Nuclear import of the myogenic factor MyoD requires cAMP-dependent protein kinase activity but not the direct phosphorylation of MyoD.
MyoD is a nuclear phosphoprotein that belongs to the family of myogenic regulatory factors and acts in the transcriptional activation of muscle-specific genes. We have investigated the role of cAMP-dependent protein kinase (A-kinase) in modulating the nuclear locale of MyoD. Purified MyoD protein microinjected into the cytoplasm of rat embryo fibroblasts is rapidly translocated into the nucleus. Inhibition of A-kinase activity through injection of the specific inhibitory peptide PKI prevents this nuclear localisation. This inhibition of nuclear location is specifically reversed by injection of purified A-kinase catalytic subunit, showing the requirement for A-kinase in the nuclear import of MyoD. Site-directed mutagenesis of all the putative sites for A-kinase-dependent phosphorylation on MyoD, substituting serine or threonine residues for the non-phosphorylatable amino acid alanine, had no effect on nuclear import of mutated MyoD. These data exclude the possibility that the effect of A-kinase on the nuclear translocation of MyoD is mediated by direct phosphorylation of MyoD and imply that A-kinase operates through phosphorylation of components involved in the nuclear transport of MyoD.
Vandromme
M
M
Cell Biology Unit, CRBM, CNRS-INSERM, Montpellier, France.
Carnac
G
G
Gauthier-Rouvière
C
C
Fesquet
D
D
Lamb
N
N
Fernandez
A
A
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D005455:Fluorescent Antibody Technique; D051379:Mice; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D017570:MyoD Protein / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D051381:Rats
D017931:DNA Primers; D017570:MyoD Protein; D017868:Cyclic AMP-Dependent Protein Kinases