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 myogeni c regulatory factors and acts in the transcriptional activation of mus cle-specific genes. We have investigated the role of cAMP-dependent pr otein kinase (A-kinase) in modulating the nuclear locale of MyoD. Puri fied MyoD protein microinjected into the cytoplasm of rat embryo fibro blasts is rapidly translocated into the nucleus. Inhibition of A-kinas e activity through injection of the specific inhibitory peptide PKI pr events this nuclear localisation. This inhibition of nuclear location is specifically reversed by injection of purified A-kinase catalytic s ubunit, showing the requirement for A-kinase in the nuclear import of MyoD. Site-directed mutagenesis of an the putative sites for A-kinase- dependent phosphorylation on MyoD, substituting serine or threonine re sidues 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 me diated by direct phosphorylation of MyoD and imply that A-kinase opera tes through phosphorylation of components involved in the nuclear tran sport of MyoD.