Abstract
Agrin, a protein that mediates nerve-induced acetylcholine receptor (AChR) aggregation at developing neuromuscular junctions, has been shown to cause an increase in phosphorylation of the beta, gamma, and delta subunits of AChRs in cultured myotubes. As a step toward understanding the mechanism of agrin-induced AChR aggregation, we examined the effects of inhibitors of protein kinases on AChR aggregation and phosphorylation in chick myotubes in culture. Staurosporine, an antagonist of both protein serine and tyrosine kinases, blocked agrin-induced AChR aggregation in a dose-dependent manner; 50% inhibition occurred at approximately 2 nM. The extent of inhibition was independent of agrin concentration, suggesting an effect downstream of the interaction of agrin with its receptor. Staurosporine blocked agrin-induced phosphorylation of the AChR beta subunit, which occurs at least in part on tyrosine residues, but did not reduce phosphorylation of the gamma and delta subunits, which occurs on serine/threonine residues. Staurosporine also prevented the agrin- induced decrease in the rate at which AChRs are extracted from intact myotubes by mild detergents. H-7, an antagonist of protein serine kinases, inhibited agrin-induced phosphorylation of the gamma and delta subunits but did not block agrin-induced phosphorylation of the AChR beta subunit, AChR aggregation, or the decrease in AChR extractability. The results provide support for the hypothesis that tyrosine phosphorylation of the beta subunit plays a role in agrin-induced AChR aggregation.
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- Akiyama T., Ishida J., Nakagawa S., Ogawara H., Watanabe S., Itoh N., Shibuya M., Fukami Y. Genistein, a specific inhibitor of tyrosine-specific protein kinases. J Biol Chem. 1987 Apr 25;262(12):5592–5595. [PubMed] [Google Scholar]
- Anthony D. T., Jacobs-Cohen R. J., Marazzi G., Rubin L. L. A molecular defect in virally transformed muscle cells that cannot cluster acetylcholine receptors. J Cell Biol. 1988 May;106(5):1713–1721. doi: 10.1083/jcb.106.5.1713. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Anthony D. T., Schuetze S. M., Rubin L. L. Transformation by Rous sarcoma virus prevents acetylcholine receptor clustering on cultured chicken muscle fibers. Proc Natl Acad Sci U S A. 1984 Apr;81(7):2265–2269. doi: 10.1073/pnas.81.7.2265. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Axelrod D., Ravdin P., Koppel D. E., Schlessinger J., Webb W. W., Elson E. L., Podleski T. R. Lateral motion of fluorescently labeled acetylcholine receptors in membranes of developing muscle fibers. Proc Natl Acad Sci U S A. 1976 Dec;73(12):4594–4598. doi: 10.1073/pnas.73.12.4594. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Baker L. P., Peng H. B. Tyrosine phosphorylation and acetylcholine receptor cluster formation in cultured Xenopus muscle cells. J Cell Biol. 1993 Jan;120(1):185–195. doi: 10.1083/jcb.120.1.185. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Berg M. M., Sternberg D. W., Parada L. F., Chao M. V. K-252a inhibits nerve growth factor-induced trk proto-oncogene tyrosine phosphorylation and kinase activity. J Biol Chem. 1992 Jan 5;267(1):13–16. [PubMed] [Google Scholar]
- Cohen M. W., Godfrey E. W. Early appearance of and neuronal contribution to agrin-like molecules at embryonic frog nerve-muscle synapses formed in culture. J Neurosci. 1992 Aug;12(8):2982–2992. doi: 10.1523/JNEUROSCI.12-08-02982.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Fischbach G. D. Synapse formation between dissociated nerve and muscle cells in low density cell cultures. Dev Biol. 1972 Jun;28(2):407–429. doi: 10.1016/0012-1606(72)90023-1. [DOI] [PubMed] [Google Scholar]
- Froehner S. C. Regulation of ion channel distribution at synapses. Annu Rev Neurosci. 1993;16:347–368. doi: 10.1146/annurev.ne.16.030193.002023. [DOI] [PubMed] [Google Scholar]
- Gazit A., Yaish P., Gilon C., Levitzki A. Tyrphostins I: synthesis and biological activity of protein tyrosine kinase inhibitors. J Med Chem. 1989 Oct;32(10):2344–2352. doi: 10.1021/jm00130a020. [DOI] [PubMed] [Google Scholar]
- Huganir R. L., Miles K. Protein phosphorylation of nicotinic acetylcholine receptors. Crit Rev Biochem Mol Biol. 1989;24(3):183–215. doi: 10.3109/10409238909082553. [DOI] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Marazzi G., Bard F., Klymkowsky M. W., Rubin L. L. Microinjection of a monoclonal antibody against a 37-kD protein (tropomyosin 2) prevents the formation of new acetylcholine receptor clusters. J Cell Biol. 1989 Nov;109(5):2337–2344. doi: 10.1083/jcb.109.5.2337. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McMahan U. J. The agrin hypothesis. Cold Spring Harb Symp Quant Biol. 1990;55:407–418. doi: 10.1101/sqb.1990.055.01.041. [DOI] [PubMed] [Google Scholar]
- McMahan U. J., Wallace B. G. Molecules in basal lamina that direct the formation of synaptic specializations at neuromuscular junctions. Dev Neurosci. 1989;11(4-5):227–247. doi: 10.1159/000111903. [DOI] [PubMed] [Google Scholar]
- Nitkin R. M., Smith M. A., Magill C., Fallon J. R., Yao Y. M., Wallace B. G., McMahan U. J. Identification of agrin, a synaptic organizing protein from Torpedo electric organ. J Cell Biol. 1987 Dec;105(6 Pt 1):2471–2478. doi: 10.1083/jcb.105.6.2471. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nye S. H., Squinto S. P., Glass D. J., Stitt T. N., Hantzopoulos P., Macchi M. J., Lindsay N. S., Ip N. Y., Yancopoulos G. D. K-252a and staurosporine selectively block autophosphorylation of neurotrophin receptors and neurotrophin-mediated responses. Mol Biol Cell. 1992 Jun;3(6):677–686. doi: 10.1091/mbc.3.6.677. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Onoda T., Iinuma H., Sasaki Y., Hamada M., Isshiki K., Naganawa H., Takeuchi T., Tatsuta K., Umezawa K. Isolation of a novel tyrosine kinase inhibitor, lavendustin A, from Streptomyces griseolavendus. J Nat Prod. 1989 Nov-Dec;52(6):1252–1257. doi: 10.1021/np50066a009. [DOI] [PubMed] [Google Scholar]
- Peng H. B., Baker L. P., Chen Q. Induction of synaptic development in cultured muscle cells by basic fibroblast growth factor. Neuron. 1991 Feb;6(2):237–246. doi: 10.1016/0896-6273(91)90359-8. [DOI] [PubMed] [Google Scholar]
- Peng H. B., Baker L. P., Dai Z. A role of tyrosine phosphorylation in the formation of acetylcholine receptor clusters induced by electric fields in cultured Xenopus muscle cells. J Cell Biol. 1993 Jan;120(1):197–204. doi: 10.1083/jcb.120.1.197. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Podleski T. R., Salpeter M. M. Acetylcholine receptor clustering and triton solubility: neural effect. J Neurobiol. 1988 Mar;19(2):167–185. doi: 10.1002/neu.480190206. [DOI] [PubMed] [Google Scholar]
- Prives J., Fulton A. B., Penman S., Daniels M. P., Christian C. N. Interaction of the cytoskeletal framework with acetylcholine receptor on th surface of embryonic muscle cells in culture. J Cell Biol. 1982 Jan;92(1):231–236. doi: 10.1083/jcb.92.1.231. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Qu Z. C., Moritz E., Huganir R. L. Regulation of tyrosine phosphorylation of the nicotinic acetylcholine receptor at the rat neuromuscular junction. Neuron. 1990 Mar;4(3):367–378. doi: 10.1016/0896-6273(90)90049-l. [DOI] [PubMed] [Google Scholar]
- Reist N. E., Werle M. J., McMahan U. J. Agrin released by motor neurons induces the aggregation of acetylcholine receptors at neuromuscular junctions. Neuron. 1992 May;8(5):865–868. doi: 10.1016/0896-6273(92)90200-w. [DOI] [PubMed] [Google Scholar]
- Ross A., Rapuano M., Prives J. Induction of phosphorylation and cell surface redistribution of acetylcholine receptors by phorbol ester and carbamylcholine in cultured chick muscle cells. J Cell Biol. 1988 Sep;107(3):1139–1145. doi: 10.1083/jcb.107.3.1139. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Rüegg U. T., Burgess G. M. Staurosporine, K-252 and UCN-01: potent but nonspecific inhibitors of protein kinases. Trends Pharmacol Sci. 1989 Jun;10(6):218–220. doi: 10.1016/0165-6147(89)90263-0. [DOI] [PubMed] [Google Scholar]
- Tamaoki T., Nomoto H., Takahashi I., Kato Y., Morimoto M., Tomita F. Staurosporine, a potent inhibitor of phospholipid/Ca++dependent protein kinase. Biochem Biophys Res Commun. 1986 Mar 13;135(2):397–402. doi: 10.1016/0006-291x(86)90008-2. [DOI] [PubMed] [Google Scholar]
- Tapley P., Lamballe F., Barbacid M. K252a is a selective inhibitor of the tyrosine protein kinase activity of the trk family of oncogenes and neurotrophin receptors. Oncogene. 1992 Feb;7(2):371–381. [PubMed] [Google Scholar]
- Uehara Y., Hori M., Takeuchi T., Umezawa H. Phenotypic change from transformed to normal induced by benzoquinonoid ansamycins accompanies inactivation of p60src in rat kidney cells infected with Rous sarcoma virus. Mol Cell Biol. 1986 Jun;6(6):2198–2206. doi: 10.1128/mcb.6.6.2198. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Uehara Y., Murakami Y., Mizuno S., Kawai S. Inhibition of transforming activity of tyrosine kinase oncogenes by herbimycin A. Virology. 1988 May;164(1):294–298. doi: 10.1016/0042-6822(88)90649-6. [DOI] [PubMed] [Google Scholar]
- Umezawa K., Hori T., Tajima H., Imoto M., Isshiki K., Takeuchi T. Inhibition of epidermal growth factor-induced DNA synthesis by tyrosine kinase inhibitors. FEBS Lett. 1990 Jan 29;260(2):198–200. doi: 10.1016/0014-5793(90)80102-o. [DOI] [PubMed] [Google Scholar]
- Wallace B. G. Agrin-induced specializations contain cytoplasmic, membrane, and extracellular matrix-associated components of the postsynaptic apparatus. J Neurosci. 1989 Apr;9(4):1294–1302. doi: 10.1523/JNEUROSCI.09-04-01294.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wallace B. G. Mechanism of agrin-induced acetylcholine receptor aggregation. J Neurobiol. 1992 Jul;23(5):592–604. doi: 10.1002/neu.480230512. [DOI] [PubMed] [Google Scholar]
- Wallace B. G., Qu Z., Huganir R. L. Agrin induces phosphorylation of the nicotinic acetylcholine receptor. Neuron. 1991 Jun;6(6):869–878. doi: 10.1016/0896-6273(91)90227-q. [DOI] [PubMed] [Google Scholar]
- Wallace B. G. Regulation of agrin-induced acetylcholine receptor aggregation by Ca++ and phorbol ester. J Cell Biol. 1988 Jul;107(1):267–278. doi: 10.1083/jcb.107.1.267. [DOI] [PMC free article] [PubMed] [Google Scholar]