Skip to main content
The Journal of Cell Biology logoLink to The Journal of Cell Biology
. 1996 Sep 1;134(5):1241–1254. doi: 10.1083/jcb.134.5.1241

Accumulation in fetal muscle and localization to the neuromuscular junction of cAMP-dependent protein kinase A regulatory and catalytic subunits RI alpha and C alpha

PMCID: PMC2120986  PMID: 8794865

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 alpha 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 epsilon subunit of nicotinic acetylcholine receptor. RI alpha 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 RI alpha protein is also localized. In addition, RI alpha recruits C alpha 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 C alpha 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 C alpha hybridization signal of muscle is weak and homogeneous.

Full Text

The Full Text of this article is available as a PDF (5.6 MB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Boshart M., Weih F., Nichols M., Schütz G. The tissue-specific extinguisher locus TSE1 encodes a regulatory subunit of cAMP-dependent protein kinase. Cell. 1991 Sep 6;66(5):849–859. doi: 10.1016/0092-8674(91)90432-x. [DOI] [PubMed] [Google Scholar]
  2. Brenner H. R., Witzemann V., Sakmann B. Imprinting of acetylcholine receptor messenger RNA accumulation in mammalian neuromuscular synapses. Nature. 1990 Apr 5;344(6266):544–547. doi: 10.1038/344544a0. [DOI] [PubMed] [Google Scholar]
  3. Cadd G., McKnight G. S. Distinct patterns of cAMP-dependent protein kinase gene expression in mouse brain. Neuron. 1989 Jul;3(1):71–79. doi: 10.1016/0896-6273(89)90116-5. [DOI] [PubMed] [Google Scholar]
  4. Chahine K. G., Baracchini E., Goldman D. Coupling muscle electrical activity to gene expression via a cAMP-dependent second messenger system. J Biol Chem. 1993 Feb 5;268(4):2893–2898. [PubMed] [Google Scholar]
  5. Duclert A., Changeux J. P. Acetylcholine receptor gene expression at the developing neuromuscular junction. Physiol Rev. 1995 Apr;75(2):339–368. doi: 10.1152/physrev.1995.75.2.339. [DOI] [PubMed] [Google Scholar]
  6. Duclert A., Savatier N., Changeux J. P. An 83-nucleotide promoter of the acetylcholine receptor epsilon-subunit gene confers preferential synaptic expression in mouse muscle. Proc Natl Acad Sci U S A. 1993 Apr 1;90(7):3043–3047. doi: 10.1073/pnas.90.7.3043. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Dürr I., Numberger M., Berberich C., Witzemann V. Characterization of the functional role of E-box elements for the transcriptional activity of rat acetylcholine receptor epsilon-subunit and gamma-subunit gene promoters in primary muscle cell cultures. Eur J Biochem. 1994 Sep 1;224(2):353–364. doi: 10.1111/j.1432-1033.1994.00353.x. [DOI] [PubMed] [Google Scholar]
  8. Fan C. M., Porter J. A., Chiang C., Chang D. T., Beachy P. A., Tessier-Lavigne M. Long-range sclerotome induction by sonic hedgehog: direct role of the amino-terminal cleavage product and modulation by the cyclic AMP signaling pathway. Cell. 1995 May 5;81(3):457–465. doi: 10.1016/0092-8674(95)90398-4. [DOI] [PubMed] [Google Scholar]
  9. Fontaine B., Changeux J. P. Localization of nicotinic acetylcholine receptor alpha-subunit transcripts during myogenesis and motor endplate development in the chick. J Cell Biol. 1989 Mar;108(3):1025–1037. doi: 10.1083/jcb.108.3.1025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Fontaine B., Klarsfeld A., Changeux J. P. Calcitonin gene-related peptide and muscle activity regulate acetylcholine receptor alpha-subunit mRNA levels by distinct intracellular pathways. J Cell Biol. 1987 Sep;105(3):1337–1342. doi: 10.1083/jcb.105.3.1337. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Fontaine B., Sassoon D., Buckingham M., Changeux J. P. Detection of the nicotinic acetylcholine receptor alpha-subunit mRNA by in situ hybridization at neuromuscular junctions of 15-day-old chick striated muscles. EMBO J. 1988 Mar;7(3):603–609. doi: 10.1002/j.1460-2075.1988.tb02853.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Goldman D., Brenner H. R., Heinemann S. Acetylcholine receptor alpha-, beta-, gamma-, and delta-subunit mRNA levels are regulated by muscle activity. Neuron. 1988 Jun;1(4):329–333. doi: 10.1016/0896-6273(88)90081-5. [DOI] [PubMed] [Google Scholar]
  13. Goldman D., Staple J. Spatial and temporal expression of acetylcholine receptor RNAs in innervated and denervated rat soleus muscle. Neuron. 1989 Aug;3(2):219–228. doi: 10.1016/0896-6273(89)90035-4. [DOI] [PubMed] [Google Scholar]
  14. Gundersen K., Sanes J. R., Merlie J. P. Neural regulation of muscle acetylcholine receptor epsilon- and alpha-subunit gene promoters in transgenic mice. J Cell Biol. 1993 Dec;123(6 Pt 1):1535–1544. doi: 10.1083/jcb.123.6.1535. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Hall Z. W., Ralston E. Nuclear domains in muscle cells. Cell. 1989 Dec 1;59(5):771–772. doi: 10.1016/0092-8674(89)90597-7. [DOI] [PubMed] [Google Scholar]
  16. Hall Z. W., Sanes J. R. Synaptic structure and development: the neuromuscular junction. Cell. 1993 Jan;72 (Suppl):99–121. doi: 10.1016/s0092-8674(05)80031-5. [DOI] [PubMed] [Google Scholar]
  17. Hammerschmidt M., Bitgood M. J., McMahon A. P. Protein kinase A is a common negative regulator of Hedgehog signaling in the vertebrate embryo. Genes Dev. 1996 Mar 15;10(6):647–658. doi: 10.1101/gad.10.6.647. [DOI] [PubMed] [Google Scholar]
  18. Huganir R. L., Delcour A. H., Greengard P., Hess G. P. Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization. Nature. 1986 Jun 19;321(6072):774–776. doi: 10.1038/321774a0. [DOI] [PubMed] [Google Scholar]
  19. Huganir R. L., Greengard P. cAMP-dependent protein kinase phosphorylates the nicotinic acetylcholine receptor. Proc Natl Acad Sci U S A. 1983 Feb;80(4):1130–1134. doi: 10.1073/pnas.80.4.1130. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Jiang J., Struhl G. Protein kinase A and hedgehog signaling in Drosophila limb development. Cell. 1995 Feb 24;80(4):563–572. doi: 10.1016/0092-8674(95)90510-3. [DOI] [PubMed] [Google Scholar]
  21. Jo S. A., Zhu X., Marchionni M. A., Burden S. J. Neuregulins are concentrated at nerve-muscle synapses and activate ACh-receptor gene expression. Nature. 1995 Jan 12;373(6510):158–161. doi: 10.1038/373158a0. [DOI] [PubMed] [Google Scholar]
  22. Jones K. W., Shapero M. H., Chevrette M., Fournier R. E. Subtractive hybridization cloning of a tissue-specific extinguisher: TSE1 encodes a regulatory subunit of protein kinase A. Cell. 1991 Sep 6;66(5):861–872. doi: 10.1016/0092-8674(91)90433-y. [DOI] [PubMed] [Google Scholar]
  23. KOELLE G. B., FRIEDENWALD J. A. A histochemical method for localizing cholinesterase activity. Proc Soc Exp Biol Med. 1949 Apr;70(4):617–622. doi: 10.3181/00379727-70-17013. [DOI] [PubMed] [Google Scholar]
  24. Klarsfeld A., Bessereau J. L., Salmon A. M., Triller A., Babinet C., Changeux J. P. An acetylcholine receptor alpha-subunit promoter conferring preferential synaptic expression in muscle of transgenic mice. EMBO J. 1991 Mar;10(3):625–632. doi: 10.1002/j.1460-2075.1991.tb07990.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Kues W. A., Brenner H. R., Sakmann B., Witzemann V. Local neurotrophic repression of gene transcripts encoding fetal AChRs at rat neuromuscular synapses. J Cell Biol. 1995 Aug;130(4):949–957. doi: 10.1083/jcb.130.4.949. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Lalli E., Sassone-Corsi P. Signal transduction and gene regulation: the nuclear response to cAMP. J Biol Chem. 1994 Jul 1;269(26):17359–17362. [PubMed] [Google Scholar]
  27. Lepage T., Cohen S. M., Diaz-Benjumea F. J., Parkhurst S. M. Signal transduction by cAMP-dependent protein kinase A in Drosophila limb patterning. Nature. 1995 Feb 23;373(6516):711–715. doi: 10.1038/373711a0. [DOI] [PubMed] [Google Scholar]
  28. Li W., Ohlmeyer J. T., Lane M. E., Kalderon D. Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development. Cell. 1995 Feb 24;80(4):553–562. doi: 10.1016/0092-8674(95)90509-x. [DOI] [PubMed] [Google Scholar]
  29. McCartney S., Little B. M., Langeberg L. K., Scott J. D. Cloning and characterization of A-kinase anchor protein 100 (AKAP100). A protein that targets A-kinase to the sarcoplasmic reticulum. J Biol Chem. 1995 Apr 21;270(16):9327–9333. doi: 10.1074/jbc.270.16.9327. [DOI] [PubMed] [Google Scholar]
  30. McKnight G. S. Cyclic AMP second messenger systems. Curr Opin Cell Biol. 1991 Apr;3(2):213–217. doi: 10.1016/0955-0674(91)90141-k. [DOI] [PubMed] [Google Scholar]
  31. Merlie J. P., Sanes J. R. Concentration of acetylcholine receptor mRNA in synaptic regions of adult muscle fibres. Nature. 1985 Sep 5;317(6032):66–68. doi: 10.1038/317066a0. [DOI] [PubMed] [Google Scholar]
  32. Mishina M., Takai T., Imoto K., Noda M., Takahashi T., Numa S., Methfessel C., Sakmann B. Molecular distinction between fetal and adult forms of muscle acetylcholine receptor. Nature. 1986 May 22;321(6068):406–411. doi: 10.1038/321406a0. [DOI] [PubMed] [Google Scholar]
  33. Morita N., Namikawa K., Kiyama H. Up-regulation of PKA RI alpha subunit mRNA in rat skeletal muscle after nerve injury. Neuroreport. 1995 May 9;6(7):1050–1052. doi: 10.1097/00001756-199505090-00025. [DOI] [PubMed] [Google Scholar]
  34. Moscoso L. M., Chu G. C., Gautam M., Noakes P. G., Merlie J. P., Sanes J. R. Synapse-associated expression of an acetylcholine receptor-inducing protein, ARIA/heregulin, and its putative receptors, ErbB2 and ErbB3, in developing mammalian muscle. Dev Biol. 1995 Nov;172(1):158–169. doi: 10.1006/dbio.1995.0012. [DOI] [PubMed] [Google Scholar]
  35. Mulle C., Benoit P., Pinset C., Roa M., Changeux J. P. Calcitonin gene-related peptide enhances the rate of desensitization of the nicotinic acetylcholine receptor in cultured mouse muscle cells. Proc Natl Acad Sci U S A. 1988 Aug;85(15):5728–5732. doi: 10.1073/pnas.85.15.5728. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Pan D., Rubin G. M. cAMP-dependent protein kinase and hedgehog act antagonistically in regulating decapentaplegic transcription in Drosophila imaginal discs. Cell. 1995 Feb 24;80(4):543–552. doi: 10.1016/0092-8674(95)90508-1. [DOI] [PubMed] [Google Scholar]
  37. Pavlath G. K., Rich K., Webster S. G., Blau H. M. Localization of muscle gene products in nuclear domains. Nature. 1989 Feb 9;337(6207):570–573. doi: 10.1038/337570a0. [DOI] [PubMed] [Google Scholar]
  38. Piette J., Bessereau J. L., Huchet M., Changeux J. P. Two adjacent MyoD1-binding sites regulate expression of the acetylcholine receptor alpha-subunit gene. Nature. 1990 May 24;345(6273):353–355. doi: 10.1038/345353a0. [DOI] [PubMed] [Google Scholar]
  39. Piette J., Huchet M., Houzelstein D., Changeux J. P. Compartmentalized expression of the alpha- and gamma-subunits of the acetylcholine receptor in recently fused myofibers. Dev Biol. 1993 May;157(1):205–213. doi: 10.1006/dbio.1993.1124. [DOI] [PubMed] [Google Scholar]
  40. Sanes J. R., Johnson Y. R., Kotzbauer P. T., Mudd J., Hanley T., Martinou J. C., Merlie J. P. Selective expression of an acetylcholine receptor-lacZ transgene in synaptic nuclei of adult muscle fibers. Development. 1991 Dec;113(4):1181–1191. doi: 10.1242/dev.113.4.1181. [DOI] [PubMed] [Google Scholar]
  41. Sassoon D. A. Myogenic regulatory factors: dissecting their role and regulation during vertebrate embryogenesis. Dev Biol. 1993 Mar;156(1):11–23. doi: 10.1006/dbio.1993.1055. [DOI] [PubMed] [Google Scholar]
  42. Sassoon D., Rosenthal N. Detection of messenger RNA by in situ hybridization. Methods Enzymol. 1993;225:384–404. doi: 10.1016/0076-6879(93)25027-y. [DOI] [PubMed] [Google Scholar]
  43. Schulman H. Protein phosphorylation in neuronal plasticity and gene expression. Curr Opin Neurobiol. 1995 Jun;5(3):375–381. doi: 10.1016/0959-4388(95)80051-4. [DOI] [PubMed] [Google Scholar]
  44. Scott J. D., McCartney S. Localization of A-kinase through anchoring proteins. Mol Endocrinol. 1994 Jan;8(1):5–11. doi: 10.1210/mend.8.1.8152430. [DOI] [PubMed] [Google Scholar]
  45. Simon A. M., Burden S. J. An E box mediates activation and repression of the acetylcholine receptor delta-subunit gene during myogenesis. Mol Cell Biol. 1993 Sep;13(9):5133–5140. doi: 10.1128/mcb.13.9.5133. [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Simon A. M., Hoppe P., Burden S. J. Spatial restriction of AChR gene expression to subsynaptic nuclei. Development. 1992 Mar;114(3):545–553. doi: 10.1242/dev.114.3.545. [DOI] [PubMed] [Google Scholar]
  47. Tang J., Jo S. A., Burden S. J. Separate pathways for synapse-specific and electrical activity-dependent gene expression in skeletal muscle. Development. 1994 Jul;120(7):1799–1804. doi: 10.1242/dev.120.7.1799. [DOI] [PubMed] [Google Scholar]
  48. Taylor S. S., Buechler J. A., Yonemoto W. cAMP-dependent protein kinase: framework for a diverse family of regulatory enzymes. Annu Rev Biochem. 1990;59:971–1005. doi: 10.1146/annurev.bi.59.070190.004543. [DOI] [PubMed] [Google Scholar]
  49. Uhler M. D., Chrivia J. C., McKnight G. S. Evidence for a second isoform of the catalytic subunit of cAMP-dependent protein kinase. J Biol Chem. 1986 Nov 25;261(33):15360–15363. [PubMed] [Google Scholar]
  50. Valenzuela D. M., Stitt T. N., DiStefano P. S., Rojas E., Mattsson K., Compton D. L., Nuñez L., Park J. S., Stark J. L., Gies D. R. Receptor tyrosine kinase specific for the skeletal muscle lineage: expression in embryonic muscle, at the neuromuscular junction, and after injury. Neuron. 1995 Sep;15(3):573–584. doi: 10.1016/0896-6273(95)90146-9. [DOI] [PubMed] [Google Scholar]
  51. Van Patten S. M., Howard P., Walsh D. A., Maurer R. A. The alpha- and beta-isoforms of the inhibitor protein of the 3',5'-cyclic adenosine monophosphate-dependent protein kinase: characteristics and tissue- and developmental-specific expression. Mol Endocrinol. 1992 Dec;6(12):2114–2122. doi: 10.1210/mend.6.12.1491692. [DOI] [PubMed] [Google Scholar]
  52. Vandromme M., Carnac G., Gauthier-Rouvière C., Fesquet D., Lamb N., Fernandez A. Nuclear import of the myogenic factor MyoD requires cAMP-dependent protein kinase activity but not the direct phosphorylation of MyoD. J Cell Sci. 1994 Feb;107(Pt 2):613–620. doi: 10.1242/jcs.107.2.613. [DOI] [PubMed] [Google Scholar]
  53. Vandromme M., Cavadore J. C., Bonnieu A., Froeschlé A., Lamb N., Fernandez A. Two nuclear localization signals present in the basic-helix 1 domains of MyoD promote its active nuclear translocation and can function independently. Proc Natl Acad Sci U S A. 1995 May 9;92(10):4646–4650. doi: 10.1073/pnas.92.10.4646. [DOI] [PMC free article] [PubMed] [Google Scholar]
  54. Walke W., Staple J., Adams L., Gnegy M., Chahine K., Goldman D. Calcium-dependent regulation of rat and chick muscle nicotinic acetylcholine receptor (nAChR) gene expression. J Biol Chem. 1994 Jul 29;269(30):19447–19456. [PubMed] [Google Scholar]
  55. Wen W., Meinkoth J. L., Tsien R. Y., Taylor S. S. Identification of a signal for rapid export of proteins from the nucleus. Cell. 1995 Aug 11;82(3):463–473. doi: 10.1016/0092-8674(95)90435-2. [DOI] [PubMed] [Google Scholar]
  56. Wilkinson D. G., Nieto M. A. Detection of messenger RNA by in situ hybridization to tissue sections and whole mounts. Methods Enzymol. 1993;225:361–373. doi: 10.1016/0076-6879(93)25025-w. [DOI] [PubMed] [Google Scholar]
  57. Winter B., Braun T., Arnold H. H. cAMP-dependent protein kinase represses myogenic differentiation and the activity of the muscle-specific helix-loop-helix transcription factors Myf-5 and MyoD. J Biol Chem. 1993 May 5;268(13):9869–9878. [PubMed] [Google Scholar]
  58. Witzemann V., Barg B., Nishikawa Y., Sakmann B., Numa S. Differential regulation of muscle acetylcholine receptor gamma- and epsilon-subunit mRNAs. FEBS Lett. 1987 Oct 19;223(1):104–112. doi: 10.1016/0014-5793(87)80518-5. [DOI] [PubMed] [Google Scholar]
  59. Witzemann V., Brenner H. R., Sakmann B. Neural factors regulate AChR subunit mRNAs at rat neuromuscular synapses. J Cell Biol. 1991 Jul;114(1):125–141. doi: 10.1083/jcb.114.1.125. [DOI] [PMC free article] [PubMed] [Google Scholar]
  60. Zhu X., Lai C., Thomas S., Burden S. J. Neuregulin receptors, erbB3 and erbB4, are localized at neuromuscular synapses. EMBO J. 1995 Dec 1;14(23):5842–5848. doi: 10.1002/j.1460-2075.1995.tb00272.x. [DOI] [PMC free article] [PubMed] [Google Scholar]

Articles from The Journal of Cell Biology are provided here courtesy of The Rockefeller University Press

RESOURCES