In fetal liver, bipotential hepatoblasts differentiate into hepatocytes and bile duct cells (cholangiocytes).

Abstract. Using probes specific for cAMP-dependent protein kinase, we have analyzed by in situ hybridization the patterns of expression of regulatory and catalytic subunits in mouse embryos and in adult muscle. RI~ transcripts are distributed in muscle fibers exactly as acetylcholinesterase, showing that this RNA is localized at the neuromuscular junction. The transcript levels increase upon denervation of the muscle, but the RNA remains localized, indicating a regulation pattern similar to that of the ~ subunit of nicotinic acetylcholine receptor. RIa transcripts have accumulated in the muscle by day 12 of mouse embryogenesis, and localization is established by day 14, at about the time of formation of junctions. This localization is maintained throughout development and in the adult. Immunocytochemical analysis has demonstrated that RIa protein is also localized. In addition, RIa recruits Ca protein to the junction, providing at this site the potential for local responsiveness to cAMP. PKA could be implicated in the establishment and/or maintenance of the unique pattern of gene expression occurring at the junction, or in the modulation of synaptic activity via protein phosphorylation.Embryonic skeletal muscle shows a high level of Ca transcripts and protein throughout the fiber; the transcripts are already present by day 12 of embryogenesis, and their elevated level is maintained only through fetal life. In the adult, the Ca hybridization signal of muscle is weak and homogeneous.T r~E cAMP-dependent protein kinase (PKA) 1 is composed of regulatory and catalytic subunits. The subunits associate in tetrameric complexes of two regulatory and two catalytic subunits, the holoenzyme, which is cytoplasmic and inactive. Intracellular synthesis of cAMP results in the binding of this second messenger to the regulatory subunits and liberation of monomeric catalytic subunits, endowed with serine-threonine kinase activity, which can phosphorylate target proteins in the cytoplasm and the nucleus (Taylor et al., 1990;McKnight, 1991).The regulatory subunits belong to two different families, RI and RII, each of which has distinct properties concern-

Expression of the phenylalanine hydroxylase gene in livers and kidneys of rodents is activated at birth and is induced by glucocorticoids and cyclic AMP in the liver. Regulatory elements in a 10-kb fragment upstream of the mouse gene have been characterized. The promoter lacks TAATA and CCAAT consensus sequences and shows only extremely weak activity in transitory expression assays with phenylalanine hydroxylase-producing hepatoma cells. No key elements for regulation of promoter activity are localized within 2 kb of upstream sequences. However, a liver-specific DNase I-hypersensitive site at kb ؊3.5 comprises a tissue-specific and hormone-inducible enhancer. This enhancer contains multiple protein binding sites, including sites for ubiquitous factors (NF1 and AP1), the glucocorticoid receptor, and the hepatocyte-enriched transcription factors hepatocyte nuclear factor 1 (HNF1) and C/EBP. Mutation revealed that the last two sites are critical not only for basal activity but also for obtaining a maximal hormone response. Efficient transcription from the highly inducible promoter shows absolute dependence upon the enhancer at kb ؊3.5, which in turn requires HNF1 and C/EBP as well as hormones. The regulatory region of the mouse phenylalanine hydroxylase gene differs totally from that of humans, even though the genes of both species are expressed essentially in the liver. Furthermore, the phenylalanine hydroxylase gene of mice shows an expression pattern very similar to those of the rodent tyrosine aminotransferase and phosphoenolpyruvate carboxykinase genes, yet each shows a different organization of its regulatory region.

In skeletal muscle, transcription of the gene encoding the mouse type I␣ (RI␣) 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 USF͞E1 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␣ protein at the neuromuscular junction. Using DNA injection into muscle of plasmids encoding segments of RI␣ or RII␣ fused to green fluorescent protein, we demonstrate that anchoring at the neuromuscular junction is specific to RI␣ subunits and requires the amino-terminal residues 1-81. Mutagenesis of Phe-54 to Ala in the full-length RI␣-green fluorescent protein template abolishes localization, indicating that dimerization of RI␣ is essential for anchoring. Moreover, two other hydrophobic residues, Val-22 and Ile-27, are crucial for localization of RI␣ at the neuromuscular junction. These amino acids are involved in the interaction of the Caenorhabditis elegans type I␣ homologue RCE with AKAPCE and for in vitro binding of RI␣ 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␣ tethering at this site.S ubcellular localization is a crucial mechanism to achieve optimal activation and substrate specificity of the cAMPdependent protein kinase (PKA, EC 2.7.1.37). PKA type II targeting to subcellular structures and organelles or assembly in signaling complexes is a result of tethering of RII regulatory (R) subunits by members of the A-kinase anchoring protein (AKAP) family, a group of structurally divergent proteins possessing a conserved RII-binding site (1, 2).Although a large proportion of type I␣ R subunit (RI␣) is dispersed in the cytosol, it also is associated with the plasma membrane of human erythrocytes (3), recruited to the ''cap site'' of activated T lymphocytes (4) and sequestered along the fibrous sheath of mammalian spermatozoa (5). In addition, we have demonstrated previously the accumulation of RI␣ at the neuromuscular junction (NMJ) of skeletal muscle (6) and its association with microtubules (7). The high affinity RII-binding sites of certain AKAPs, named dual (D)-AKAPs, also sequester RI␣ in vitro but with a 25-500 lower affinity (8, 9). Thus, type I PKA also could be anchored in intact cells through specific AKAP-RI␣ interactions. In fact, Angelo and Rubin (10) have identified AKAP CE from Caenorhabditis elegans, which binds the R CE subunit. Because R CE is closely related to mammalian RI␣, AKAP CE is the first eukaryotic RI-specific teth...