Differentiated myotubes were stimulated with agrin for 30 min (+, A4B8; −, A0B0) and incubated with anti-HA antibodies to label surface MuSK. Cells were lysed and HA-bound MuSK protein was purified by immunoprecipitation (IP). Samples were analysed by immunoblotting using anti-Myc antibodies. Total lysates were used as loading controls and immunoblotted with antibodies against Myc and Tubulin, respectively. (B) Quantification of MuSK surface expression normalized to total MuSK protein is shown. Cell lines express similar levels of surface MuSK. MuSK wild-type and MuSK S751D show slightly increased MuSK surface expression upon agrin treatment compared to MuSK S751A. Values are presented as the mean ± S.E.M. (one-way ANOVA with Tukey’s multiple comparison test, n = 4) (C) C2C12 myotubes expressing MuSK wild-type (WT), S751D or S751A with a N-terminal HA-Tag and a C-terminal Myc-Tag were incubated with cycloheximide (CHX) for different time periods. Cells were lysed and total lysates were analysed by immunoblotting using antibodies against anti-Myc and anti-Tubulin antibodies, respectively. Note that MuSK expression is highly decreased after 6 h of cycloheximide incubation whereas Tubulin is stable. (D) Quantification of MuSK expression upon cycloheximide treatment is shown (normalized against Tubulin). MuSK-S751A is significantly faster degraded within the first hour of cycloheximide treatment compared to MuSK–WT (p = 0.044) and MuSK-S751D (p = 0.017). In contrast, expression level is similar for all tested MuSK proteins at later time points. Values are presented as the mean ± S.E.M. (two-way ANOVA with Tukey’s multiple comparison test, n = 3 in duplicates). Full-length blots are presented in Supplementary Fig. S10.
Mentions: Serine phosphorylation of RTKs not only regulates downstream signalling but also endocytosis and internalization of RTKs22232425. To examine whether S751 phosphorylation influences MuSK surface expression we expressed MuSK wild-type, MuSK S751A and MuSK S751D with a N-terminal HA-Tag in C2C12 muscle cells. Surface MuSK was isolated from myotubes untreated or treated with agrin via the HA-Tag. Subsequent analysis of protein expression by immunoblotting revealed that similar amounts of MuSK wild-type, MuSK S751A and MuSK S751D were present on the cell surface (Fig. 5A,B). This observation is further supported by determining MuSK surface expression using biotinylation of surface proteins (Supplementary Fig. S5). Establishing that mutations in S751 did not affect surface expression, we next tested whether MuSK dimerization is affected. Here we took advantage of the fact that our cell system expressed endogenous wild-type MuSK and exogenous MuSK either wild-type, S751A or S751D, which carry, in addition to the N-terminal HA-Tag, a C-terminal Myc-Tag. We used an antibody that recognizes only endogenous MuSK for immunoprecipitation. Samples were subsequently analysed by immunoblotting against endogenous MuSK using anti-MuSK antibodies and exogenous MuSK using antibodies against the Myc-Tag. Quantification showed that similar amounts of wild-type and mutant MuSK were isolated together with endogenous MuSK suggesting that MuSK dimerization is not affected by S751 (Supplementary Fig. S6).

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