Earliest Holozoan expansion of phosphotyrosine signaling

Abstract

Phosphotyrosine (pTyr) signaling is involved in development and maintenance of metazoans' multicellular body through cell-to-cell communication. Tyrosine kinases (TKs), tyrosine phosphatases, and other proteins relaying the signal compose the cascade. Domain architectures of the pTyr signaling proteins are diverse in metazoans, reflecting their complex intercellular communication. Previous studies had shown that the metazoan-type TKs, as well as other pTyr signaling proteins, were already diversified in the common ancestor of metazoans, choanoflagellates, and filastereans (which are together included in the clade Holozoa) whereas they are absent in fungi and other nonholozoan lineages. However, the earliest-branching holozoans Ichthyosporea and Corallochytrea, as well as the two fungi-related amoebae Fonticula and Nuclearia, have not been studied. Here, we analyze the complete genome sequences of two ichthyosporeans and Fonticula, and RNAseq data of three additional ichthyosporeans, one corallochytrean, and Nuclearia. Both the ichthyosporean and corallochytrean genomes encode a large variety of receptor TKs (RTKs) and cytoplasmic TKs (CTKs), as well as other pTyr signaling components showing highly complex domain architectures. However, Nuclearia and Fonticula have no TK, and show much less diversity in other pTyr signaling components. The CTK repertoires of both Ichthyosporea and Corallochytrea are similar to those of Metazoa, Choanoflagellida, and Filasterea, but the RTK sets are totally different from each other. The complex pTyr signaling equipped with positive/negative feedback mechanism likely emerged already at an early stage of holozoan evolution, yet keeping a high evolutionary plasticity in extracellular signal reception until the co-option of the system for cell-to-cell communication in metazoans.

Keywords: corallochytreans; evolution; ichthyosporeans; multicellularity; tyrosine kinase.

FIG. 1

Phylogeny of eukaryotes. Schema of a widely accepted eukaryotic phylogeny. Genera described in this study are indicated after the hyphens. Blue and orange boxes represent the Opisthokonta and the Holozoa, respectively.

FIG. 2

Ichthyosporean and corallochytrean TKs. TKs found in the whole genome data of two ichthyosporeans (Creo, Creolimax; Spha, Sphaeroforma) and the RNAseq data (asterisks) from three other ichthyosporeans (Abeo, Abeoforma; Piru, Pirum; Amoe, Amoebidium), and a corallochytrean (Cora, Corallochytrium) are classified according to their domain architectures and phylogenetic positions. CTKs are classified into ichthyosporean-specific families (IchCTK1-4) or corallochytrean-specific families (CorCTK1) unless they are homologous to metazoan CTKs. The domain architecture of a family member is schematically shown. The number of genes within each family is shown on the right (red and blue shades for ichthyosporeans and corallochytreans, respectively). See supplementary figure S1, Supplementary Material online, for the full list of annotated TKs. ANF, atrial natriuretic factor receptor-like ligand binding region; ANK, ankyrin repeats; BTK, Bruton’s tyrosine kinase Cys-rich motif; CLECT, C-type lectin (CTL) or carbohydrate-recognition domain (CRD); EF-hand, EF-hand-like domain; EGF, epidermal growth factor-like domain; ENTH, epsin N-terminal homology domain; FCH, Fes/CIP4 homology domain; I1, I1 domain; I3, I3 domain; Kelch, Kelch motif; LRR, leucine-rich repeat; Myr, predicted myristoylation site; PH, Pleckstrin homology domain; PLCXc, phospholipase C catalytic domain X; SH2, Src homology 2 domain; SH3, Src homology 3 domain; Sig, signal peptide; TM, transmembrane segment; TNFR, tumor necrosis factor receptor/nerve growth factor receptor repeat; TSP1, thrombospondin type 1 repeats; TyrKc, tyrosine kinase catalytic domain. Incomplete sequences are shown by dotted lines.

FIG. 3

Complex architecture of pTyr signaling proteins in holozoans. Domain is represented by a circle, whose size is proportional to the domain count (more than 25 counts are shown in identical size). Each line directly connecting two domains represents their co-occurrence in single proteins, and its width is proportional to the frequency of co-occurrence (more than 25 connections are shown in identical width). Duplicated domains in a protein were not counted. The four core pTyr signaling modules are in red, while the group B TK catalytic domain is in turquoise. SMART or Pfam domain names are shown, except for TK-A (group A TK catalytic domain), TK-B (group B TK catalytic domain), and TM (transmembrane segment). Names of extracellular and intracellular domains are shown in violet and green, respectively. Holozoan and nonholozoan data are divided by a dotted line. Asterisk, RNAseq data.

FIG. 4

Phylogenetic tree of the TK family. Branches with low support (bootstrap values <10%) are collapsed and represented by multifurcated nodes. The full tree showing each gene name and bootstrap value is in the supplementary figure S6, Supplementary Material online. Seven STKs are used as an outgroup. TK families are highlighted by grey shading. Group A and group B TKs are shaded in green and red, respectively. The subtree containing all RTKs and a few CTKs of ichthyosporeans is represented by a red triangle, which is fully shown in supplementary figure S8, Supplementary Material online. Presence of orthologs of 10 common metazoan CTK families in ichthyosporeans (I), a corallochytrean (Co), filastereans (F), and choanoflagellates (Ch) are shown on the right. Orthologs of metazoan RTK were not identified in premetazoans, as shown in the abbreviated row “Metazoan RTK families”. †Ortholog not found in the Monosiga brevicollis genome, but identified in Codonosiga gracilis (Suga, Sasaki, et al. 2008). §A CTK with domain architecture similar to Jak is present in M. brevicollis, but its TK domain is phylogentically close to that of Syk (Suga et al. 2012).

FIG. 5

Clade-specific RTK expansion in the Ichthyosporea. The phylogenetic tree was inferred from the TK sequences of Creolimax (C) and Sphaeroforma (S). The family classification is represented by symbols. A typical structure of each family is shown at the bottom. Likely orthologous divergences of Creolimax and Sphaeroforma are labeled by black dots. One of the most parsimonious scenarios of domain architecture alteration was manually inferred based on the assumption that the ancestral gene belongs to the most dispersed RTK1 family. Red bars indicate branches where an architectural alteration is assumed. The full figure with bootstrap values and domain architectures is in supplementary figure S9, Supplementary Material online.

FIG. 6

Evolution of the holozoan TK diversity. The history of family diversification of the metazoan-type (group A) TK is schematically represented. The tree is based on the time-calibrated phylogeny by Bayesian inference. Blue bars represent the 95% range of highest probability density (Drummond et al. 2012) of the node age. Red and blue circles represent extensive TK diversification and massive reduction of TK diversity, respectively. The period of RTK diversification in Abeoforma whisleri and Pirum gemmata remains unclear (red dotted circle), because RNAseq data may represent a minor fraction of TK diversity. The open arrowhead represents the evolution of the complex pTyr-mediated signaling system with the diversified pTyr signaling modules, as well as their enhanced combination. Note that we do not exclude the possibility of basal holozoan expansion of group A RTKs (green dotted circle), which may be obscured today by their rapid turnover during the holozoan evolution. Amoe, Amoebidium; Abeo, Abeoforma; Creo, Creolimax; Cora, Corallochytrium; Caps, Capsaspora; Font, Fonticula; Mini, Ministeria; Mono, Monosiga brevicollis; Nucl., Nuclearia; Piru, Pirum; Spha, Sphaeroforma; Salp, Salpingoeca. Species that were newly analyzed in this study are underlined, with the RNAseq data labeled with asterisks.