• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of biochemjBJ Latest papers and much more!
Biochem J. Jul 1, 1994; 301(Pt 1): 305–310.
PMCID: PMC1137176

Characterization of 14-3-3 proteins in adrenal chromaffin cells and demonstration of isoform-specific phospholipid binding.


Isoform-specific antisera were used to examine which 14-3-3 isoforms were present in bovine adrenal chromaffin cells. The eta, tau and sigma isoforms were not detectable, and the epsilon isoform was present at only low levels. 14-3-3 isoforms were readily detected with antisera against the beta, gamma and zeta isoforms. The latter isoforms were found to leak from digitonin-permeabilized chromaffin cells, as expected for cytosolic proteins, but a proportion of each isoform was retained. In subcellular fractionation studies isoforms recognized by the beta and zeta antisera were found in the cytosol and Triton-insoluble cytoskeletal fractions, while the gamma isoform was found in cytosol and also in microsomal and chromaffin granule membrane fractions. The gamma 14-3-3 protein associated with granule membranes was partially removed by a high-salt/carbonate wash, and the membranes could bind further gamma from cytosol or from a purified brain 14-3-3 protein mixture. The binding of gamma 14-3-3 was not Ca(2+)-dependent, nor was it affected by phorbol ester, GTP analogues or cyclic AMP. Using pure phospholipid vesicles it was found that gamma and also epsilon 14-3-3 proteins bound directly to phospholipids. Little binding of brain beta, eta or zeta to phospholipid vesicles was detected. Brain 14-3-3 proteins were also able to aggregate phospholipid vesicles. Recombinant 14-3-3 isoforms (tau and the Xenopus protein) were able to stimulate Ca(2+)-dependent exocytosis in digitonin-permeabilized chromaffin cells. The Xenopus proteins lacks part of the extreme N-terminus, indicating that this domain is not essential for function in exocytosis.

Full text

Full text is available as a scanned copy of the original print version. Get a printable copy (PDF file) of the complete article (1.6M), or click on a page image below to browse page by page. Links to PubMed are also available for Selected References.

Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Aitken A, Amess B, Howell S, Jones D, Martin H, Patel Y, Robinson K, Toker A. The role of specific isoforms of 14-3-3 protein in regulating protein kinase activity in the brain. Biochem Soc Trans. 1992 Aug;20(3):607–611. [PubMed]
  • 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. [PMC free article] [PubMed]
  • Toker A, Ellis CA, Sellers LA, Aitken A. Protein kinase C inhibitor proteins. Purification from sheep brain and sequence similarity to lipocortins and 14-3-3 protein. Eur J Biochem. 1990 Jul 31;191(2):421–429. [PubMed]
  • Watanabe M, Isobe T, Okuyama T, Ichimura T, Kuwano R, Takahashi Y, Kondo H. Molecular cloning of cDNA to rat 14-3-3 eta chain polypeptide and the neuronal expression of the mRNA in the central nervous system. Brain Res Mol Brain Res. 1991 May;10(2):151–158. [PubMed]
  • 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. [PubMed]
  • Toker A, Sellers LA, 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. [PubMed]
  • Ichimura-Ohshima Y, Morii K, Ichimura T, Araki K, Takahashi Y, Isobe T, Minoshima S, Fukuyama R, Shimizu N, Kuwano R. cDNA cloning and chromosome assignment of the gene for human brain 14-3-3 protein eta chain. J Neurosci Res. 1992 Apr;31(4):600–605. [PubMed]
  • 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. [PubMed]
  • Zupan LA, Steffens DL, Berry CA, Landt M, Gross RW. 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]
  • Watanabe M, Isobe T, Ichimura T, Kuwano R, Takahashi Y, Kondo H. Molecular cloning of rat cDNAs for beta and gamma subtypes of 14-3-3 protein and developmental changes in expression of their mRNAs in the nervous system. Brain Res Mol Brain Res. 1993 Jan;17(1-2):135–146. [PubMed]
  • Fu H, Coburn J, Collier RJ. The eukaryotic host factor that activates exoenzyme S of Pseudomonas aeruginosa is a member of the 14-3-3 protein family. Proc Natl Acad Sci U S A. 1993 Mar 15;90(6):2320–2324. [PMC free article] [PubMed]
  • Leffers H, Madsen P, Rasmussen HH, Honoré B, Andersen AH, Walbum E, Vandekerckhove J, Celis JE. Molecular cloning and expression of the transformation sensitive epithelial marker stratifin. A member of a protein family that has been involved in the protein kinase C signalling pathway. J Mol Biol. 1993 Jun 20;231(4):982–998. [PubMed]
  • van Heusden GP, Wenzel TJ, Lagendijk EL, de Steensma HY, van den Berg JA. Characterization of the yeast BMH1 gene encoding a putative protein homologous to mammalian protein kinase II activators and protein kinase C inhibitors. FEBS Lett. 1992 May 11;302(2):145–150. [PubMed]
  • 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. [PubMed]
  • Brandt J, Thordal-Christensen H, Vad K, Gregersen PL, Collinge DB. A pathogen-induced gene of barley encodes a protein showing high similarity to a protein kinase regulator. Plant J. 1992 Sep;2(5):815–820. [PubMed]
  • Swanson KD, Ganguly R. Characterization of a Drosophila melanogaster gene similar to the mammalian genes encoding the tyrosine/tryptophan hydroxylase activator and protein kinase C inhibitor proteins. Gene. 1992 Apr 15;113(2):183–190. [PubMed]
  • McConnell JE, Hodges PE. The alternative 5'-end of the Drosophila melanogaster epidermal growth factor receptor cDNA (DER) is part of the D14-3-3 cDNA. Gene. 1993 Apr 30;126(2):293–294. [PubMed]
  • Martens GJ, Piosik PA, Danen EH. Evolutionary conservation of the 14-3-3 protein. Biochem Biophys Res Commun. 1992 May 15;184(3):1456–1459. [PubMed]
  • Martin H, Patel Y, Jones D, Howell S, Robinson K, Aitken A. Antibodies against the major brain isoforms of 14-3-3 protein. An antibody specific for the N-acetylated amino-terminus of a protein. FEBS Lett. 1993 Oct 4;331(3):296–303. [PubMed]
  • Nielsen PJ. Primary structure of a human protein kinase regulator protein. Biochim Biophys Acta. 1991 Mar 26;1088(3):425–428. [PubMed]
  • Prasad GL, Valverius EM, McDuffie E, Cooper HL. Complementary DNA cloning of a novel epithelial cell marker protein, HME1, that may be down-regulated in neoplastic mammary cells. Cell Growth Differ. 1992 Aug;3(8):507–513. [PubMed]
  • 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]
  • Morgan A, Roth D, Martin H, Aitken A, Burgoyne RD. Identification of cytosolic protein regulators of exocytosis. Biochem Soc Trans. 1993 May;21(2):401–405. [PubMed]
  • Robinson K, Jones D, Patel Y, Martin H, Madrazo J, Martin S, Howell S, Elmore M, Finnen MJ, Aitken A. Mechanism of inhibition of protein kinase C by 14-3-3 isoforms. 14-3-3 isoforms do not have phospholipase A2 activity. Biochem J. 1994 May 1;299(Pt 3):853–861. [PMC free article] [PubMed]
  • Morgan A, Burgoyne RD. Exo1 and Exo2 proteins stimulate calcium-dependent exocytosis in permeabilized adrenal chromaffin cells. Nature. 1992 Feb 27;355(6363):833–836. [PubMed]
  • Morgan A, Burgoyne RD. 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. [PMC free article] [PubMed]
  • Wu YN, Vu ND, Wagner PD. 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. [PMC free article] [PubMed]
  • Blackwood RA, Ernst JD. Characterization of Ca2(+)-dependent phospholipid binding, vesicle aggregation and membrane fusion by annexins. Biochem J. 1990 Feb 15;266(1):195–200. [PMC free article] [PubMed]
  • Burgoyne RD, Morgan A, O'Sullivan AJ. A major role for protein kinase C in calcium-activated exocytosis in permeabilised adrenal chromaffin cells. FEBS Lett. 1988 Sep 26;238(1):151–155. [PubMed]
  • Dunn LA, Holz RW. Catecholamine secretion from digitonin-treated adrenal medullary chromaffin cells. J Biol Chem. 1983 Apr 25;258(8):4989–4993. [PubMed]
  • Wilson SP, Kirshner N. Calcium-evoked secretion from digitonin-permeabilized adrenal medullary chromaffin cells. J Biol Chem. 1983 Apr 25;258(8):4994–5000. [PubMed]
  • Burgoyne RD, Morgan A, O'Sullivan AJ. The control of cytoskeletal actin and exocytosis in intact and permeabilized adrenal chromaffin cells: role of calcium and protein kinase C. Cell Signal. 1989;1(4):323–334. [PubMed]
  • Ali SM, Geisow MJ, Burgoyne RD. A role for calpactin in calcium-dependent exocytosis in adrenal chromaffin cells. Nature. 1989 Jul 27;340(6231):313–315. [PubMed]
  • Morgan A, Wilkinson M, Burgoyne RD. Identification of Exo2 as the catalytic subunit of protein kinase A reveals a role for cyclic AMP in Ca(2+)-dependent exocytosis in chromaffin cells. EMBO J. 1993 Oct;12(10):3747–3752. [PMC free article] [PubMed]
  • Burgoyne RD, Morgan A. Regulated exocytosis. Biochem J. 1993 Jul 15;293(Pt 2):305–316. [PMC free article] [PubMed]
  • Cheek TR, Burgoyne RD. Nicotine-evoked disassembly of cortical actin filaments in adrenal chromaffin cells. FEBS Lett. 1986 Oct 20;207(1):110–114. [PubMed]
  • Roth D, Morgan A, Burgoyne RD. Identification of a key domain in annexin and 14-3-3 proteins that stimulate calcium-dependent exocytosis in permeabilized adrenal chromaffin cells. FEBS Lett. 1993 Apr 12;320(3):207–210. [PubMed]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society


Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...


Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...