A Combined Proteome and Ultrastructural Localization Analysis of 14-3-3 Proteins in Transformed Human Amnion (AMA) Cells

Abstract

The 14-3-3 proteins constitute a family of highly conserved and broadly expressed multifunctional polypeptides that are involved in a variety of important cellular processes that include cell cycle progression, growth, differentiation, and apoptosis. Although the exact cellular function(s) of 14-3-3 proteins is not fully elucidated, as a rule these proteins act by binding to protein ligands, thus regulating their activity; so far more than 300 cellular proteins have been reported to interact with 14-3-3 proteins. Binding to cognate interacting partners is isoform-specific, but redundancy also exists as several binding peptides can be recognized by all isoforms, and some functions can be carried out by any isoform indistinctly. Moreover by interacting with different ligands in a spatially and temporally regulated fashion the same isoform can play multiple possibly even opposing roles where the resultant cellular outcome will be determined by the integration of the various effects. Although there is a large body of literature on specific aspects of 14-3-3 biology, not much is known on the coordinated aspects of 14-3-3 isoform expression, post-translational modifications, and subcellular localization. To address the question of isoform-specific differences, we carried out a comparative analysis of the patterns of expression, phosphorylation, and subcellular localization of the 14-3-3 ␤, , , , and protein isoforms in transformed human amnion (AMA) cells. To validate as well as broaden our observations we analyzed the occurrence of the various isoforms in a large number of established cell lines and mammary and urothelial tissue specimens. Given the systematic approach we undertook and our application of isoform-discriminating technologies to the analysis of various cellular systems, we expect the data presented in this study to serve as an enabling resource for researchers working with 14-3-3

14-3-3 Proteins are Regulators of Autophagy

“…A large number of 14-3-3 binding motifs have been established [5], and these have recently been reviewed [6]. Seven mammalian 14-3-3 isoforms have been reported (α/β, ε, γ, σ, ζ/δ, τ/θ and η) that are encoded by seven different genes and vary in their expression levels between tissues [7,8,9]. Moreover, highly redundant functions exist between the different 14-3-3 isoforms.…”

Three-way Interaction between 14-3-3 Proteins, the N-terminal Region of Tyrosine Hydroxylase, and Negatively Charged Membranes

“…In addition, different expression levels of 14-3-3 isoforms (seven in humans) (7) have been reported (e.g. the 14-3-3 isoform is nearly absent in many tissues, including brain, but expressed at the level of actin in keratinocytes) (8). The 14-3-3 proteins exert their function as homo-or heterodimers (1,2,9), often by binding one or two partners, which are phosphorylated at Ser/Thr residues within specific sequence motifs (10 -12).…”

Protein intrinsic disorder and network connectivity. The case of 14-3-3 proteins

“…Although originally the different isoforms of the 14-3-3 family were thought to be redundant, the fact that seven functional paralogs are strictly conserved within all mammalian species raised questions about their roles and specificity (Moreira et al, 2008; Paul et al, 2012). Yeast and human isoforms failed to reveal any isotype-specific phosphopeptide-binding activity in a full in vitro assay (Panni et al, 2011); however, there are many reports containing examples of in vivo isoform specificity (see Table 1 in Sun et al, 2009).…”

“…Because of their ubiquitous conservation, it seems reasonable to think that each 14-3-3 isoform has (at least one) specific function, probably product of subfunctionalization or subneofunctionalization [a combination of neofunctionalization and subfunctionalization (He and Zhang, 2005)]. Several studies have shown tissue and/or cell cycle phase specific expression of 14-3-3 isoforms (Moreira et al, 2008). Structural data show little divergence in the phosphopeptide-binding pockets of the 14-3-3 paralogs (Yang et al, 2006), and because most 14-3-3 binding motifs conform to a few consensus sequences, it seems that isoform specificity does not reside in the binding site of the 14-3-3 partners (Uhart et al, 2011).…”

“…The preservative role of subfunctionalization in humans and other higher eukaryotes is the result of mild mutations likely to cause a differential expression, regulation, and/or function in gene duplicates (Fernández et al, 2012), which could be the case within the 14-3-3 protein family. 14-3-3 paralogs differential expression has been documented for specific tissues, cell types, subcellular localization, and even cell cycle phase (Moreira et al, 2008). Additionally, the isoforms are differentially regulated by specific kinases (Kjarland et al, 2006), which in combination with the regulation of their expression (through time, relative amounts, and tissue, cell or subcellular localization) all contribute to fine tune their specific functions.…”