EXPRESSION AND STRUCTURAL-ANALYSIS OF 14-3-3-PROTEINS

The 14-3-3 family of proteins plays a role in a wide variety of cellul ar 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 d omain of the protein. Two isoforms of 14-3-3, tau and epsilon, were ex pressed in E. coli and their secondary structure was shown by circular dichroism to be identical to wild-type protein, and expression of N-t erminally-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 inh ibit PKC activity Site-specific mutagenesis of several regions in the tau isoform of 14-3-3, including the mutation of a putative pseudosubs trate site to a potential substrate sequence, did not alter its inhibi tory activity. Intact 14-3-3 proteins are phosphorylated by protein ki nase C with a low stoichiometry, but truncated isoforms are phosphoryl ated much more efficiently by this kinase. This may imply that the pro teins may adopt a different structural conformation, possibly upon bin ding to the membrane, which could modulate their activity 14-3-3 prote ins are found at high concentration on synaptic plasma membranes and t his binding is mediated through the N-terminal 12 kDa of 14-3-3.