A receptor tyrosine kinase from choanoflagellates: molecular insights into early animal evolution

Abstract

The evolution of the Metazoa from protozoans is one of the major milestones in life's history. The genetic and developmental events involved in this evolutionary transition are unknown but may have involved the evolution of genes required for signaling and gene regulation in metazoans. The genome of animal ancestors may be reconstructed by identification of animal genes that are shared with related eukaryotes, particularly those that share a more recent ancestry and cell biology with animals. The choanoflagellates have long been suspected to be closer relatives of animals than are fungi, the closest outgroup of animals for which comparative genomic information is available. Phylogenetic analyses of choanoflagellate and animal relationships based on small subunit rDNA sequence, however, have yielded ambiguous and conflicting results. We find that analyses of four conserved proteins from a unicellular choanoflagellate, Monosiga brevicollis, provide robust support for a close relationship between choanoflagellates and Metazoa, suggesting that comparison of the complement of expressed genes from choanoflagellates and animals may be informative concerning the early evolution of metazoan genomes. We have discovered in M. brevicollis the first receptor tyrosine kinase (RTK), to our knowledge, identified outside of the Metazoa, MBRTK1. The architecture of MBRTK1, which includes multiple extracellular ligand-binding domains, resembles that of RTKs in sponges and humans and suggests the ability to receive and transduce signals. Thus, choanoflagellates express genes involved in animal development that are not found in other eukaryotes and that may be linked to the origin of the Metazoa.

Figure 1

Congruent evidence from four proteins for a close relationship between choanoflagellates and Metazoa. Phylogenetic trees inferred by MP analyses of EF-2 (a), β-tubulin (b), α-tubulin (c), and actin (d) sequences from M. brevicollis (underlined), metazoans (red), fungi (blue), plants (green), and protists (black). For the nodes defining the Metazoan clade in a and b, bootstrap values for MP (1,000 replicates), ME (1,000 replicates), and reliability values for quartet puzzling (QP; 10,000 puzzling steps) are indicated in the open box. Nodes defining choanoflagellates + Metazoa are supported by values shown in the closed box. For nodes defining Fungi, Metazoa + Fungi, and Plants or Plants/Green Algae, bootstrap values for MP and ME are shown above and below the nodes, respectively. For b–d, branches with <50% bootstrap support are collapsed.

Figure 2

Mbrtk1 encodes a receptor tyrosine kinase. (a) Conceptual translation of a 3,419-bp Mbrtk1 cDNA sequence predicts a 1,061-aa polypeptide chain containing a Ca-binding EGF motif (green), an Ig/MHC motif (gray), two EGF-like repeats (violet), and a tyrosine kinase domain (red), in addition to a single transmembrane domain (yellow) and six tandem MR repeats (blue). The positions of three short introns (77–150 bp) are indicated (arrowhead). Introns interrupt sequences encoding the tyrosine kinase domain and sequences 3′ of the MR repeats. The amino terminus of MBRTK1 is not full length. (b) M. brevicollis MBRTK1 contains modules found in sponge and human receptor tyrosine kinases. Modules shared between MBRTK1 and RTKs from sponges (G.c., Geodia cydonium; E.f., Ephydatia fluviatilis; S.r., Sycon raphanus) and human (H.s.) are colored as in a, along with Fibronectin III domains (pink) and a SAM domain (black). (c) Alignment of select EGF-like repeats. (Top) Residues 11–59 of MBRTK1 aligned with Ca-binding EGF-motifs from Rattus norvegicus (R.n.), Eimeria tenella (E.t.), and human (H.s.). (Middle) Residues 145–190 of MBRTK1 aligned with EGF-like motifs from Mus musculus (M.m.), and Stronglyocentrotus purpuratus (S.p.). (Bottom) Residues 214–234 of MBRTK1 aligned with EGF-like motifs from Caenorhabditis elegans (C.e.), Sus scrofa (S.s.), and Branchiostoma floridae (B.f.). Highly conserved residues are indicated in color, similar residues are highlighted in gray, and diagnostic residues are boxed. (d) Alignment of the tyrosine kinase domain from M. brevicollis MBRTK1 with tyrosine kinase domains from human (H.s.) and Drosophila melanogaster (D.m.) RTKs. Highly conserved residues are indicated in red; similar residues are highlighted in gray. (e) Alignment of six tandem MR (for Monosiga rtk) repeats. Amino acid positions are indicated on the left. The last repeat (residues 955–988) corresponds to only the first half of the motif. Arrows indicate the positions of conserved tyrosine residues that may be autophosphorylated.

Figure 3

Model of choanoflagellate evolution and the assembly of receptor tyrosine kinases. Choanoflagellates (“choanos”) are more closely related to Metazoa than are Fungi or Plants. Because the genomes of Metazoa and a choanoflagellate encode members of the receptor tyrosine kinase family of signaling molecules, the first RTK likely arose before their divergence (green and red box). Although RTKs have been found only in animals and choanoflagellates, components of RTKs arose earlier. Cytoplasmic tyrosine kinases are found throughout eukaryotes and are thought to have evolved before the divergence of the plant, fungal, and animal lineages (red). The EGF-like motif has also been found in animals, plants, and protists and may have arisen before the plant/animal/fungal divergence. However, EGF-like motifs have only previously been found joined to tyrosine kinase domains in Metazoa and, now, in M. brevicollis.