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

Abstract

In skeletal muscle, transcription of the gene encoding the mouse type Ialpha (RIalpha) subunit of the cAMP-dependent protein kinase is initiated from the alternative noncoding first exons 1a and 1b. Here, we report that activity of the promoter upstream of exon 1a (Pa) depends on two adjacent E boxes (E1 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 E1. 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 RIalpha protein at the neuromuscular junction. Using DNA injection into muscle of plasmids encoding segments of RIalpha or RIIalpha fused to green fluorescent protein, we demonstrate that anchoring at the neuromuscular junction is specific to RIalpha subunits and requires the amino-terminal residues 1-81. Mutagenesis of Phe-54 to Ala in the full-length RIalpha-green fluorescent protein template abolishes localization, indicating that dimerization of RIalpha is essential for anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27, are crucial for localization of RIalpha at the neuromuscular junction. These amino acids are involved in the interaction of the Caenorhabditis elegans type Ialpha homologue R(CE) with AKAP(CE) and for in vitro binding of RIalpha 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 RIalpha tethering at this site.