RESUMEN
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. CONCLUSION: 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.
Asunto(s)
Péptidos y Proteínas de Señalización Intracelular/metabolismo , Unión Neuromuscular/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Agregación de Receptores/fisiología , Receptores Colinérgicos/metabolismo , Membranas Sinápticas/metabolismo , Familia-src Quinasas/metabolismo , Acetilcolina/metabolismo , Agrina/metabolismo , Agrina/farmacología , Animales , Células COS , Chlorocebus aethiops , Regulación hacia Abajo/fisiología , Inhibidores Enzimáticos/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Péptidos y Proteínas de Señalización Intracelular/genética , Ratones , Ratones Noqueados , Fibras Musculares Esqueléticas/metabolismo , Fibras Musculares Esqueléticas/ultraestructura , Músculo Esquelético/inervación , Músculo Esquelético/metabolismo , Técnicas de Cultivo de Órganos , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Proteínas Tirosina Fosfatasas/antagonistas & inhibidores , Proteínas Tirosina Fosfatasas/genética , Interferencia de ARN , Agregación de Receptores/efectos de los fármacos , Proteínas Tirosina Quinasas Receptoras/metabolismo , Transmisión Sináptica/fisiología , Familia-src Quinasas/antagonistas & inhibidoresRESUMEN
Agrin triggers signaling mechanisms of high temporal and spatial specificity to achieve phosphorylation, clustering, and stabilization of postsynaptic acetylcholine receptors (AChRs). Agrin transiently activates the kinase MuSK; MuSK activation has largely vanished when AChR clusters appear. Thus, a tyrosine kinase cascade acts downstream from MuSK, as illustrated by the agrin-evoked long-lasting activation of Src family kinases (SFKs) and their requirement for AChR cluster stabilization. We have investigated this cascade and report that pharmacological inhibition of SFKs reduces early but not later agrin-induced phosphorylation of MuSK and AChRs, while inhibition of Abl kinases reduces late phosphorylation. Interestingly, SFK inhibition applied selectively during agrin-induced AChR cluster formation caused rapid cluster dispersal later upon agrin withdrawal. We also report that a single 5-min agrin pulse, followed by extensive washing, triggered long-lasting MuSK and AChR phosphorylation and efficient AChR clustering. Following the pulse, MuSK phosphorylation increased and, beyond a certain level, caused maximal clustering. These data reveal novel temporal aspects of tyrosine kinase action in agrin signaling. First, during AChR cluster formation, SFKs initiate early phosphorylation and an AChR stabilization program that acts much later. Second, a kinase mechanism rapidly activated by agrin acts thereafter autonomously in agrin's absence to further increase MuSK phosphorylation and cluster AChRs.