Characterization and Modification of Light-Sensitive Phosphodiesterases from Choanoflagellates.

Enzyme rhodopsins, including cyclase opsins (Cyclops) and rhodopsin phosphodiesterases (RhoPDEs), were recently discovered in fungi, algae and protists. In contrast to the well-developed light-gated guanylyl/adenylyl cyclases as optogenetic tools, ideal light-regulated phosphodiesterases are still in demand. Here, we investigated and engineered the RhoPDEs from Salpingoeca rosetta, Choanoeca flexa and three other protists. All the RhoPDEs (fused with a cytosolic N-terminal YFP tag) can be expressed in Xenopus oocytes, except the AsRhoPDE that lacks the retinal-binding lysine residue in the last (8th) transmembrane helix. An N296K mutation of YFP::AsRhoPDE enabled its expression in oocytes, but this mutant still has no cGMP hydrolysis activity. Among the RhoPDEs tested, SrRhoPDE, CfRhoPDE1, 4 and MrRhoPDE exhibited light-enhanced cGMP hydrolysis activity. Engineering SrRhoPDE, we obtained two single point mutants, L623F and E657Q, in the C-terminal catalytic domain, which showed ~40 times decreased cGMP hydrolysis activity without affecting the light activation ratio. The molecular characterization and modification will aid in developing ideal light-regulated phosphodiesterase tools in the future.

Structural insights into the mechanism of rhodopsin phosphodiesterase.

Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.

Genomic innovation of ATD alleviates mistranslation associated with multicellularity in Animalia.

The emergence of multicellularity in Animalia is associated with increase in ROS and expansion of tRNA-isodecoders. tRNA expansion leads to misselection resulting in a critical error of L-Ala mischarged onto tRNAThr, which is proofread by Animalia-specific-tRNA Deacylase (ATD) in vitro. Here we show that in addition to ATD, threonyl-tRNA synthetase (ThrRS) can clear the error in cellular scenario. This two-tier functional redundancy for translation quality control breaks down during oxidative stress, wherein ThrRS is rendered inactive. Therefore, ATD knockout cells display pronounced sensitivity through increased mistranslation of threonine codons leading to cell death. Strikingly, we identify the emergence of ATD along with the error inducing tRNA species starting from Choanoflagellates thus uncovering an important genomic innovation required for multicellularity that occurred in unicellular ancestors of animals. The study further provides a plausible regulatory mechanism wherein the cellular fate of tRNAs can be switched from protein biosynthesis to non-canonical functions.

The first animals evolved around 750 million years ago from single-celled ancestors that were most similar to modern-day organisms called the Choanoflagellates. As animals evolved they developed more complex body plans consisting of multiple cells organized into larger structures known as tissues and organs. Over time cells also evolved increased levels of molecules called reactive oxygen species, which are involved in many essential cell processes but are toxic at high levels. Animal cells also contain more types of molecules known as transfer ribonucleic acids, or tRNAs for short, than Choanoflagellate cells and other single-celled organisms. These molecules deliver building blocks known as amino acids to the machinery that produces new proteins. To ensure the proteins are made correctly, it is important that tRNAs deliver specific amino acids to the protein-building machinery in the right order. Each type of tRNA usually only pairs with a specific type of amino acid, but sometimes the enzymes involved in this process can make mistakes. Therefore, cells contain proofreading enzymes that help remove incorrect amino acids on tRNAs. One such enzyme ­ called ATD ­ is only found in animals. Experiments in test tubes reported that ATD removes an amino acid called alanine from tRNAs that are supposed to carry threonine, but its precise role in living cells remained unclear. To address this question, Kuncha et al. studied proofreading enzymes in human kidney cells. The experiments showed that, in addition to ATD, a second enzyme known as ThrRS was also able to correct alanine substitutions for threonines on tRNAs. However, reactive oxygen species inactivated the proofreading ability of ThrRS, suggesting ATD plays an essential role in correcting errors in cells containing high levels of reactive oxygen species. These findings suggest that as organisms evolved multiple cells and the levels of tRNA and oxidative stress increased, this led to the appearance of a new proofreading enzyme. Further studies found that ATD originated around 900 million years ago, before Choanoflagellates and animals diverged, indicating these enzymes might have helped to shape the evolution of animals. The next step following on from this work will be to understand the role of ATD in the cells of organs that are known to have particularly high levels of reactive oxygen species, such as testis and ovaries.

Divergent Evolution of Eukaryotic CC- and A-Adding Enzymes.

Synthesis of the CCA end of essential tRNAs is performed either by CCA-adding enzymes or as a collaboration between enzymes restricted to CC- and A-incorporation. While the occurrence of such tRNA nucleotidyltransferases with partial activities seemed to be restricted to Bacteria, the first example of such split CCA-adding activities was reported in Schizosaccharomyces pombe. Here, we demonstrate that the choanoflagellate Salpingoeca rosetta also carries CC- and A-adding enzymes. However, these enzymes have distinct evolutionary origins. Furthermore, the restricted activity of the eukaryotic CC-adding enzymes has evolved in a different way compared to their bacterial counterparts. Yet, the molecular basis is very similar, as highly conserved positions within a catalytically important flexible loop region are missing in the CC-adding enzymes. For both the CC-adding enzymes from S. rosetta as well as S. pombe, introduction of the loop elements from closely related enzymes with full activity was able to restore CCA-addition, corroborating the significance of this loop in the evolution of bacterial as well as eukaryotic tRNA nucleotidyltransferases. Our data demonstrate that partial CC- and A-adding activities in Bacteria and Eukaryotes are based on the same mechanistic principles but, surprisingly, originate from different evolutionary events.

Spectroscopic study of the transmembrane domain of a rhodopsin-phosphodiesterase fusion protein from a unicellular eukaryote.

The choanoflagellate Salpingoeca rosetta contains a chimeric rhodopsin protein composed of an N-terminal rhodopsin (Rh) domain and a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. The Rh-PDE enzyme light-dependently decreases the concentrations of cyclic nucleotides such as cGMP and cAMP. Photoexcitation of purified full-length Rh-PDE yields an "M" intermediate with a deprotonated Schiff base, and its recovery is much faster than that of the enzyme domain. To gain structural and mechanistic insights into the Rh domain, here we expressed and purified the transmembrane domain of Rh-PDE, Rh-PDE(TMD), and analyzed it with transient absorption, light-induced difference UV-visible, and FTIR spectroscopy methods. These analyses revealed that the "K" intermediate forms within 0.005 ms and converts into the M intermediate with a time constant of 4 ms, with the latter returning to the original state within 4 s. FTIR spectroscopy revealed that all-trans to 13-cis photoisomerization occurs as the primary event during which chromophore distortion is located at the middle of the polyene chain, allowing the Schiff base to form a stronger hydrogen bond. We also noted that the peptide backbone of the α-helix becomes deformed upon M intermediate formation. Results from site-directed mutagenesis suggested that Glu-164 is protonated and that Asp-292 acts as the only Schiff base counterion in Rh-PDE. A strong reduction of enzymatic activity in a D292N variant, but not in an E164Q variant, indicated an important catalytic role of the negative charge at Asp-292. Our findings provide further mechanistic insights into rhodopsin-mediated, light-dependent regulation of second-messenger levels in eukaryotic microbes.

Analyzing the function of the insert region found between the α and ß-subunits in the eukaryotic nitrile hydratase from Monosiga brevicollis.

The functional roles of the (His)17 region and an insert region in the eukaryotic nitrile hydratase (NHase, EC 4.2.1.84) from Monosiga brevicollis (MbNHase), were examined. Two deletion mutants, MbNHaseΔ238-257 and MbNHaseΔ219-272, were prepared in which the (His)17 sequence and the entire insert region were removed. Each of these MbNHase enzymes provided an α2ß2 heterotetramer, identical to that observed for prokaryotic NHases and contains their full complement of cobalt ions. Deletion of the (His)17 motif provides an MbNHase enzyme that is ∼55% as active as the WT enzyme when expressed in the absence of the Co-type activator (ε) protein from Pseudonocardia thermophila JCM 3095 (PtNHaseact) but ∼28% more active when expressed in the presence of PtNHaseact. MbNHaseΔ219-272 exhibits ∼55% and ∼89% of WT activity, respectively, when expressed in the absence or presence of PtNHaseact. Proteolytic cleavage of MbNHase provides an α2ß2 heterotetramer that is modestly more active compared to WT MbNHase (kcat = 163 ±â€¯4 vs 131 ±â€¯3 s-1). Combination of these data establish that neither the (His)17 nor the insert region are required for metallocentre assembly and maturation, suggesting that Co-type eukaryotic NHases utilize a different mechanism for metal ion incorporation and post-translational activation compared to prokaryotic NHases.

Phosphorylation control of the ubiquitin ligase Cbl is conserved in choanoflagellates.

Cbl proteins are E3 ubiquitin ligases specialized for the regulation of tyrosine kinases by ubiquitylation. Human Cbl proteins are activated by tyrosine phosphorylation, thus setting up a feedback loop whereby the activation of tyrosine kinases triggers their own degradation. Cbl proteins are targeted to their substrates by a phosphotyrosine-binding SH2 domain. Choanoflagellates, unicellular eukaryotes that are closely related to metazoans, also contain Cbl. The tyrosine kinase complement of choanoflagellates is distinct from that of metazoans, and it is unclear if choanoflagellate Cbl is regulated similarly to metazoan Cbl. Here, we performed structure-function studies on Cbl from the choanoflagellate species Salpingoeca rosetta and found that it undergoes phosphorylation-dependent activation. We show that S. rosetta Cbl can be phosphorylated by S. rosetta Src kinase, and that it can ubiquitylate S. rosetta Src. We also compared the substrate selectivity of human and S. rosetta Cbl by measuring ubiquitylation of Src constructs in which Cbl-recruitment sites are placed in different contexts with respect to the kinase domain. Our results indicate that for both human and S. rosetta Cbl, ubiquitylation depends on proximity and accessibility, rather than being targeted toward specific lysine residues. Our results point to an ancient interplay between phosphotyrosine and ubiquitin signaling in the metazoan lineage.

A novel rhodopsin phosphodiesterase from Salpingoeca rosetta shows light-enhanced substrate affinity.

It is since many years textbook knowledge that the concentration of the second messenger cGMP is regulated in animal rod and cone cells by type II rhodopsins via a G-protein signaling cascade. Microbial rhodopsins with enzymatic activity for regulation of cGMP concentration were only recently discovered: in 2014 light-activated guanylyl-cyclase opsins in fungi and in 2017 a novel rhodopsin phosphodiesterase (RhoPDE) in the protist Salpingoeca rosetta (SrRhoPDE). The light regulation of SrRhoPDE, however, seemed very weak or absent. Here, we present strong evidence for light regulation by studying SrRhoPDE, expressed in Xenopus laevis oocytes, at different substrate concentrations. Hydrolysis of cGMP shows an ∼100-fold higher turnover than that of cAMP. Light causes a strong decrease in the Km value for cGMP from 80 to 13 µM but increases the maximum turnover only by ∼30%. The PDE activity for cAMP is similarly enhanced by light at low substrate concentrations. Illumination does not affect the cGMP degradation of Lys296 mutants that are not able to form a covalent bond of Schiff base type to the chromophore retinal. We demonstrate that SrRhoPDE shows cytosolic N- and C-termini, most likely via an eight-transmembrane helix structure. SrRhoPDE is a new optogenetic tool for light-regulated cGMP manipulation which might be further improved by genetic engineering.

Purification and Characterization of RhoPDE, a Retinylidene/Phosphodiesterase Fusion Protein and Potential Optogenetic Tool from the Choanoflagellate Salpingoeca rosetta.

RhoPDE is a type I rhodopsin/phosphodiesterase gene fusion product from the choanoflagellate Salpingoeca rosetta. The gene was discovered around the time that a similar type I rhodopsin/guanylyl cyclase fusion protein, RhoGC, was shown to control phototaxis of an aquatic fungus through a cGMP signaling pathway. RhoPDE has potential as an optogenetic tool catalyzing the hydrolysis of cyclic nucleotides. Here we provide an expression and purification system for RhoPDE, as well as a crystal structure of the C-terminal phosphodiesterase catalytic domain. We show that RhoPDE contains an even number of transmembrane segments, with N- and C-termini both located on the cytoplasmic surface of the cell membrane. The purified protein exhibits an absorption maximum at 490 nm in the dark state, which shifts to 380 nm upon exposure to light. The protein acts as a cGMP-selective phosphodiesterase. However, the activity does not appear to be modulated by light. The protein is also active with cAMP as a substrate, but with a roughly 5-7-fold lower kcat. A truncation consisting solely of the phosphodiesterase domain is also active with a kcat for cGMP roughly 6-9-fold lower than that of the full-length protein. The isolated PDE domain was crystallized, and the X-ray structure showed the protein to be a dimer similar to human PDE9. We anticipate that the purification system introduced here will enable further structural and biochemical experiments to improve our understanding of the function and mechanism of this unique fusion protein.

EroS Enzyme from Aliivibrio fischeri Plays Cupid to Choanoflagellates.

Cupid's bow: A collaborative effort by the King and Clardy laboratories has serendipitously identified a bacterial chondroitinase that triggers the choanoflagellate S. rosetta to swarm and sexually reproduce. This unprecedented interaction between a bacterium and a choanoflagellate could give insights into a key evolutionary leap-sexual reproduction.

Early emergence of negative regulation of the tyrosine kinase Src by the C-terminal Src kinase.

Stringent regulation of tyrosine kinase activity is essential for normal cellular function. In humans, the tyrosine kinase Src is inhibited via phosphorylation of its C-terminal tail by another kinase, C-terminal Src kinase (Csk). Although Src and Csk orthologs are present across holozoan organisms, including animals and protists, the Csk-Src negative regulatory mechanism appears to have evolved gradually. For example, in choanoflagellates, Src and Csk are both active, but the negative regulatory mechanism is reportedly absent. In filastereans, a protist clade closely related to choanoflagellates, Src is active, but Csk is apparently inactive. In this study, we use a combination of bioinformatics, in vitro kinase assays, and yeast-based growth assays to characterize holozoan Src and Csk orthologs. We show that, despite appreciable differences in domain architecture, Csk from Corallochytrium limacisporum, a highly diverged holozoan marine protist, is active and can inhibit Src. However, in comparison with other Csk orthologs, Corallochytrium Csk displays broad substrate specificity and inhibits Src in an activity-independent manner. Furthermore, in contrast to previous studies, we show that Csk from the filasterean Capsaspora owczarzaki is active and that the Csk-Src negative regulatory mechanism is present in Csk and Src proteins from C. owczarzaki and the choanoflagellate Monosiga brevicollis Our results suggest that negative regulation of Src by Csk is more ancient than previously thought and that it might be conserved across all holozoan species.

A unique choanoflagellate enzyme rhodopsin exhibits light-dependent cyclic nucleotide phosphodiesterase activity.

Photoactivated adenylyl cyclase (PAC) and guanylyl cyclase rhodopsin increase the concentrations of intracellular cyclic nucleotides upon illumination, serving as promising second-generation tools in optogenetics. To broaden the arsenal of such tools, it is desirable to have light-activatable enzymes that can decrease cyclic nucleotide concentrations in cells. Here, we report on an unusual microbial rhodopsin that may be able to meet the demand. It is found in the choanoflagellate Salpingoeca rosetta and contains a C-terminal cyclic nucleotide phosphodiesterase (PDE) domain. We examined the enzymatic activity of the protein (named Rh-PDE) both in HEK293 membranes and whole cells. Although Rh-PDE was constitutively active in the dark, illumination increased its hydrolytic activity 1.4-fold toward cGMP and 1.6-fold toward cAMP, as measured in isolated crude membranes. Purified full-length Rh-PDE displayed maximal light absorption at 492 nm and formed the M intermediate with the deprotonated Schiff base upon illumination. The M state decayed to the parent spectral state in 7 s, producing long-lasting activation of the enzyme domain with increased activity. We discuss a possible mechanism of the Rh-PDE activation by light. Furthermore, Rh-PDE decreased cAMP concentration in HEK293 cells in a light-dependent manner and could do so repeatedly without losing activity. Thus, Rh-PDE may hold promise as a potential optogenetic tool for light control of intracellular cyclic nucleotides (e.g. to study cyclic nucleotide-associated signal transduction cascades).

A cobalt-containing eukaryotic nitrile hydratase.

Nitrile hydratase (NHase), an industrially important enzyme that catalyzes the hydration of nitriles to their corresponding amides, has only been characterized from prokaryotic microbes. The putative NHase from the eukaryotic unicellular choanoflagellate organism Monosiga brevicollis (MbNHase) was heterologously expressed in Escherichia coli. The resulting enzyme expressed as a single polypeptide with fused α- and ß-subunits linked by a seventeen-histidine region. Size-exclusion chromatography indicated that MbNHase exists primarily as an (αß)2 homodimer in solution, analogous to the α2ß2 homotetramer architecture observed for prokaryotic NHases. The NHase enzyme contained its full complement of Co(III) and was fully functional without the co-expression of an activator protein or E. coli GroES/EL molecular chaperones. The homology model of MbNHase was developed identifying Cys400, Cys403, and Cys405 as active site ligands. The results presented here provide the first experimental data for a mature and active eukaryotic NHase with fused subunits. Since this new member of the NHase family is expressed from a single gene without the requirement of an activator protein, it represents an alternative biocatalyst for industrial syntheses of important amide compounds.

Evolutionary dynamics of triosephosphate isomerase gene intron location pattern in Metazoa: A new perspective on intron evolution in animals.

Intron evolution, including its dynamics in the evolutionary transitions and diversification of eukaryotes, remains elusive. Inadequate taxon sampling due to data shortage, unclear phylogenetic framework, and inappropriate outgroup application might be among the causes. Besides, the integrity of all the introns within a gene was often neglected previously. Taking advantage of the ancient conserved triosephosphate isomerase gene (tim), the relatively robust phylogeny of Metazoa, and choanoflagellates as outgroup, the evolutionary dynamics of tim intron location pattern (ILP) in Metazoa was investigated. From 133 representative species of ten phyla, 30 types of ILPs were identified. A most common one, which harbors the maximum six intron positions, is deduced to be the common ancestral tim ILP of Metazoa, which almost had formed in their protozoan ancestor and was surprisingly retained and passed down till to each ancestors of metazoan phyla. In the subsequent animal diversification, it underwent different evolutionary trajectories: within Deuterostomia, it was almost completely retained only with changes in a few species with relatively recently fast-evolving histories, while within the rapidly radiating Protostomia, besides few but remarkable retention, it usually displayed extensive intron losses and a few gains. Therefore, a common ancestral exon-intron arrangement pattern of an animal gene is definitely discovered; besides the 'intron-rich view' of early animal genes being confirmed, the novel insight that high exon-intron re-arrangements of genes seem to be associated with the relatively recently rapid evolution of lineages/species/genomes but have no correlation with the ancient major evolutionary transitions in animal evolution, is revealed.

Constitutive Activity in an Ancestral Form of Abl Tyrosine Kinase.

The c-abl proto-oncogene encodes a nonreceptor tyrosine kinase that is found in all metazoans, and is ubiquitously expressed in mammalian tissues. The Abl tyrosine kinase plays important roles in the regulation of mammalian cell physiology. Abl-like kinases have been identified in the genomes of unicellular choanoflagellates, the closest relatives to the Metazoa, and in related unicellular organisms. Here, we have carried out the first characterization of a premetazoan Abl kinase, MbAbl2, from the choanoflagellate Monosiga brevicollis. The enzyme possesses SH3, SH2, and kinase domains in a similar arrangement to its mammalian counterparts, and is an active tyrosine kinase. MbAbl2 lacks the N-terminal myristoylation and cap sequences that are critical regulators of mammalian Abl kinase activity, and we show that MbAbl2 is constitutively active. When expressed in mammalian cells, MbAbl2 strongly phosphorylates cellular proteins on tyrosine, and transforms cells much more potently than mammalian Abl kinase. Thus, MbAbl2 appears to lack the autoinhibitory mechanism that tightly constrains the activity of mammalian Abl kinases, suggesting that this regulatory apparatus arose more recently in metazoan evolution.

Molecular cloning and characterization of a tyrosine phosphatase from Monosiga brevicollis.

Protein tyrosine phosphorylation is thought to be a unique feature of multicellular animals. Interestingly, the genome of the unicellular protist Monosiga brevicollis reveals a surprisingly high number and diversity of protein tyrosine kinases, protein tyrosine phosphatases (PTPs), and phosphotyrosine-binding domains. Our study focuses on a hypothetical SH2 domain-containing PTP (SHP), which interestingly has a predicted structure that is distinct from SHPs found in animals. In this study, we isolated cDNA of the enzyme and discovered that its actual sequence was different from the predicted sequence as a result of non-consensus RNA splicing. Contrary to the predicted structure with one SH2 domain and a disrupted phosphatase domain, Monosiga brevicollis SHP (MbSHP) contains two SH2 domains and an intact PTP domain, closely resembling SHP enzymes found in animals. We further expressed the full-length and SH2 domain-truncated forms of the enzyme in Escherichiacoli cells and characterized their enzymatic activities. The double-SH2 domain-truncated form of the enzyme effectively dephosphorylated a common PTP substrate with a specific activity among the highest in characterized PTPs, while the full-length and the N-terminal SH2 domain-truncated forms of the enzyme showed much lower activity with altered pH dependency and responses to ionic strength and common PTP inhibitors. This indicates that SH2 domains suppress the catalytic activity. SHP represents a highly conserved ancient PTP, and studying MbSHP should provide a better understanding about the evolution of tyrosine phosphorylation.

Enzymatically active 2',5'-oligoadenylate synthetases are widely distributed among Metazoa, including protostome lineage.

2',5'-Oligoadenylate synthetases (OASs) belong to the nucleotidyl transferase family together with poly(A) polymerases, CCA-adding enzymes and the recently discovered cyclic-GMP-AMP synthase (cGAS). Mammalian OASs have been thoroughly characterized as components of the interferon-induced antiviral system. The OAS activity and the respective genes were also discovered in marine sponges where the interferon system is absent. In this study the recombinant OASs from several multicellular animals and their closest unicellular relative, a choanoflagellate, were expressed in a bacterial expression system and their enzymatic activities were examined. We demonstrated 2-5A synthesizing activities of OASs from the marine sponge Tedania ignis, a representative of the phylogenetically oldest metazoan phylum (Porifera), from an invertebrate of the protostome lineage, the mollusk Mytilus californianus (Mollusca), and from a vertebrate species, a cartilaginous fish Leucoraja erinacea (Chordata). However, the expressed proteins from an amphibian, the salamander Ambystoma mexicanum (Chordata), and from a protozoan, the marine choanoflagellate Monosiga brevicollis (Choanozoa), did not show 2-5A synthesizing activity. Differently from other studied OASs, OAS from the marine sponge T. ignis was able to catalyze the formation of oligomers having both 2',5'- and 3',5'-phosphodiester linkages. Our data suggest that OASs from sponges and evolutionarily higher animals have similar activation mechanisms which still include different affinities and possibly different structural requirements for the activating RNAs. Considering their 2'- and 3'-specificities, sponge OASs could represent a link between evolutionarily earlier nucleotidyl transferases and 2'-specific OASs from higher animals.

Earliest Holozoan expansion of phosphotyrosine signaling.

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.

Metazoan-like signaling in a unicellular receptor tyrosine kinase.

BACKGROUND: Receptor tyrosine kinases (RTKs) are crucial components of signal transduction systems in multicellular animals. Surprisingly, numerous RTKs have been identified in the genomes of unicellular choanoflagellates and other protists. Here, we report the first biochemical study of a unicellular RTK, namely RTKB2 from Monosiga brevicollis. RESULTS: We cloned, expressed, and purified the RTKB2 kinase, and showed that it is enzymatically active. The activity of RTKB2 is controlled by autophosphorylation, as in metazoan RTKs. RTKB2 possesses six copies of a unique domain (designated RM2) in its C-terminal tail. An isolated RM2 domain (or a synthetic peptide derived from the RM2 sequence) served as a substrate for RTKB2 kinase. When phosphorylated, the RM2 domain bound to the Src homology 2 domain of MbSrc1 from M. brevicollis. NMR structural studies of the RM2 domain indicated that it is disordered in solution. CONCLUSIONS: Our results are consistent with a model in which RTKB2 activation stimulates receptor autophosphorylation within the RM2 domains. This leads to recruitment of Src-like kinases (and potentially other M. brevicollis proteins) and further phosphorylation, which may serve to increase or dampen downstream signals. Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.

PTB domain-directed substrate targeting in a tyrosine kinase from the unicellular choanoflagellate Monosiga brevicollis.

Choanoflagellates are considered to be the closest living unicellular relatives of metazoans. The genome of the choanoflagellate Monosiga brevicollis contains a surprisingly high number and diversity of tyrosine kinases, tyrosine phosphatases, and phosphotyrosine-binding domains. Many of the tyrosine kinases possess combinations of domains that have not been observed in any multicellular organism. The role of these protein interaction domains in M. brevicollis kinase signaling is not clear. Here, we have carried out a biochemical characterization of Monosiga HMTK1, a protein containing a putative PTB domain linked to a tyrosine kinase catalytic domain. We cloned, expressed, and purified HMTK1, and we demonstrated that it possesses tyrosine kinase activity. We used immobilized peptide arrays to define a preferred ligand for the third PTB domain of HMTK1. Peptide sequences containing this ligand sequence are phosphorylated efficiently by recombinant HMTK1, suggesting that the PTB domain of HMTK1 has a role in substrate recognition analogous to the SH2 and SH3 domains of mammalian Src family kinases. We suggest that the substrate recruitment function of the noncatalytic domains of tyrosine kinases arose before their roles in autoinhibition.