The 14-3-3 protein is an essential component of cyclic AMP signaling for regulation of chemotaxis and development in Dictyostelium

Highlights

• 14-3-3 is a critical component of cAMP signaling in Dictyostelium.

• 14-3-3 regulates cAMP signaling through binding to a hundred of proteins.

• The 14-3-3 interactome controls chemotaxis and Dictyostelium development.

Abstract

The evolutionarily-conserved 14-3-3 proteins regulate many cellular processes through binding to various phosphorylated targets in eukaryotes. It first appears in Dictyostelium, however its role in this organism is poorly understood. Here we show that down-regulation of the 14-3-3 impairs chemotaxis and causes multiple-tip formation in Dictyostelium. Mechanistically, the 14-3-3 is a critical component of cyclic AMP (cAMP) signaling and binds to nearly a hundred of proteins in Dictyostelium, including a number of evolutionarily-conserved proteins. 14-3-3 − interaction with its targets is up-regulated in response to developmental cues/regulators including starvation, osmotic stress and cAMP. cAMP stimulates 14-3-3 − binding to phospho-Ser431 on a guanine nucleotide exchange factor Gef-Q. Interestingly, overexpression of Gef-Q^Ser431Ala mutant but not wild-type Gef-Q protein causes a multiple-tip phenotype in Dictyostelium, which partially resembles phenotypes of the 14-3-3 − deficient mutant. Collectively, these data demonstrate that the 14-3-3 plays an important role in Dictyostelium and may help to deepen our understanding of the evolution of 14-3-3 − interactomes in eukaryotes.

Introduction

Protein phosphorylation is an important type of protein post-translational modification and controls cellular activities in response to various internal and external cues [1]. Phosphorylation of proteins often recruits adaptor/regulatory proteins that can bind to phosphorylated residues. These phosphorylation-dependent interactions are key nodes in signaling transduction [2].

14-3-3 proteins are a group of conserved regulatory proteins that form dimers and bind to phosphorylated serine/threonine residues on target proteins [3]. Upon binding to their targets, 14-3-3 proteins can modulate location, or conformational changes, or enzymatic alterations of target proteins [4]. In human, 14-3-3 − binding phosphoproteins are highly enriched in 2R-ohnologue families in which protein-coding gene duplicates stem from two rounds of whole genome duplication (2R-WGD) when the vertebrates emerged. The 14-3-3 − binding to these 2R-ohnologues may facilitate their evolution into signal multiplexing systems through a ‘lynchpin hypothesis’ [5]. In this hypothesis, an ancestral 14-3-3 − binding protein duplicated during the 2R-WGD, and the resultant 2R-ohnologues kept one 14-3-3 − binding site on them unchanged (a so-called ‘lynchpin’) and allowed a second 14-3-3 − binding site to evolve to respond to a different upstream protein kinase. One example for such a 14-3-3 − binding 2R-ohnologue family consists of two related Rab GTPase activating proteins AS160 and TBC1D1. The lynchpin on AS160 mediating 14-3-3 − binding is under control of protein kinase B (PKB) and required for insulin-stimulated glucose uptake [6]. In addition to the lynchpin, TBC1D1 contains a second 14-3-3 − binding site that is regulated by AMP-activated protein kinase (AMPK) and involved in regulation of secretion of insulin-like growth factor-1 [7].

14-3-3 proteins are only found in eukaryotic organisms, and themselves belong to 2R-ohnologue families [8]. More 14-3-3 isoforms emerged in the vertebrates than those in lower eukaryotic organisms such as yeast and Drosophila in which only two 14-3-3 isoforms exist. Dictyostelium discoideum is a soil amoeba, whose ancestor diverged from the lineage leading to animals at about the same time as the plant/animal divergence [9]. Dictyostelium encodes only one 14-3-3 isoform in its genome [10]. These features make Dictyostelium a very useful system for understanding of 14-3-3 biology and evolution of 14-3-3 interactome. In the life cycle of Dictyostelium, it can exist as a unicellular amoeba when nutrients are rich, and develops into a multicellular fruiting body when nutrients are sparse. Upon starvation, Dictyostelium cells aggregate towards a signaling center that produces pulsatile cAMP waves, and aggregated cells then form a mound that subsequently develops into a slug-shaped structure. Prestalk cells in the slug differentiate into a basal disk and a stalk, which lift up encapsulated spores that are differentiated from prespore cells in the slug. This process of culmination results in the formation of the fruiting body that helps Dictyostelium to survive upon adverse environments such as nutrient deficiency [11]. cAMP plays key roles during Dictyostelium development through regulating protein kinase A (PKA) [12] and PKB [13,14]. It not only guides cellular aggregation as a chemoattractant to form the signaling center, but also functions to regulate prespore differentiation [12]. In higher eukaryotes, both PKA and PKB can phosphorylate target proteins and promote 14-3-3 − binding to its phosphorylated substrates [5]. This suggests that 14-3-3 proteins may be involved in Dictyostelium development via regulating cAMP signaling.

Here, we present evidence showing that Dictyostelium 14-3-3 (d14-3-3) plays important roles in cAMP signaling during Dictyostelium development. Our data also reveal both unique and conserved features of 14-3-3 interactome in Dictyostelium as compared to that in human, highlighting the importance of Dictyostelium in understanding the evolution of 14-3-3 interactome.