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Evolution of Key Cell Signaling and Adhesion Protein Families Predates Animal Origins

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Science  18 Jul 2003:
Vol. 301, Issue 5631, pp. 361-363
DOI: 10.1126/science.1083853

Abstract

The evolution of animals from a unicellular ancestor involved many innovations. Choanoflagellates, unicellular and colonial protozoa closely related to Metazoa, provide a potential window into early animal evolution. We have found that choanoflagellates express representatives of a surprising number of cell signaling and adhesion protein families that have not previously been isolated from nonmetazoans, including cadherins, C-type lectins, several tyrosine kinases, and tyrosine kinase signaling pathway components. Choanoflagellates have a complex and dynamic tyrosine phosphoprotein profile, and cell proliferation is selectively affected by tyrosine kinase inhibitors. The expression in choanoflagellates of proteins involved in cell interactions in Metazoa demonstrates that these proteins evolved before the origin of animals and were later co-opted for development.

A central question in animal evolution is how multicellular animals evolved from a protozoan ancestor. One approach to animal origins is to determine which developmental proteins predated the origin of animals and were subsequently co-opted for animal development. Comparative genomics can identify the minimal set of genes in place at the outset of animal evolution by revealing those shared by all animals and their nearest relatives (1). The choanoflagellates, a group of unicellular and colonial flagellates that resemble cells found only in Metazoa, have emerged as an important model for studies of early animal evolution (2, 3). Analyses of nuclear and mitochondrial genes consistently support a close phylogenetic relationship between choanoflagellates and Metazoa (47). Specifically, the presence in a choanoflagellate mitochondrial genome of multiple genes lost from a conserved portion of animal (including sponge and cnidarian) mitochondrial genomes suggests that choanoflagellates are an outgroup of animals (811) (fig. S1 and tables S1 and S2). Therefore, genes expressed by choanoflagellates and animals to the exclusion of other eukaryotes define the minimal complexity of the eukaryotic genome before the emergence of multicellular animals.

To sample the diversity of genes expressed by choanoflagellates, we collected more than 5000 expressed sequence tags (ESTs) from two choanoflagellate species, Monosiga brevicollis and Proterospongia-like sp. ATCC50818 (11, 12). Under laboratory conditions, Monosiga is strictly unicellular, whereas ATCC50818 occasionally forms small colonies of apparently undifferentiated cells; both may exhibit additional behaviors in the natural environment. In marked contrast to their simple lifestyle, choanoflagellates express members of a wide variety of protein families involved in animal cell interactions, including cadherins, C-type lectins, tyrosine kinases (TKs), and a G protein–coupled receptor (GPCR), as well as several multidomain polypeptides that contain protein-protein interaction domains involved in signaling and adhesion in animals [such as the epidermal growth factor (EGF) motif, Src homology 2 (SH2) domain, tumor necrosis factor receptor (TNFR) domain, and sushi or complement control protein (CCP) domain (Fig. 1)].

Fig. 1.

Choanoflagellates express a variety of multidomain proteins that contain animal-type protein motifs (11). Cadherin domains are found as tandem repeats in predicted proteins from ATCC50818 (PRCDH1) and Monosiga(MBCDH1). Choanoflagellates also express members of the C-type lectin (MBCTL1 and MBCTL2) and TK (MBSRC1, MBRTK2, and MBRTK1) protein families. Additional multidomain proteins from choanoflagellates (such as MB3515) contain previously uncharacterized combinations of protein domains that are commonly found in animal proteins that mediate cell adhesion and signaling. MB7TM1 resembles secretin-like GPCRs, and MBSPEC1 resembles spectrin-containing proteins, both families of which are used in animals for signaling and cytoskeletal structure respectively. The source species is indicated by the first two letters of each gene name; “PR” indicates predicted proteins from ATCC50818 and “MB” indicates predicted proteins from Monosiga.

The phylogenetic distribution of each protein or domain of interest was determined by a combination of strategies. First, the occurrence of each domain in eukaryotes, Bacteria, Archaea, and viruses was examined with two protein domain annotation programs, SMART and PFAM (13, 14). Second, we queried all available sequences from representative nonanimals, including Fungi, Plantae, Dictyostelium, and diverse bacteria, using BlastX and keyword searches. Third, we examined certain sequences more closely for adherence to the conserved traits of the protein domain family (fig. S2). Proteins containing cadherin, fibrinogen, GPCR proteolytic site (GPS), somatomedin, and CCP domains were not detected in Fungi, Plantae, or other eukaryotes (11). Although putative TK and C-type lectin domains were detected in plant genomes by the SMART and PFAM annotation sites, further scrutiny revealed these sequences to have been classified incorrectly (11). We conclude that choanoflagellates and animals share, to the exclusion of other eukaryotes whose genomes have been analyzed, proteins that contain secretin-like GPCR, GPS, fibrinogen, somatomedin, and CCP domains, as well as members of the cadherin, C-type lectin, and TK protein families.

In animals, cadherins mediate cell-cell adhesion and signaling through homophilic interactions (15). Both Monosiga and ATCC50818 were found to express a cadherin-encoding gene (Fig. 1). MBCDH1 from Monosiga encodes at least two cadherin repeats and PRCDH1 from ATCC50818 encodes at least four. Comparisons of cadherin repeat sequences and the domain architectures of cadherin-containing proteins have revealed at least six subfamilies within the cadherin family (16). Phylogenetic analysis revealed the choanoflagellate cadherins to be most similar to protocadherins and to the Flamingo class of cadherins (11) (fig. S3).

C-type lectins mediate cell adhesion and signaling through calcium-dependent recognition and binding of specific sugars. Within the superfamily that contains C-type lectin–like domains, those containing carbohydrate recognition domains (CRDs) have previously been identified only within animals (17). We found two CRD-containing C-type lectin genes in Monosiga, one containing three C-type lectin domains (MBCTL1) and a second containing a C-type lectin domain fused to three CCP domains (MBCTL2) (Fig. 1 and fig. S2).

We have also isolated five homologs of animal TKs, four from Monosiga and one from ATCC50818, including MBRTK1, which we have previously reported (5) (fig. S2). Two choanoflagellate TKs, MBRTK1 and MBRTK2, conform to the general structure of receptor TKs, with a C-terminal TK enzymatic core connected through a transmembrane span to an N-terminal extracellular domain (Fig. 1). The remaining three choanoflagellate TKs (MBSRC1, MBTK3, and PRTK1) lack transmembrane domains and are predicted to operate downstream in TK signaling pathways. For instance, MBSRC1 contains an N-terminal SH2 domain linked to a TK domain and resembles Src TKs from animals (Fig. 1 and fig. S3).

Because components of TK signaling pathways appear highly conserved from sponges to vertebrates, we sought to determine whether TK signaling, although absent from fungi and plants, evolved before the origin of animals. We identified ESTs for many components of TK signaling pathways in Monosiga and ATCC50818, including phosphoinositide 3-kinase, growth factor receptor–bound protein 2, Vav proteins, PDZ-containing sequences, tyrosine phosphatases, Raf kinase, and Ras, in addition to mitogen-activated protein kinase and Src kinase (1820). The expression of these signal transduction components suggests that choanoflagellates may use tyrosine phosphorylation for signaling in a manner similar to animals.

To investigate the extent of TK signaling in choanoflagellates, we probed Monosiga cell lysates with antibodies specific to phosphotyrosine. In cells maintained under conditions of nutrient depletion, approximately 15 to 18 specific phosphotyrosine-containing proteins were detected (Fig. 2, A and B), a similar number to that seen in unstimulated animal cells (21, 22). In animal cells, TK signaling is rapidly stimulated by the addition of serum to cultures of serumstarved cells (21, 23). We compared the tyrosine phosphoprotein profile of nutrient-depleted cells cultured in seawater with the profile in cells exposed to seawater enriched with either a cereal grass infusion (1525) or 1525 supplemented with Enterobacter (1525/Ea). No change was seen in the relative abundance of tyrosine-phosphorylated proteins from starved cells treated with fresh seawater compared to untreated starved cells (data not shown). In contrast, treatment of starved cells with either 1525 or 1525/Ea resulted in rapid modifications to the tyrosine phosphorylation status of at least four proteins (Fig. 2C). An apparently phosphotyrosine-free 95-kD protein (P95) became tyrosine-phosphorylated within 5 min of treatment, indicating the probable activation of one or more tyrosine kinases, and the tyrosine phosphorylation levels of three protein species (P38, P80, and P83) diminished in response to stimulation with enriched media (Fig. 2C).

Fig. 2.

The spectrum of tyrosine phosphoproteins in Monosiga. Monosiga cell lysates were electrophoresed, blotted onto membranes, and probed with antibodies specific to phosphotyrosine (11). (A) The profile of tyrosine-phosphorylated proteins in nutrient-deprived cells. About 15 to 18 tyrosine-phosphorylated protein species, ranging in size from 40 kD to >200 kD, were detected in lysates from Monosiga cultured in seawater. (B) All but one of the detected tyrosine phosphoproteins in (A) derive from Monosiga. We compared the tyrosine phosphoproteins in protein extracts from Monosiga cultured with Enterobacter (M.b. + E.a.) with those from Enterobacter (E.a.) protein extracts (left panel). Two bacterial protein species reacted with the antibodies to phosphotyrosine, but only the larger molecular weight band (approximately 78 kD) is specific as determined by inhibition with the hapten p-nitrophenyl phosphate (PNPP) (right panel). (C) Protein tyrosine phosphorylation status is affected by environmental conditions. Cells were incubated in seawater as in (A) and (B), then treated for 5, 10, or 30 min with either seawater (S.W.), a cereal grass infusion of seawater (1525), or 1525 supplemented with Enterobacter at 108 cells/mL (1525/Ea). Within 5 min, a 95-kD protein species (P95) was detected in lysates treated with cereal grass infusion either with or without Enterobacter, but not in lysates treated with seawater. In contrast, the antibodies to phosphotyrosine showed diminished reactivity to protein species of 38, 80, and 83 kD (P38 and P80/83) in the presence of cereal grass infusion.

To examine the functional significance of TK signaling in choanoflagellate biology, we measured the proliferation and maximal cell density of Monosiga cultures treated with specific inhibitors of TKs at concentrations previously shown to be effective in cultured animal cells (11, 20, 24). Control cultures of Monosiga grown in 1525/Ea conformed to the typical growth characteristics of cultured microbes, exhibiting lag, log, and stationary phases (Fig. 3). The doubling time of untreated cells during log phase was approximately 6 hours and cells reached a maximum density of ∼107 cells/mL. In contrast, cultures treated with genistein, a general inhibitor of TKs, were delayed in their entry into log phase and reached a maximum density of 2 × 106 cells/mL, one-fifth that of untreated cells. In addition, treatment of Monosiga cultures with PP2, an inhibitor of Src kinases, entirely blocked cell proliferation. Treatment of Monosiga cultures with inactive analogs of genistein and PP2, the control compounds genistin and PP3, had no significant effect on cell density (not shown). Importantly, genistein- and PP2-treated cells displayed normal levels of motility and feeding behavior. The dependence of Monosiga cells on TK activity indicates a functional link between TK signaling and the cell cycle or cell survival.

Fig. 3.

TK activity is required for proliferation of Monosiga cultures. In contrast with cultures grown in the presence of a control solvent [dimethyl sulfoxide (DMSO), diamonds], the increase in cell density of cultures treated with TK inhibitors [25 μM genistein (circles) or 500 nM PP2 (squares) in DMSO] is significantly reduced (11).

The discovery of multiple signaling and adhesion gene family members in choanoflagellates demonstrates that key proteins required for animal development evolved before the origin of animals. Other multicellular eukaryotes, and even those with complex modes of development (e.g., Dictyostelium, fungi, and plants), apparently lack many proteins used by animals for signaling and adhesion. Therefore, choanoflagellates, with their closer evolutionary relationship to animals (47) and their expression of signaling and cell adhesion protein homologs, are more informative for studies of animal origins.

The existence in unicellular choanoflagellates of proteins used for cell adhesion and signal transduction in animals prompts the question of their ancestral function in the progenitor of animals and choanoflagellates. Despite the apparent simplicity of the choanoflagellate lifestyle, it is possible that choanoflagellate homologs of animal proteins perform similar biochemical functions within a unicellular context. For instance, TKs may act in choanoflagellates to detect changes in the extracellular environment, as we have demonstrated through their response to nutrient availability. In addition, animal cell adhesion proteins, such as the cadherins, may derive from ancestral proteins that stabilized the interactions between protozoan cells during conjugation or colony formation. Proteins that mediate cell attachment or defense against pathogens in animals may have evolved from proteins required for prey recognition and capture. C-type lectins might allow choanoflagellates to distinguish between and capture different bacterial species by binding specific sugar groups displayed on bacterial cell walls. Targeted manipulations of gene function in choanoflagellates will be necessary to test hypotheses about the ancestral roles of these conserved molecules.

We have sampled just a fraction of the choanoflagellate proteome. The diversity of choanoflagellate proteins predicted to function in cell interactions suggests that additional proteins shared exclusively with animals will be discovered through sequencing the entire choanoflagellate genome. Of particular interest will be the repertoire of transcription factors and the potential representation of families of proteins that regulate cell differentiation and development in animals. It may then be possible to determine whether entire regulatory pathways linking receptor-based signaling inputs to gene regulation and cell behavior predate the origin of animals.

Supporting Online Material

www.sciencemag.org/cgi/content/full/301/5631/361/DC1

Materials and Methods

SOM Text

Figs. S1 to S4

Tables S1 and S2

References and Notes

References and Notes

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