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. 1994 Apr;6(4):501–510. doi: 10.1105/tpc.6.4.501

Phosphorylation and calcium binding properties of an Arabidopsis GF14 brain protein homolog.

G Lu 1, P C Sehnke 1, R J Ferl 1
PMCID: PMC160453  PMID: 8205002

Abstract

Arabidopsis GF14 omega was originally described because of its apparent association with a DNA-protein complex; it is a member of the 14-3-3 kinase regulatory protein family that is conserved throughout eukaryotes. Here, we demonstrated that recombinant GF14 omega is expressed in Escherichia coli as a dimer. Blot binding and electrophoretic mobility shift analyses indicated that GF14 omega binds calcium. Equilibrium dialysis further demonstrated that GF14 omega binds an equimolar amount of calcium with an apparent binding constant of 5.5 x 10(4) M-1 under physiological conditions. The C-terminal domain, which contains a potential EF hand motif, is responsible for the calcium binding. The C-terminal domain also cross-reacted with the anti-GF14 omega monoclonal antibody. In addition, GF14 omega is phosphorylated by Arabidopsis protein kinase activity at a serine residue(s) in vitro. Therefore, GF14 omega protein has biochemical properties consistent with potential signaling roles in plants. The presence of a potential EF hand-like motif in the highly conserved C terminus of 14-3-3 proteins together with the calcium-dependent multiple functions attributed to the 14-3-3 proteins indicate that the C terminus EF hand is a common functional element of this family of proteins.

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Selected References

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  1. Aitken A., Collinge D. B., van Heusden B. P., Isobe T., Roseboom P. H., Rosenfeld G., Soll J. 14-3-3 proteins: a highly conserved, widespread family of eukaryotic proteins. Trends Biochem Sci. 1992 Dec;17(12):498–501. doi: 10.1016/0968-0004(92)90339-b. [DOI] [PubMed] [Google Scholar]
  2. Aitken A., Ellis C. A., Harris A., Sellers L. A., Toker A. Kinase and neurotransmitters. Nature. 1990 Apr 12;344(6267):594–594. doi: 10.1038/344594a0. [DOI] [PubMed] [Google Scholar]
  3. Babu Y. S., Sack J. S., Greenhough T. J., Bugg C. E., Means A. R., Cook W. J. Three-dimensional structure of calmodulin. Nature. 1985 May 2;315(6014):37–40. doi: 10.1038/315037a0. [DOI] [PubMed] [Google Scholar]
  4. Battey N. H. Calcium-activated protein kinase from soluble and membrane fractions of maize coleoptiles. Biochem Biophys Res Commun. 1990 Jul 16;170(1):17–22. doi: 10.1016/0006-291x(90)91234-j. [DOI] [PubMed] [Google Scholar]
  5. Block A., Dangl J. L., Hahlbrock K., Schulze-Lefert P. Functional borders, genetic fine structure, and distance requirements of cis elements mediating light responsiveness of the parsley chalcone synthase promoter. Proc Natl Acad Sci U S A. 1990 Jul;87(14):5387–5391. doi: 10.1073/pnas.87.14.5387. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Donald R. G., Cashmore A. R. Mutation of either G box or I box sequences profoundly affects expression from the Arabidopsis rbcS-1A promoter. EMBO J. 1990 Jun;9(6):1717–1726. doi: 10.1002/j.1460-2075.1990.tb08295.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Ellis P. D., Strang P., Potter J. D. Cadmium-substituted skeletal troponin C. Cadmium-113 NMR spectroscopy and metal binding investigations. J Biol Chem. 1984 Aug 25;259(16):10348–10356. [PubMed] [Google Scholar]
  8. Emori Y., Kawasaki H., Sugihara H., Imajoh S., Kawashima S., Suzuki K. Isolation and sequence analyses of cDNA clones for the large subunits of two isozymes of rabbit calcium-dependent protease. J Biol Chem. 1986 Jul 15;261(20):9465–9471. [PubMed] [Google Scholar]
  9. Haigler H. T., Schlaepfer D. D., Burgess W. H. Characterization of lipocortin I and an immunologically unrelated 33-kDa protein as epidermal growth factor receptor/kinase substrates and phospholipase A2 inhibitors. J Biol Chem. 1987 May 15;262(14):6921–6930. [PubMed] [Google Scholar]
  10. Harper J. F., Sussman M. R., Schaller G. E., Putnam-Evans C., Charbonneau H., Harmon A. C. A calcium-dependent protein kinase with a regulatory domain similar to calmodulin. Science. 1991 May 17;252(5008):951–954. doi: 10.1126/science.1852075. [DOI] [PubMed] [Google Scholar]
  11. Hirsch S., Aitken A., Bertsch U., Soll J. A plant homologue to mammalian brain 14-3-3 protein and protein kinase C inhibitor. FEBS Lett. 1992 Jan 20;296(2):222–224. doi: 10.1016/0014-5793(92)80384-s. [DOI] [PubMed] [Google Scholar]
  12. Huang L., Franklin A. E., Hoffman N. E. Primary structure and characterization of an Arabidopsis thaliana calnexin-like protein. J Biol Chem. 1993 Mar 25;268(9):6560–6566. [PubMed] [Google Scholar]
  13. Ichimura T., Isobe T., Okuyama T., Takahashi N., Araki K., Kuwano R., Takahashi Y. Molecular cloning of cDNA coding for brain-specific 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases. Proc Natl Acad Sci U S A. 1988 Oct;85(19):7084–7088. doi: 10.1073/pnas.85.19.7084. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Isobe T., Hiyane Y., Ichimura T., Okuyama T., Takahashi N., Nakajo S., Nakaya K. Activation of protein kinase C by the 14-3-3 proteins homologous with Exo1 protein that stimulates calcium-dependent exocytosis. FEBS Lett. 1992 Aug 17;308(2):121–124. doi: 10.1016/0014-5793(92)81257-m. [DOI] [PubMed] [Google Scholar]
  15. Isobe T., Ichimura T., Sunaya T., Okuyama T., Takahashi N., Kuwano R., Takahashi Y. Distinct forms of the protein kinase-dependent activator of tyrosine and tryptophan hydroxylases. J Mol Biol. 1991 Jan 5;217(1):125–132. doi: 10.1016/0022-2836(91)90616-e. [DOI] [PubMed] [Google Scholar]
  16. Kilhoffer M. C., Haiech J., Demaille J. G. Ion binding to calmodulin. A comparison with other intracellular calcium-binding proteins. Mol Cell Biochem. 1983;51(1):33–54. doi: 10.1007/BF00215584. [DOI] [PubMed] [Google Scholar]
  17. Lu G., DeLisle A. J., de Vetten N. C., Ferl R. J. Brain proteins in plants: an Arabidopsis homolog to neurotransmitter pathway activators is part of a DNA binding complex. Proc Natl Acad Sci U S A. 1992 Dec 1;89(23):11490–11494. doi: 10.1073/pnas.89.23.11490. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Marcotte W. R., Jr, Russell S. H., Quatrano R. S. Abscisic acid-responsive sequences from the em gene of wheat. Plant Cell. 1989 Oct;1(10):969–976. doi: 10.1105/tpc.1.10.969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Michalak M., Milner R. E., Burns K., Opas M. Calreticulin. Biochem J. 1992 Aug 1;285(Pt 3):681–692. doi: 10.1042/bj2850681. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Morgan A., Burgoyne R. D. Exo1 and Exo2 proteins stimulate calcium-dependent exocytosis in permeabilized adrenal chromaffin cells. Nature. 1992 Feb 27;355(6363):833–836. doi: 10.1038/355833a0. [DOI] [PubMed] [Google Scholar]
  21. Morgan A., Burgoyne R. D. Interaction between protein kinase C and Exo1 (14-3-3 protein) and its relevance to exocytosis in permeabilized adrenal chromaffin cells. Biochem J. 1992 Sep 15;286(Pt 3):807–811. doi: 10.1042/bj2860807. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Pla M., Vilardell J., Guiltinan M. J., Marcotte W. R., Niogret M. F., Quatrano R. S., Pagès M. The cis-regulatory element CCACGTGG is involved in ABA and water-stress responses of the maize gene rab28. Plant Mol Biol. 1993 Jan;21(2):259–266. doi: 10.1007/BF00019942. [DOI] [PubMed] [Google Scholar]
  23. Sassone-Corsi P., Ransone L. J., Lamph W. W., Verma I. M. Direct interaction between fos and jun nuclear oncoproteins: role of the 'leucine zipper' domain. Nature. 1988 Dec 15;336(6200):692–695. doi: 10.1038/336692a0. [DOI] [PubMed] [Google Scholar]
  24. Sehnke P. C., Johnson J. E. Crystallization and preliminary X-ray characterization of tobacco streak virus and a proteolytically modified form of the capsid protein. Virology. 1993 Sep;196(1):328–331. doi: 10.1006/viro.1993.1483. [DOI] [PubMed] [Google Scholar]
  25. Toker A., Sellers L. A., Amess B., Patel Y., Harris A., Aitken A. Multiple isoforms of a protein kinase C inhibitor (KCIP-1/14-3-3) from sheep brain. Amino acid sequence of phosphorylated forms. Eur J Biochem. 1992 Jun 1;206(2):453–461. doi: 10.1111/j.1432-1033.1992.tb16946.x. [DOI] [PubMed] [Google Scholar]
  26. Williams M. E., Foster R., Chua N. H. Sequences flanking the hexameric G-box core CACGTG affect the specificity of protein binding. Plant Cell. 1992 Apr;4(4):485–496. doi: 10.1105/tpc.4.4.485. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Wu Y. N., Vu N. D., Wagner P. D. Anti-(14-3-3 protein) antibody inhibits stimulation of noradrenaline (norepinephrine) secretion by chromaffin-cell cytosolic proteins. Biochem J. 1992 Aug 1;285(Pt 3):697–700. doi: 10.1042/bj2850697. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Yamamoto K. K., Gonzalez G. A., Biggs W. H., 3rd, Montminy M. R. Phosphorylation-induced binding and transcriptional efficacy of nuclear factor CREB. Nature. 1988 Aug 11;334(6182):494–498. doi: 10.1038/334494a0. [DOI] [PubMed] [Google Scholar]
  29. Yamauchi T., Nakata H., Fujisawa H. A new activator protein that activates tryptophan 5-monooxygenase and tyrosine 3-monooxygenase in the presence of Ca2+-, calmodulin-dependent protein kinase. Purification and characterization. J Biol Chem. 1981 Jun 10;256(11):5404–5409. [PubMed] [Google Scholar]
  30. Zupan L. A., Steffens D. L., Berry C. A., Landt M., Gross R. W. Cloning and expression of a human 14-3-3 protein mediating phospholipolysis. Identification of an arachidonoyl-enzyme intermediate during catalysis. J Biol Chem. 1992 May 5;267(13):8707–8710. [PubMed] [Google Scholar]
  31. de Vetten N. C., Lu G., Feri R. J. A maize protein associated with the G-box binding complex has homology to brain regulatory proteins. Plant Cell. 1992 Oct;4(10):1295–1307. doi: 10.1105/tpc.4.10.1295. [DOI] [PMC free article] [PubMed] [Google Scholar]

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