Biotecnologia Aplicada Elfos Scientiae ISSN: 0684-4551 Vol. 12, Num. 3, 1995, pp. 166-167

REPORTE CORTO / SHORT REPORT

EXPRESSION AND STRUCTURAL ANALYSIS OF 14-3-3 PROTEINS

Joel Madrazo,^1 David Jones,^2 Harry Martin,^2 Karen Robinson,^3 Peter Nielsen,^3 Patrick Roseboom,^4 Yasmina Patel,^5 Steven Howell^5 and Alastair Aitken^5.

^1CIGB, P.O. Box 6162, La Habana 6, C.P.10600, Cuba.^ 2Laboratory of Protein Structure, National Institute for Medical Research, Mill Hill, London NW7 1AA, UK. ^3Current Address, Proteus Molecular Design Ltd., Lyme Green Business Park, Macclesfield, Cheshire, SK11 OJL, UK. ^ 4MPI fur Immunobiologie, Stubeweg 51, P.O. Box 1169, D-7800 Freiburg-Zahringen, Germany.^ 5National Institute for Child Health and Human Development, NIH, Bethesda, MD 208992, USA.

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In this study we have used isoform-specific antibodies to analyse the domain structure of members of the 14-3-3 family after digestion with proteases. We concentrated on two isoforms of 14-3-3: tau, which is found at low levels in all tissues tested to date, and epsilon, which is found at high levels in brain and other tissues. Intact tau isoform and various deleted forms of tau were expressed in E. coli. Regions of the protein involved in dimerisation and membrane attachment were determined, and the nature of the phosphorilation by protein kinase C was analysed. In this way we have started to dissect the structure of 14-3-3 proteins and their function as regulators of protein kinase C.

INTRODUCTION

The 14-3-3 family of proteins was so named due to its migration on position on two-dimensional DEAE and gel electrophoresis (1). These proteins all have a molecular mass of around 30 kDa and exist as dimmers (2). To date, seven to eight mammalian brain isoforms of 14-3-3 have been described, named alpha-eta after their respective elution positions on HPLC (3). Five of these have been sequenced (4) and the alpha and delta isoforms are identical in primary structure to the beta and zeta isoforms respectively, but differ only in a post-translational modifications (5). The 14-3-3 family is highly conserved and individual isoforms differ by 1 to 5 mainly conservative amino acid substitutions. Isoforms have also been described from other mammalian tissues which are absent or present at low levels in the brain. These include an isoform found in T-cells (6) and one found in epithelial cells (7, 8). These have been named tau and epsilon respectively (5).

In this multi-disciplinary study we have used isoform-specific antibodies to analyse the domain structure of members of the 14-3-3 family after digestion with proteases. We concentrated on two isoforms of 14-3-3: tau, which is found at low levels in all tissues tested to date, and epsilon, which is found at high levels in brain and other tissues. Intact tau isoform and various deleted forms of tau were expressed in E. coli. Regions of the protein involved in dimerisation and membrane attachment were determined, and the nature of the phosphorilation by protein kinase C was analysed. In this way we have started to dissect the structure of 14-3-3 proteins and their function as regulators of protein kinase C.

RESULTS AND DISCUSSION

Using antisera specific for the N-termini of 14-3-3 isoforms described previously and an additional antiserum specific for the C-terminus of epsilon isoform, protease digestion of intact 14-3-3 showed that the N-terminal half of 14-3-3 (a 16 kDa fragment) was an intact, dimeric domain of the protein. This was confirmed by electrospray mass spectrometry.

Two isoforms of 14-3-3, tau and epsilon, were expressed in E. coli and secondary structure was shown by circular dichroism to be identical to wild-type protein. Expression of N-terminally-deleted epsilon 14-3-3 protein showed that the N-terminal 26 amino acids are important for dimerisation. Intact 14-3-3 is a potent inhibitor of protein kinase C, but the N-terminal domain does not inhibit PKC activity. Site-specific mutagenesis of several regions in the N-terminal of the tau isoform of 14-3-3 did not alter its inhibitory activity. 14-3-3 proteins are found at high concentration on synaptic plasma membranes. This binding is mediated through the N-terminal 12 kDa of 14-3-3. Intact 14-3-3 are phosphorilated by protein kinase C with a low stoichiometry, but truncated isoforms are phosphorilated much more efficiently by this kinase. This may imply that the proteins may adopt a different structural conformation, possibly upon binding to the membrane, which could modulate their activity.

REFERENCES

1. MOORE, B. W.; and V. J. PEREZ (1968) In: Physiological and Biochemical Aspects of Nervous Integration (Carlson, F. D. ed) 343-359, Prentice-Hall.

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

3. ICHIMURA, T.; T. ISOBE; T. OKUYAMA; N. TAKAHASHI; K. ARAKI; R. KUWANO and Y. TAKAHASHI. (1988). Proc. Natl. Acad. Sci. USA 85: 7084-7088.

4. AITKEN, A.; D. B COLLINGE; G. P. H VAN HEUSDEN; P. H. ROSEBOOM; T. ISOBE; G. ROSENFELD and J. SOLL (1992) Trends in Biochemical Science 17: 498-501.

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

6. NIELSEN;P. J.; (1991) BIOCHIM. BIOPHYS. ACTA 1088: 425-428.

7. LEFFER, H.; P. MADSEN et al. (1993) J. Mol. Biol. 231: 982-998.

8. PRASAD, G. L.; E. M. VALVARIUS; E. MCDUFFIE and H. L. COOPER (1992) Cell Growh Differ. 3: 507-513.

Copyright 1995 Sociedad Iberolatinamericana de Biotecnologia Aplicada a la Salud