MAINTENANCE OF THE DIFFERENTIATED STATE IN SKELETAL-MUSCLE - ACTIVATION OF V-SRC DISRUPTS SARCOMERES IN QUAIL MYOTUBES

We have used quail skeletal myotubes expressing a temperature-sensitiv e allele of the v-src oncogene to address the issue of the homeostasis of sarcomeric myofibrils in differentiated muscle cells. Reactivation of the v-Src tyrosine kinase by shifting the cultures to the permissi ve temperature leads within minutes to the formation of F-actin-contai ning bodies (ABs), that originate in the ventral region of the myotube s and increase in number concomitantly with the dismantling of the I-Z -I complex of the sarcomeres. This process is detailed by confocal and electron microscopy. Indirect immunofluorescence reveals that ABs con tain muscle-specific protein isoforms associated with the I-Z-I comple xes and vinculin, a component of the cytoskeletal network. Anti-phosph o tyro sine antibodies label proteins in ABs and Z-discs. Evidence is presented indicating that this phenomenon specifically depends on the persistent activation of v-Src, rather than on a general increase in p hosphotyrosine content such as that induced by vanadate. AB formation is prevented by activation of protein kinase C by phorbol ester or by treatment with the kinase inhibitor 2-aminopurine, without any detecta ble effect on tyrosine phosphorylation. Taken together these findings indicate that phosphorylation of specific target proteins by v-Src, al though necessary, is not sufficient per se to induce AB formation. In addition, the signal transduction cascade that culminates in MAP kinas e activation and its nuclear translocation is activated both by v-Src and phorbol ester, and is relatively unaffected by 2-aminopurine. Thes e findings imply that both phorbol esters and 2-aminopurine operate, a t least in part, at the level of alternative pathways that may diverge upstream of the MAP kinase and are presumably mediating the early eff ects of v-Src on the differentiated phenotype.