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-sensitive 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 permissive temperature leads within minutes to the formation of F-actin-containing bodies (ABs), that originate in the ventral region of the myotubes 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 contain muscle-specific protein isoforms associated with the I-Z-I complexes and vinculin, a component of the cytoskeletal network. Anti-phosphotyrosine 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 phosphotyrosine 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 detectable effect on tyrosine phosphorylation. Taken together these findings indicate that phosphorylation of specific target proteins by v-Src, although necessary, is not sufficient per se to induce AB formation. In addition, the signal transduction cascade that culminates in MAP kinase activation and its nuclear translocation is activated both by v-Src and phorbol ester, and is relatively unaffected by 2-aminopurine. These findings imply that both phorbol esters and 2-aminopurine operate, at least in part, at the level of alternative pathways that may diverge upstream of the MAP kinase and are presumably mediating the early effects of v-Src on the differentiated phenotype.
Castellani
L
L
Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Università di Roma Tor Vergata, Italy.
Reedy
M C
MC
Gauzzi
M C
MC
Provenzano
C
C
Alemà
S
S
Falcone
G
G
D000199:Actins / Q000378:metabolism; D000818:Animals; D001358:Avian Sarcoma Viruses / Q000235:genetics; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism; D002451:Cell Compartmentation; D002454:Cell Differentiation; D002478:Cells, Cultured; D003370:Coturnix; D004789:Enzyme Activation; D005455:Fluorescent Antibody Technique; D006706:Homeostasis; D008840:Microfilament Proteins / Q000378:metabolism; D008854:Microscopy, Electron; D024510:Muscle Development; D009124:Muscle Proteins / Q000378:metabolism*; D018482:Muscle, Skeletal / Q000254:growth & development / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D015688:Oncogene Protein pp60(v-src) / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D010750:Phosphoproteins / Q000378:metabolism; D011493:Protein Kinase C / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000096:biosynthesis; D012518:Sarcomeres / Q000378:metabolism* / Q000473:pathology / Q000648:ultrastructure; D013997:Time Factors; D014170:Transformation, Genetic
D000199:Actins; D008840:Microfilament Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; D011994:Recombinant Proteins; D011505:Protein-Tyrosine Kinases; D015688:Oncogene Protein pp60(v-src); D011493:Protein Kinase C; D017871:Calcium-Calmodulin-Dependent Protein Kinases