Tyrosine phosphatases such as SHP-2 act in a balance with Src-family kinases in stabilization of postsynaptic clusters of acetylcholine receptors.
BACKGROUND
Development of neural networks requires that synapses are formed, eliminated and stabilized. At the neuromuscular junction (NMJ), agrin/MuSK signaling, by triggering downstream pathways, causes clustering and phosphorylation of postsynaptic acetylcholine receptors (AChRs). Postnatally, AChR aggregates are stabilized by molecular pathways that are poorly characterized. Gain or loss of function of Src-family kinases (SFKs) disassembles AChR clusters at adult NMJs in vivo, whereas AChR aggregates disperse rapidly upon withdrawal of agrin from cultured src-/-;fyn-/- myotubes. This suggests that a balance between protein tyrosine phosphatases (PTPs) and protein tyrosine kinases (PTKs) such as those of the Src-family may be essential in stabilizing clusters of AChRs.


RESULTS
We have analyzed the role of PTPs in maintenance of AChR aggregates, by adding and then withdrawing agrin from cultured myotubes in the presence of PTP or PTK inhibitors and quantitating remaining AChR clusters. In wild-type myotubes, blocking PTPs with pervanadate caused enhanced disassembly of AChR clusters after agrin withdrawal. When added at the time of agrin withdrawal, SFK inhibitors destabilized AChR aggregates but concomitant addition of pervanadate rescued cluster stability. Likewise in src-/-;fyn-/- myotubes, in which agrin-induced AChR clusters form normally but rapidly disintegrate after agrin withdrawal, pervanadate addition stabilized AChR clusters. The PTP SHP-2, known to be enriched at the NMJ, associated and colocalized with MuSK, and agrin increased this interaction. Specific SHP-2 knockdown by RNA interference reduced the stability of AChR clusters in wild-type myotubes. Similarly, knockdown of SHP-2 in adult mouse soleus muscle by electroporation of RNA interference constructs caused disassembly of pretzel-shaped AChR-rich areas in vivo. Finally, we found that src-/-;fyn-/- myotubes contained elevated levels of SHP-2 protein.


CONCLUSIONS
Our data are the first to show that the fine balance between PTPs and SFKs is a key aspect in stabilization of postsynaptic AChR clusters. One phosphatase that acts in this equilibrium is SHP-2. Thus, PTPs such as SHP-2 stabilize AChR clusters under normal circumstances, but when these PTPs are not balanced by SFKs, they render clusters unstable.
Camilleri
Alain A
AA
Brain Research Institute, University of Zurich, Zurich, Switzerland. camilleri@hifo.uzh.ch <camilleri@hifo.uzh.ch>
Willmann
Raffaella
R
Sadasivam
Gayathri
G
Lin
Shuo
S
Rüegg
Markus A
MA
Gesemann
Matthias
M
Fuhrer
Christian
C
D000109:Acetylcholine / Q000378:metabolism; D018171:Agrin / Q000378:metabolism / Q000494:pharmacology; D000818:Animals; D019556:COS Cells; D002522:Chlorocebus aethiops; D015536:Down-Regulation / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D018485:Muscle Fibers, Skeletal / Q000378:metabolism / Q000648:ultrastructure; D018482:Muscle, Skeletal / Q000294:innervation / Q000378:metabolism; D009469:Neuromuscular Junction / Q000378:metabolism*; D009924:Organ Culture Techniques; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D034622:RNA Interference; D011940:Receptor Aggregation / Q000187:drug effects / Q000502:physiology*; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism; D011950:Receptors, Cholinergic / Q000378:metabolism*; D013570:Synaptic Membranes / Q000378:metabolism*; D009435:Synaptic Transmission / Q000502:physiology; D019061:src-Family Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*
D018171:Agrin; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; D011950:Receptors, Cholinergic; C511695:MuSK protein, mouse; D020794:Receptor Protein-Tyrosine Kinases; D019061:src-Family Kinases; D054592:Protein Tyrosine Phosphatase, Non-Receptor Type 11; D017027:Protein Tyrosine Phosphatases; C516725:Ptpn11 protein, mouse; D000109:Acetylcholine