Jones D H, Martin H, Madrazo J, Robinson K A, Nielsen P, Roseboom P H, Patel Y, Howell S A, Aitken A
Laboratory of Protein Structure, National Institute for Medical Research, London, U.K.
J Mol Biol. 1995 Jan 27;245(4):375-84. doi: 10.1006/jmbi.1994.0031.
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.
14-3-3蛋白家族在多种细胞功能中发挥作用，包括蛋白激酶C的调节和胞吐作用。使用先前描述的针对14-3-3亚型N端的抗血清以及另一种针对ε亚型C端的抗血清，对完整的14-3-3进行蛋白酶消化，结果表明14-3-3的N端半段（一个16 kDa的片段）是该蛋白的完整二聚体结构域。14-3-3的两种亚型，即tau和ε，在大肠杆菌中表达，通过圆二色性显示它们的二级结构与野生型蛋白相同，并且N端缺失的ε 14-3-3蛋白的表达表明N端的26个氨基酸对二聚化很重要。完整的14-3-3是蛋白激酶C的有效抑制剂，但N端结构域不抑制PKC活性。对14-3-3的tau亚型中的几个区域进行位点特异性诱变，包括将一个假定的假底物位点突变为潜在的底物序列，并没有改变其抑制活性。完整的14-3-3蛋白被蛋白激酶C以低化学计量比磷酸化，但截短的亚型被该激酶磷酸化的效率要高得多。这可能意味着这些蛋白可能在与膜结合时采取不同的结构构象，从而调节它们的活性。在突触质膜上发现高浓度的14-3-3蛋白，这种结合是通过14-3-3的N端12 kDa介导的。
