In Vivo Functional Specificity and Homeostasis of Drosophila 14-3-3 Proteins

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The functional specialization or redundancy of the ubiquitous 14-3-3 proteins constitutes a fundamental question in their biology and stems from their highly conserved structure and multiplicity of coexpressed isotypes.We address this question in vivo using mutations in the two Drosophila 14-3-3 genes, leonardo (14-3-3ζ) and D14-3-3ε. We demonstrate that D14-3-3ε is essential for embryonic hatching. Nevertheless, D14-3-3ε null homozygotes survive because they upregulate transcripts encoding the LEOII isoform at the time of hatching, compensating D14-3-3ε loss. This novel homeostatic response explains the reported functional redundancy of the Drosophila 14-3-3 isotypes and survival of D14-3-3ε mutants. The response appears unidirectional, as D14-3-3ε elevation upon LEO loss was not observed and elevation of leo transcripts was stage and tissue specific. In contrast, LEO levels are not changed in the wing disks, resulting in the aberrant wing veins characterizing D14-3-3ε mutants. Nevertheless, conditional overexpression of LEOI, but not of LEOII, in the wing disk can partially rescue the venation deficits. Thus, excess of a particular LEO isoform can functionally compensate for D14-3-3ε loss in a cellular-context-specific manner. These results demonstrate functional differences both among Drosophila 14-3-3 proteins and between the two LEO isoforms in vivo, which likely underlie differential dimer affinities toward 14-3-3 targets.

A fundamental issue concerning members of highly conserved protein families is the extent to which they are functionally redundant or exhibit specialized biological functions. The 14-3-3 proteins compose a highly conserved family of acidic molecules present in all eukaryotes (Aitken 1995; Wang and Shakes 1996; Rosenquist et al. 2000). 14-3-3's share a common structure composed of nine antiparallel a-helices forming a horseshoe shape with a negatively charged interior surface (Fu et al. 2000; Tzivion et al. 2001; Aitken et al. 2002; Bridges and Moorehead 2005; Van Heudsen 2005; Coblitz et al. 2006). Interactions among particular amino acids in the first helix, with ones in helix 2 and helix 3 of another monomer, promote dimerization (Luo et al. 1995; Xiao et al. 1995; Fu et al. 2000; Van Heudsen 2005).Dimerization generates a tandem binding surface, which can simultaneously bind to one or two sites on one target protein or to sites on two different client molecules. The dimers bind clients containing phosphoserine- or phosphothreonine-containing motifs via highly conserved amino acids within the groove (Muslin et al. 1996; Yaffe and Elia 2001; Tzivion and Avruch 2002). 14-3-3 proteins can also bind targets with surfaces outside the conserved phosphopeptidebinding cleft (Benton et al. 2002; Wilker et al. 2005). 14-3-3 binding may allosterically stabilize conformational changes, leading to activation or deactivation of the target or to interaction between two proteins (Yaffe 2002). Furthermore, 14-3-3 binding may mask or expose interaction sites, often leading to changes in the subcellular localization of client proteins (Van Hemert et al. 2001; Aitken et al. 2002; Bridges andMoorehead 2005; Van Heudsen 2005).

An extraordinary feature of this protein family is the high sequence conservation among isotypes, characterized by long stretches of invariant amino acids (Wang and Shakes 1996; Gardino et al. 2006), suggesting functional redundancy. However, despite this extensive sequence identity, multiple 14-3-3 proteins exist in metazoans, indicating at least some functional specificity. Vertebrates contain seven distinct protein isotypes, b, e, z, g, h, u, and s (Aitken et al. 1995). In vertebrate brains where these proteins are highly abundant, there is some specificity in isotype distribution, but generally 14-3-3's are expressed in complex overlapping patterns (Martin et al. 1994; Baxter et al. 2002). In addition, multiple heterodimers are possible in tissues that contain more than one isotype (Jones et al. …