Muscle-regulated expression and determinants for neuromuscular junctional localization of the mouse RIα regulatory subunit of cAMP-dependent protein kinase

In skeletal muscle, transcription of the gene encoding the mouse type I alpha (RI alpha) subunit of the cAMP-dependent protein kinase is initiated from the alternative noncoding first exons la and Ib, Here, we report that activity of the promoter upstream of exon la (Pa) depends on two adjacent E boxes (El and E2) in NIH 3T3-transfected fibroblasts as well as in intact muscle. Both basal activity and MyoD transactivation of the Pa promoter require binding of the upstream stimulating factors (UsF) to El. E2 binds either an unknown protein in a USF/E1 complex-dependent manner or MyoD, Both E2-bound proteins seem to function as repressors. but with different strengths, of the UsF transactivation potential. Previous work has shown localization of the RI alpha protein at the neuromuscular junction. Using DNA injection into muscle of plasmids encoding segments of RI alpha or RII alpha fused to green fluorescent protein. we demonstrate that anchoring at the neuromuscular junction is specific to RI alpha subunits and requires the amino-terminal residues 1-81, Mutagenesis of Phe-54 to Ala in the full-length RI alpha -green fluorescent protein template abolishes localization, indicating that dimerization of RI alpha is essential for anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27, are crucial for localization of RI alpha at the neuromuscular junction. These amino acids are involved in the interaction of the Caenorhabditis elegans type I alpha homologue R-CE with AKAP(CE) and for in vitro binding of RI alpha to dual A-kinase anchoring protein 1, We also show enrichment of dual A-kinase anchoring protein 1 at the neuromuscular junction, suggesting that it could be responsible for RI alpha tethering at this site.