Skip to main content
NCBI home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1991 Apr 1;88(7):2643–2647. doi: 10.1073/pnas.88.7.2643

Phosphorylation of a Ras-related GTP-binding protein, Rap-1b, by a neuronal Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr.

N Sahyoun 1, O B McDonald 1, F Farrell 1, E G Lapetina 1
PMCID: PMC51294  PMID: 1901412

Abstract

A neuron-specific Ca2+/calmodulin-dependent protein kinase, CaM kinase Gr, phosphorylates selectively a Ras-related GTP-binding protein (Rap-1b) that is enriched in brain tissue. The phosphorylation reaction achieves a stoichiometry of about 1 and involves a serine residue near the carboxyl terminus of the substrate. Both CaM kinase Gr and cAMP-dependent protein kinase, but not CaM kinase II, phosphorylate identical or contiguous serine residues in Rap-1b. The rate of phosphorylation of Rap-1b by CaM kinase Gr is enhanced following autophosphorylation of the protein kinase. Other low molecular weight GTP-binding proteins belonging to the Ras superfamily, including Rab-3A, Rap-2b, and c-Ha-ras p21, are not phosphorylated by CaM kinase Gr. The phosphorylation of Rap-1b itself can be reversed by an endogenous brain phosphoprotein phosphatase. These observations provide a potential connection between a neuronal Ca2(+)-signaling pathway and a specific low molecular weight GTP-binding protein that may regulate neuronal transmembrane signaling, vesicle transport, or neurotransmitter release.

Full text

PDF
2643

Images in this article

2644Image
on p.2644
2644Image
on p.2644
2644Image
on p.2644
2645Image
on p.2645

Selected References

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

  1. Ballester R., Furth M. E., Rosen O. M. Phorbol ester- and protein kinase C-mediated phosphorylation of the cellular Kirsten ras gene product. J Biol Chem. 1987 Feb 25;262(6):2688–2695. [PubMed] [Google Scholar]
  2. Bender A., Pringle J. R. Multicopy suppression of the cdc24 budding defect in yeast by CDC42 and three newly identified genes including the ras-related gene RSR1. Proc Natl Acad Sci U S A. 1989 Dec;86(24):9976–9980. doi: 10.1073/pnas.86.24.9976. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Bourne H. R., Sanders D. A., McCormick F. The GTPase superfamily: a conserved switch for diverse cell functions. Nature. 1990 Nov 8;348(6297):125–132. doi: 10.1038/348125a0. [DOI] [PubMed] [Google Scholar]
  4. Bradford M. M. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976 May 7;72:248–254. doi: 10.1016/0003-2697(76)90527-3. [DOI] [PubMed] [Google Scholar]
  5. Burgoyne R. D. Small GTP-binding proteins. Trends Biochem Sci. 1989 Oct;14(10):394–396. doi: 10.1016/0968-0004(89)90281-8. [DOI] [PubMed] [Google Scholar]
  6. Bähler M., Greengard P. Synapsin I bundles F-actin in a phosphorylation-dependent manner. Nature. 1987 Apr 16;326(6114):704–707. doi: 10.1038/326704a0. [DOI] [PubMed] [Google Scholar]
  7. Cohen P., Cohen P. T. Protein phosphatases come of age. J Biol Chem. 1989 Dec 25;264(36):21435–21438. [PubMed] [Google Scholar]
  8. Colbran R. J., Schworer C. M., Hashimoto Y., Fong Y. L., Rich D. P., Smith M. K., Soderling T. R. Calcium/calmodulin-dependent protein kinase II. Biochem J. 1989 Mar 1;258(2):313–325. doi: 10.1042/bj2580313. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Colbran R. J., Soderling T. R. Calcium/calmodulin-independent autophosphorylation sites of calcium/calmodulin-dependent protein kinase II. Studies on the effect of phosphorylation of threonine 305/306 and serine 314 on calmodulin binding using synthetic peptides. J Biol Chem. 1990 Jul 5;265(19):11213–11219. [PubMed] [Google Scholar]
  10. Erondu N. E., Kennedy M. B. Regional distribution of type II Ca2+/calmodulin-dependent protein kinase in rat brain. J Neurosci. 1985 Dec;5(12):3270–3277. doi: 10.1523/JNEUROSCI.05-12-03270.1985. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Gilman A. G. G proteins: transducers of receptor-generated signals. Annu Rev Biochem. 1987;56:615–649. doi: 10.1146/annurev.bi.56.070187.003151. [DOI] [PubMed] [Google Scholar]
  12. Hall A. The cellular functions of small GTP-binding proteins. Science. 1990 Aug 10;249(4969):635–640. doi: 10.1126/science.2116664. [DOI] [PubMed] [Google Scholar]
  13. Hoshijima M., Kikuchi A., Kawata M., Ohmori T., Hashimoto E., Yamamura H., Takai Y. Phosphorylation by cyclic AMP-dependent protein kinase of a human platelet Mr 22,000 GTP-binding protein (smg p21) having the same putative effector domain as the ras gene products. Biochem Biophys Res Commun. 1988 Dec 30;157(3):851–860. doi: 10.1016/s0006-291x(88)80953-7. [DOI] [PubMed] [Google Scholar]
  14. Huttner W. B., DeGennaro L. J., Greengard P. Differential phosphorylation of multiple sites in purified protein I by cyclic AMP-dependent and calcium-dependent protein kinases. J Biol Chem. 1981 Feb 10;256(3):1482–1488. [PubMed] [Google Scholar]
  15. Jacobs S., Cuatrecasas P. Phosphorylation of receptors for insulin and insulin-like growth factor I. Effects of hormones and phorbol esters. J Biol Chem. 1986 Jan 15;261(2):934–939. [PubMed] [Google Scholar]
  16. Jeng A. Y., Srivastava S. K., Lacal J. C., Blumberg P. M. Phosphorylation of ras oncogene product by protein kinase C. Biochem Biophys Res Commun. 1987 Jun 15;145(2):782–788. doi: 10.1016/0006-291x(87)91033-3. [DOI] [PubMed] [Google Scholar]
  17. Kawata M., Matsui Y., Kondo J., Hishida T., Teranishi Y., Takai Y. A novel small molecular weight GTP-binding protein with the same putative effector domain as the ras proteins in bovine brain membranes. Purification, determination of primary structure, and characterization. J Biol Chem. 1988 Dec 15;263(35):18965–18971. [PubMed] [Google Scholar]
  18. Kennedy M. B., Greengard P. Two calcium/calmodulin-dependent protein kinases, which are highly concentrated in brain, phosphorylate protein I at distinct sites. Proc Natl Acad Sci U S A. 1981 Feb;78(2):1293–1297. doi: 10.1073/pnas.78.2.1293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kikuchi A., Yamashita T., Kawata M., Yamamoto K., Ikeda K., Tanimoto T., Takai Y. Purification and characterization of a novel GTP-binding protein with a molecular weight of 24,000 from bovine brain membranes. J Biol Chem. 1988 Feb 25;263(6):2897–2904. [PubMed] [Google Scholar]
  20. Kim S., Kikuchi A., Mizoguchi A., Takai Y. Intrasynaptosomal distribution of the ras, rho and smg-25A GTP-binding proteins in bovine brain. Brain Res Mol Brain Res. 1989 Nov;6(2-3):167–176. doi: 10.1016/0169-328x(89)90051-x. [DOI] [PubMed] [Google Scholar]
  21. Kim S., Mizoguchi A., Kikuchi A., Takai Y. Tissue and subcellular distributions of the smg-21/rap1/Krev-1 proteins which are partly distinct from those of c-ras p21s. Mol Cell Biol. 1990 Jun;10(6):2645–2652. doi: 10.1128/mcb.10.6.2645. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. 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]
  23. Lai Y., Nairn A. C., Greengard P. Autophosphorylation reversibly regulates the Ca2+/calmodulin-dependence of Ca2+/calmodulin-dependent protein kinase II. Proc Natl Acad Sci U S A. 1986 Jun;83(12):4253–4257. doi: 10.1073/pnas.83.12.4253. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Lapetina E. G., Lacal J. C., Reep B. R., Molina y Vedia L. A ras-related protein is phosphorylated and translocated by agonists that increase cAMP levels in human platelets. Proc Natl Acad Sci U S A. 1989 May;86(9):3131–3134. doi: 10.1073/pnas.86.9.3131. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Lapetina E. G. The signal transduction induced by thrombin in human platelets. FEBS Lett. 1990 Aug 1;268(2):400–404. doi: 10.1016/0014-5793(90)81293-w. [DOI] [PubMed] [Google Scholar]
  26. Lazarowski E. R., Winegar D. A., Nolan R. D., Oberdisse E., Lapetina E. G. Effect of protein kinase A on inositide metabolism and rap 1 G-protein in human erythroleukemia cells. J Biol Chem. 1990 Aug 5;265(22):13118–13123. [PubMed] [Google Scholar]
  27. LeVine H., 3rd, Sahyoun N. E., Cuatrecasas P. Calmodulin binding to the cytoskeletal neuronal calmodulin-dependent protein kinase is regulated by autophosphorylation. Proc Natl Acad Sci U S A. 1985 Jan;82(2):287–291. doi: 10.1073/pnas.82.2.287. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lin J. W., Sugimori M., Llinás R. R., McGuinness T. L., Greengard P. Effects of synapsin I and calcium/calmodulin-dependent protein kinase II on spontaneous neurotransmitter release in the squid giant synapse. Proc Natl Acad Sci U S A. 1990 Nov;87(21):8257–8261. doi: 10.1073/pnas.87.21.8257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Llinás R., McGuinness T. L., Leonard C. S., Sugimori M., Greengard P. Intraterminal injection of synapsin I or calcium/calmodulin-dependent protein kinase II alters neurotransmitter release at the squid giant synapse. Proc Natl Acad Sci U S A. 1985 May;82(9):3035–3039. doi: 10.1073/pnas.82.9.3035. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. Matsui Y., Kikuchi A., Kondo J., Hishida T., Teranishi Y., Takai Y. Nucleotide and deduced amino acid sequences of a GTP-binding protein family with molecular weights of 25,000 from bovine brain. J Biol Chem. 1988 Aug 15;263(23):11071–11074. [PubMed] [Google Scholar]
  31. Miller S. G., Kennedy M. B. Regulation of brain type II Ca2+/calmodulin-dependent protein kinase by autophosphorylation: a Ca2+-triggered molecular switch. Cell. 1986 Mar 28;44(6):861–870. doi: 10.1016/0092-8674(86)90008-5. [DOI] [PubMed] [Google Scholar]
  32. Mizoguchi A., Ueda T., Ikeda K., Shiku H., Mizoguti H., Takai Y. Localization and subcellular distribution of cellular ras gene products in rat brain. Brain Res Mol Brain Res. 1989 Jan;5(1):31–44. doi: 10.1016/0169-328x(89)90015-6. [DOI] [PubMed] [Google Scholar]
  33. Ohmstede C. A., Farrell F. X., Reep B. R., Clemetson K. J., Lapetina E. G. RAP2B: a RAS-related GTP-binding protein from platelets. Proc Natl Acad Sci U S A. 1990 Sep;87(17):6527–6531. doi: 10.1073/pnas.87.17.6527. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Ohmstede C. A., Jensen K. F., Sahyoun N. E. Ca2+/calmodulin-dependent protein kinase enriched in cerebellar granule cells. Identification of a novel neuronal calmodulin-dependent protein kinase. J Biol Chem. 1989 Apr 5;264(10):5866–5875. [PubMed] [Google Scholar]
  35. Ouimet C. C., McGuinness T. L., Greengard P. Immunocytochemical localization of calcium/calmodulin-dependent protein kinase II in rat brain. Proc Natl Acad Sci U S A. 1984 Sep;81(17):5604–5608. doi: 10.1073/pnas.81.17.5604. [DOI] [PMC free article] [PubMed] [Google Scholar]
  36. Patton B. L., Miller S. G., Kennedy M. B. Activation of type II calcium/calmodulin-dependent protein kinase by Ca2+/calmodulin is inhibited by autophosphorylation of threonine within the calmodulin-binding domain. J Biol Chem. 1990 Jul 5;265(19):11204–11212. [PubMed] [Google Scholar]
  37. Roskoski R., Jr Assays of protein kinase. Methods Enzymol. 1983;99:3–6. doi: 10.1016/0076-6879(83)99034-1. [DOI] [PubMed] [Google Scholar]
  38. Rostas J. A., Weinberger R. P., Dunkley P. R. Multiple pools and multiple forms of calmodulin-stimulated protein kinase during development: relationship to postsynaptic densities. Prog Brain Res. 1986;69:355–371. doi: 10.1016/s0079-6123(08)61070-5. [DOI] [PubMed] [Google Scholar]
  39. Schiebler W., Jahn R., Doucet J. P., Rothlein J., Greengard P. Characterization of synapsin I binding to small synaptic vesicles. J Biol Chem. 1986 Jun 25;261(18):8383–8390. [PubMed] [Google Scholar]
  40. Schulman H., Lou L. L. Multifunctional Ca2+/calmodulin-dependent protein kinase: domain structure and regulation. Trends Biochem Sci. 1989 Feb;14(2):62–66. doi: 10.1016/0968-0004(89)90045-5. [DOI] [PubMed] [Google Scholar]
  41. Schworer C. M., Colbran R. J., Soderling T. R. Reversible generation of a Ca2+-independent form of Ca2+(calmodulin)-dependent protein kinase II by an autophosphorylation mechanism. J Biol Chem. 1986 Jul 5;261(19):8581–8584. [PubMed] [Google Scholar]
  42. Siess W., Winegar D. A., Lapetina E. G. Rap1-B is phosphorylated by protein kinase A in intact human platelets. Biochem Biophys Res Commun. 1990 Jul 31;170(2):944–950. doi: 10.1016/0006-291x(90)92182-y. [DOI] [PubMed] [Google Scholar]
  43. Sihra T. S., Wang J. K., Gorelick F. S., Greengard P. Translocation of synapsin I in response to depolarization of isolated nerve terminals. Proc Natl Acad Sci U S A. 1989 Oct;86(20):8108–8112. doi: 10.1073/pnas.86.20.8108. [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Soderling T. R. Protein kinases. Regulation by autoinhibitory domains. J Biol Chem. 1990 Feb 5;265(4):1823–1826. [PubMed] [Google Scholar]
  45. Südhof T. C., Czernik A. J., Kao H. T., Takei K., Johnston P. A., Horiuchi A., Kanazir S. D., Wagner M. A., Perin M. S., De Camilli P. Synapsins: mosaics of shared and individual domains in a family of synaptic vesicle phosphoproteins. Science. 1989 Sep 29;245(4925):1474–1480. doi: 10.1126/science.2506642. [DOI] [PubMed] [Google Scholar]
  46. Wolf M., Sahyoun N., LeVine H., 3rd, Cuatrecasas P. Protein kinase C: rapid enzyme purification and substrate-dependence of the diacylglycerol effect. Biochem Biophys Res Commun. 1984 Aug 16;122(3):1268–1275. doi: 10.1016/0006-291x(84)91229-4. [DOI] [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES