Expression and structural analysis of 14-3-3 proteins.
The 14-3-3 family of proteins plays a role in a wide variety of cellular functions including regulation of protein kinase C and exocytosis. 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. Two isoforms of 14-3-3, tau and epsilon, were expressed in E. coli and their secondary structure was shown by circular dichroism to be identical to wild-type protein, and expression of N-terminally-deleted epsilon 14-3-3 protein showed that the N-terminal 26 amino acids are important for dimerization. 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 tau isoform of 14-3-3, including the mutation of a putative pseudosubstrate site to a potential substrate sequence, did not alter its inhibitory activity. Intact 14-3-3 proteins are phosphorylated by protein kinase C with a low stoichiometry, but truncated isoforms are phosphorylated 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. 14-3-3 proteins are found at high concentration on synaptic plasma membranes and this binding is mediated through the N-terminal 12 kDa of 14-3-3.
Laboratory of Protein Structure, National Institute for Medical Research, London, U.K.
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Jones
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Martin
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Madrazo
K A
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Robinson
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Nielsen
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Roseboom
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Patel
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Howell
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A
Aitken
D048948:14-3-3 Proteins; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers; D004926:Escherichia coli / Q000235:genetics; D008565:Membrane Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D010766:Phosphorylation; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011493:Protein Kinase C / Q000378:metabolism; D017433:Protein Structure, Secondary; D011506:Proteins / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins; D017384:Sequence Deletion; D012756:Sheep; D014446:Tyrosine 3-Monooxygenase
D048948:14-3-3 Proteins; D017931:DNA Primers; D008565:Membrane Proteins; D011506:Proteins; D011994:Recombinant Proteins; D014446:Tyrosine 3-Monooxygenase; D011493:Protein Kinase C