Evolution of signal multiplexing by 14-3-3-binding 2R-ohnologue protein families in the vertebrates

+Author Affiliations

¹MRC Protein Phosphorylation Unit, College of Life Sciences, James Black Centre, University of Dundee, Dow Street, Dundee DD1 5EH, UK
²Evolutionary Developmental Genomics Group, The Scottish Oceans Institute, University of St Andrews, East Sands, St Andrews KY16 8LB, UK

e-mail: c.mackintosh@dundee.ac.uk

↵† These authors contributed equally to this study.

Abstract

14-3-3 proteins regulate cellular responses to stimuli by docking onto pairs of phosphorylated residues on target proteins. The present study shows that the human 14-3-3-binding phosphoproteome is highly enriched in 2R-ohnologues, which are proteins in families of two to four members that were generated by two rounds of whole genome duplication at the origin of the vertebrates. We identify 2R-ohnologue families whose members share a ‘lynchpin’, defined as a 14-3-3-binding phosphosite that is conserved across members of a given family, and aligns with a Ser/Thr residue in pro-orthologues from the invertebrate chordates. For example, the human receptor expression enhancing protein (REEP) 1–4 family has the commonest type of lynchpin motif in current datasets, with a phosphorylatable serine in the –2 position relative to the 14-3-3-binding phosphosite. In contrast, the second 14-3-3-binding sites of REEPs 1–4 differ and are phosphorylated by different kinases, and hence the REEPs display different affinities for 14-3-3 dimers. We suggest a conceptual model for intracellular regulation involving protein families whose evolution into signal multiplexing systems was facilitated by 14-3-3 dimer binding to lynchpins, which gave freedom for other regulatory sites to evolve. While increased signalling complexity was needed for vertebrate life, these systems also generate vulnerability to genetic disorders.

2. Introduction

Around 500 Ma, the vertebrates emerged from a massive evolutionary upheaval that involved two rounds of whole genome duplication (2R-WGD), with additional subsequent WGDs in certain lineages of bony fish and amphibians. Compelling evidence for these events emerged only recently, when the genomic signatures of the 2R-WGD were traced from invertebrates through to humans and other vertebrates [1,2]. A key new data source is the genome sequence of amphioxus (lancelet, Branchiostoma), the least-derived living invertebrate relative of the vertebrates within the phylum Chordata. Protein-coding gene duplicates that stem from the 2R-WGD are termed 2R-ohnologues. Generally, amphioxus has one ‘ancestral’ protein for each human 2R-ohnologue family. However, losses mean that only 15 to 30 per cent of genes in modern-day humans still belong to 2R-ohnologue families containing two to four members [1,2]. This raises several important questions: why did only certain gene duplicates survive? How did they shape vertebrate evolution? And what is their impact on human health and diseases?

Lists of human 2R-ohnologues were compiled recently and mapped onto datasets of genes that underpin biochemical events and diseases [2–4]. The human 2R-ohnologues were found to be less likely than non-ohnologues to have undergone subsequent small-scale duplications. This finding is consistent with the concept that present-day 2R-ohnologues have been maintained in dosage-balanced sets. Each of these sets is thought to contribute to a common process or structure that would be upset by changing the level of one or a few components [2]. Strikingly, many 2R-ohnologue families include Mendelian disease genes, which is also in line with the gene–dosage balance hypothesis [2,4,5]. Human 2R-ohnologues are also enriched in components of growth factor and developmental signalling pathways, and preferentially expressed in the nervous system and in vertebrate-specific organs [3]. The overall impression is that balanced sets of 2R-ohnologue families supported the evolution of vertebrate specialities, while also introducing vulnerability to genetic diseases.

In addition to the hypothesized retention of 2R-ohnologues owing to dosage-balance, it is thought that duplicate genes are often retained when they diverge to gain new functions (neofunctionalization) or partition subfunctions of their ancestral gene between the duplicates [6,7]. However, domain architectures are often conserved across 2R-ohnologue families, so it seems likely that functional genetic divergence may occur in the linker sequences between the domains, which tend to evolve faster than the functional domains and are enriched in regulatory phosphorylation sites [8].

Phosphorylated motifs are conserved to different degrees within protein families and across species. Some regulations require a precisely positioned phosphorylation, whereas in other cases the density of charge matters more than position [8–13]. Many phosphorylated residues dock onto regulatory proteins whose specificities may further constrain the evolution of the phosphoprotein.

The eukaryotic 14-3-3s comprise one such family of phosphoprotein-binding proteins. Their name refers to their discovery as proteins in fraction 14-3-3 in a sequential DEAE–cellulose and starch–gel separation of brain extract. 14-3-3s are dimers that dock onto specific pairs of phosphorylated serine and threonine residues on many proteins. These targets include human proteins that are linked to metabolic and neurological disorders, and to cancer [14]. By docking onto two phosphorylated residues that may be phosphorylated by different kinases, a 14-3-3 dimer can act as a logic gate that integrates two inputs. The bound 14-3-3 dimer may mask a functional domain in the target protein, or induce a conformational change [14–17]. Thus, a 14-3-3 dimer is a protein device that integrates two kinase signalling inputs and exerts a mechanical action on the target.

14-3-3-binding motifs generally have at least one basic residue in the −3 to −5 positions relative to the phosphorylated serine or threonine, and never a +1 proline [14,17]. Such sequences are phosphorylated by AGC (protein kinase A/protein kinase G/protein kinase C family) and CAMK (Ca^2+/calmodulin-dependent protein kinase) protein kinases, including PKA (protein kinase A), Akt/PKB (protein kinase B), SGK (serum and glutocorticoid-regulated kinase), p90RSK (90 kDa ribosomal protein S6 kinase), PKCs (protein kinase C family members) and AMPK (AMP-activated protein kinase) [18]. Therefore, 14-3-3s mediate cellular responses to insulin, growth factors and other stimuli that activate these kinases [14,19].

Recent studies of two sister Rab-GTPase activating proteins (AS160/TBC1D4 and TBC1D1) that regulate glucose uptake into muscles inspired speculation that 14-3-3 dimers could provide an evolutionary mechanism for the regulatory divergence of their targets [14]. AS160 and TBC1D1 each contain two 14-3-3-binding sites: one site is similar in both proteins, but the second site differs between them. The 14-3-3-binding site common to each protein is an insulin-regulated Akt/PKB-phosphorylated site. The second site on AS160 is phosphorylated by Akt/PKB and p90RSK, whereas on TBC1D1 it is phosphorylated by kinases including AMPK, which is activated in energy-depleted cells [20–22]. It is therefore inferred that AS160 and TBC1D1 have complementary roles in regulating glucose homoeostasis in response to insulin and energy stress, respectively [23].

Accordingly, we proposed the lynchpin hypothesis. Suppose that an ancestral 14-3-3-binding protein were duplicated. Then, if one 14-3-3-binding site remained unchanged, it could provide a ‘lynchpin’ whose binding to a 14-3-3 dimer might provide sufficient intracellular control to permit the second 14-3-3-binding site to evolve into a consensus for phosphorylation by a different protein kinase. The result would be two proteins with the same function, but regulated by different signalling inputs [14].

Here, we wished to progress from an anecdotal to a more systematic analysis. Therefore, we classified human proteins for which 14-3-3-binding sites have been reported, and discovered that the majority are also 2R-ohnologues. Sequence alignments indicated interesting patterns of conservation and divergence of 14-3-3-binding sites across 2R-ohnologue families. We investigated these in the REEP protein family, which includes the Hereditary Spastic Paraplegia 31 protein REEP1. Our findings have implications for understanding the evolution of vertebrates, and also for certain neurodevelopmental disorders, metabolic diseases and cancer. Further, we suggest a conceptual model for considering intracellular regulation in terms of multiple-input multiple-output (MIMO) array systems.

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Michele Tinti1,†, Catherine Johnson1,†, Rachel Toth1, David E. K. Ferrier2 and Carol MacKintosh1 (2012). Evolution of signal multiplexing by 14-3-3-binding 2R-ohnologue protein families in the vertebrates Open Biology : 10.1098

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Evolution of signal multiplexing by 14-3-3-binding 2R-ohnologue protein families in the vertebrates.