Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 12, Num. 3, 1995, pp. 178-179

REPORTE CORTO / SHORT REPORT

STRUCTURE AND FUNCTION OF 14-3-3 ISOFORMS

A. Aitken^1, Y. Patel^1, H.Martin^1, D. Jones^1, K. Robinson^1, J. Madrazo^2 and S. Howell^1.

^1Laboratory of Protein Structure, National Institute for Medical Research, The Ridgeway, Mill Hill, London, NW7 1AA, UK. ^2Center for Genetic Engineering and Biotechnology, P.O. Box 6162, La Habana 6, Cuba.

Code Number: BA95071
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ESMS of 14-3-3 proteins gave results which are in very close agreement to the theoretical values and verified the presence of N-acetylation. Two sets of sheep and chicken brain isoforms differ in mass by 80 Da. This suggests the presence of phosphorylation in specific isoforms which is conserved across a wide range of species. We are currently identifying this site of phosphorylation which is present specifically on isoforms that have been shown to interact with the oncogene-related protein, c- Raf. Two dimensional gel electrophoretic analysis of truncated 14-3-3 recombinant proteins has enabled us to identify the site of dimerisation at the amino terminus. Crystals of recombinant 14-3-3 and another protein kinase inhibitor (a Zn^2+-binding protein) have been obtained for X-ray structure analysis.

INTRODUCTION

There are seven major mammalian brain isoforms of 14-3-3 (named due to their migration position on DEAE cellulose chromatography and starch gel electrophoresis). Epithelial cells contain a specific isoform called HME1 or stratifin (Leffers et al., 1993) and a distinct isoform has been identified in T cells. The 14-3-3 family is highly conserved (homologues are found in plants, insects, amphibians, yeast, and the nematode worm, C. elegans. Many functions have been suggested for this widely distributed family of dimeric, eukaryotic proteins (reviewed in Aitken et al., 1992).

The studies of our own group have focused on 14-3-3 as a protein kinase regulator (Toker et al., 1992; Robinson et al., 1994) and analysis of subcellular localisation and function of brain 14-3-3 (Martin et al., 1994). Recent developments in our own research and in the studies of group of Frank McCormick (personal communication) have strongly implicated the involvement of 14-3-3 proteins in the MAP kinase cascade.

METHODS

We have expressed a number of these isoforms as recombinant proteins and made a variety of mutants and truncated proteins. We have raised antisera specific for acetylated synthetic peptides, based on the N-terminal sequence of mammalian 14-3-3 isoforms (Martin et al., 1993). Electrospray mass spectrometry (ESMS) on a Fisons VG Platform instrument has been used to identify post-translational modifications in specific isoforms and the site of dimerisation. On-line trapping was used to purify/desalt proteins before introduction to the ESMS source (Kay and Mallet, 1993). This comprised a Polymer Labs. (UK) poly (styrene/divinyl-benzene) PLRP-S, 8 mm particle, 300A pore size, 0.75 mm microbore column (slurry-packed in-house).

The sample was loaded on this trapping column in a low concentration of organic modifier, washed free of interfering salts, etc, with acetonitrile/water/acetic acid 15:84:1 (v/v/v) at a flow rate of 2-500 uL min^-1. Proteins were eluted with acetonitrile/water/acetic acid 50:49:1 (v/v/v) at a flow rate of 10 uL min^-1 by switching a Rheodyne valve to put this column on-line with the source.

RESULTS AND DISCUSSION

ESMS of 14-3-3 proteins gave results which are in very close agreement to the theoretical values and verified the presence of N-acetylation. Two sets of sheep and chicken brain isoforms differ in mass by 80 Da. This suggests the presence of phosphorylation in specific isoforms which is conserved across a wide range of species.

In addition, species of sheep alpha and beta with masses c.a. 100 Da higher were detected, suggesting that they are additionally expressed (as proposed by Leffers et al., 1993) using an alternative initator methionine codon six nucleotides upstream of the major initation site. With Thr (residue mass 101 Da) as the second amino acid, this initiator Met is predicted to be removed (Aitken, 1990) resulting in alternative species commencing with the amino terminal sequence, N-Ac.Thr-Met-Lys-Ser-, instead of N-Ac.Met-Lys-Ser-. The presence of the former has been verified by our new anti-peptide antiserum. We are currently identifying this site of phosphorylation which is present specifically on isoforms that have been shown to interact with the oncogene-related protein, c- Raf.

Two dimensional gel electrophoretic analysis of truncated 14-3-3 recombinant proteins has enabled us to identify the site of dimerisation at the amino terminus. Crystals of recombinant 14-3-3 and another protein kinase inhibitor (a Zn^2+-binding protein) have been obtained for X-ray structure analysis. Initial diffraction data has been obtained on the latter by the group of G. Dodson, NIMR.

ACKNOWLEDGEMENTS

This work was funded by the Medical Research Council, U.K. and the Wellcome Trust (to JM).

REFERENCES

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4. MARTIN, J.; Y. PATEL; D. JONES; S. HOWELL; K. ROBINSON; and A. AITKEN. (1993) FEBS Lett. 331: 296-303.

5. MARTIN, J; J. ROSTAS; Y. PATEL; and A. AITKEN. (1994). J. Neurochem. (in press.).

6. PATEL, Y; H. MARTIN; S. HOWELL; D. JONES; K. ROBINSON; and A. AITKEN. (1994) Biochim. Biophys. Acta 1222: 405- 409.

7. ROBINSON, K; D. JONES; Y. PATEL; H. MARTIN; J. MADRAZO; S. MARTIN; S. HOWELL; M. ELMORE; M. FINNEN; and A. AITKEN. (1994). Biochem. J. 299: 853-861.

8. TOKER, A; L. A. SELLERS; Y. PATEL; A. HARRIS; and A. AITKEN (1992). Eur. J. Biochem. 206: 453-461.

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