Metazoan-like signaling in a unicellular receptor tyrosine kinase
© Schultheiss et al.; licensee BioMed Central Ltd. 2013
Received: 4 October 2012
Accepted: 4 February 2013
Published: 12 February 2013
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© Schultheiss et al.; licensee BioMed Central Ltd. 2013
Received: 4 October 2012
Accepted: 4 February 2013
Published: 12 February 2013
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.
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.
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.
Metazoan receptor tyrosine kinases (RTKs) respond to a variety of extracellular stimuli, initiating signals that regulate important cellular and developmental processes [1–4]. The phosphotyrosine-based signaling system (consisting of tyrosine kinases, tyrosine phosphatases, and pTyr-binding modules) evolved relatively recently [5–7]; pTyr signaling was originally thought to be unique to metazoans. Remarkably, recent genomic analyses have demonstrated that several unicellular organisms possess numbers of receptor and nonreceptor tyrosine kinases that are comparable to higher metazoans. Choanoflagellates are free-living aquatic protists that represent the closest unicellular relatives to metazoans [8, 9]. Tyrosine kinases are abundant in the choanoflagellates Monosiga brevicollis, Monosiga ovata, and Salpingoeca rosetta[10–13]. Tyrosine kinases are also found in even more ancient opisthokonts, including the filasterean Capsaspora owczarzaki. Although the physiological functions of these unicellular tyrosine kinases are not yet known, they are presumably involved in the responses to extracellular signals such as the presence of nutrients, ions, or chemical messengers.
The genome of Monosiga brevicollis is estimated to encode as many as 128 tyrosine kinases and approximately 380 total protein kinases . (For comparison, the human kinome contains 518 protein kinases, of which 90 are tyrosine kinases). Most of the Monosiga brevicollis tyrosine kinases have no identifiable metazoan homologs (the exceptions are nonreceptor tyrosine kinases of the Src, Csk, Abl, and Tec families). As in metazoans, Monosiga brevicollis tyrosine kinases never appear as isolated catalytic domains; instead, each tyrosine kinase possesses a repertoire of associated signaling domains. Many of the domain combinations are distinct from any observed in metazoans. There are predicted to be 88 RTKs in M. brevicollis; 73 of the RTKs cluster into 15 families (designated RTKA, RTKB, and so on) . While these RTKs possess many of the same features as metazoan RTKs (putative extracellular ligand-binding modules, conserved catalytic residues, and potential sites of autophosphorylation), their overall sequences show limited homology to the families of metazoan RTKs. It is not yet known whether the M. brevicollis RTKs (or any unicellular RTKs) are enzymatically active as tyrosine kinases.
Monosiga brevicollis RTKB2 is predicted to be a type I transmembrane protein, with a single membrane-spanning region  (Figure 1A). The extracellular domain organization of RTKB2 has several features in common with families of metazoan RTKs, although the exact combination of domains has not been observed in any metazoan family. RTKB2 contains a Cys-rich domain similar to those seen in the TNF receptor and to the furin-like domains of EGFR and insulin receptor . Two divergent repeats similar to hyalin are present; these modules are structurally related to the immunoglobulin-like fold seen in many metazoan RTKs . RTKB2 contains a short consensus repeat (SCR)/complement control protein domain, as seen in a variety of complement and adhesion proteins. Towards the C-terminus of the extracellular domain, RTKB2 contains five EGF-like modules, as seen in (for example) the Tie and Eph families of metazoan RTKs. As is true for all Monosiga brevicollis RTKs, the extracellular ligand is unknown.
The predicted intracellular portion of RTKB2 contains a single tyrosine kinase catalytic domain. The kinase domain of RTKB2 has limited homology to known families of metazoan RTKs, but possesses the catalytically important residues found in all tyrosine kinases (Figure 1B). RTKB2 has a single tyrosine residue in the predicted activation loop, C-terminal to the kinase-conserved DFG motif (Figure 1B). The C-terminus of RTKB2 contains six copies of a unique domain designated RM2. RM2 domains are composed of approximately 80 amino acid residues. They are found in the cytoplasmic tails of four of the nine Monosiga brevicollis RTKB tyrosine kinases, but they are not present in any other sequence in the protein database, whether from Monosiga, metazoans, or any other organism . RM2 domains contain tyrosine residues that are predicted to serve as phosphorylation sites as well as SH2-binding sites (described in more detail below).
The unique sequences of the RM2 domains suggest that they may serve as novel signaling modules. Many eukaryotic signaling domains adopt defined structures, even when removed from their parental proteins . In order to evaluate the solution structure of 15N labeled RM2, a 1H-15N HSQC spectrum was acquired (Additional file 4: Figure S4). The spectrum of RM2 illustrates that the chemical shift of the backbone amide resonances display narrow dispersion, cluster and overlap around the center of the spectrum, while the line shapes are sharp. These features are typical of a disordered protein. Furthermore, the number of backbone amide resonances is lower than the expected count of 103 (excluding the amino terminal residue and prolines) for His10 tagged RM2, presumably because the resonances are extensively overlapped due to the disordered state of the RM2 domain. We attempted to model the structure of the RM2-6 domain using the web-based Protein Homology/Analogy Recognition Engine (Phyre) (http://www.sbg.bio.ic.ac.uk/phyre2). Phyre predicted that 52% of the RM2-6 domain is disordered, and secondary structural elements were predicted with low confidence. Collectively, our data suggest that the RM2 domains of RTKB2 serve as kinase/SH2 binding sites, and that pTyr-SH2 binding is the main determinant for interaction, rather than a well-defined protein-protein interface.
The abundance and diversity of receptor and nonreceptor tyrosine kinases in the unicellular choanoflagellate Monosiga brevicollis rival that of any metazoan [10, 11, 23]. The 88 RTKs found in the genome of M. brevicollis possess a wide variety of domain organizations. The divergent architectures of the choanoflagellate RTKs were likely generated by gene duplication and domain shuffling [24, 25]. The M. brevicollis RTKs have no direct homologs in multicellular organisms. In contrast, sponges, which are regarded as the oldest surviving metazoan lineage, possess most of the RTK families found in higher metazoans [26–28]. The genome of the sponge Amphimedon queenslandica contains 150 RTK genes, including kinase domains from six known animal families: epidermal growth factor receptor (EGFR), Met, discoidin domain receptor (DDR), ROR, Eph, and Sevenless . The sponge Oscarella carmela possesses a similar array of homologs, and RTKs with homology to the receptors for insulin-like growth factor I and fibroblast growth factor . This is consistent with a model in which the common ancestor between choanoflagellates and metazoans had RTKs, but the animal cell-specific families of RTKs developed after the split between the two groups .
RTKB2, the tyrosine kinase studied here, is one of the nine RTKB-family kinases from M. brevicollis. As the most primitive RTK to be yet studied, these results shed light on the evolution of biochemical function in the receptor tyrosine kinase superfamily. RTKB2 is active as a tyrosine kinase, and the intrinsic enzymatic function of the RTKB2 catalytic domain is high towards synthetic peptides, particularly the IR/IGF1R family peptide substrate E4YM4 (Figure 2). RTKB2 also catalyzes autophosphorylation, an event that increases the activity of the enzyme (Figure 3). RTKB2 possesses a single tyrosine residue within the predicted activation loop (Figure 1B). Activation loops are one of the distinguishing features of eukaryotic protein kinases . They are flexible, dynamic segments that are often stabilized in the active conformation by addition of one or more phosphates (either through autophosphorylation or through the action of another kinase). The control of RTKB2 activity by autophosphorylation indicates that this mode of regulation was present in primitive RTKs before the evolution of multicellular animals over 600 million years ago.
The C-terminal tail of RTKB2 contains 6 copies of the RM2 domain, a unique region of ≈ 80 residues that has not been found outside of the RTKB family in M. brevicollis (Figure 4). RTKB1, RTKB3, and RTKB4 each have one copy of the RM2 domain C-terminal to their tyrosine kinase domains . Each of the RM2 domains has a tyrosine residue preceded by one or more negatively-charged amino acids. Scansite prediction indicated that these could serve as Src phosphorylation sites and/or SH2-binding sites . Our data show that an isolated RM2 domain is phosphorylated by the RTKB2 kinase or by MbSrc1, a Src-family nonreceptor tyrosine kinase from M. brevicollis (Figure 5). Small synthetic peptides derived from the potential phosphorylation sites from two of the RTKB2 RM2 domains are also substrates (Figure 2). The tyrosine phosphorylated RM2 domain binds specifically to the SH2 domain of MbSrc1 (Figure 5B), suggesting that phosphorylation of one or more RM2 domains can recruit cytoplasmic tyrosine kinases and other cellular proteins to propagate the RTKB2 signal. In contrast to other modular signaling domains (e.g., SH2 or SH3), the isolated RM2 domain appears to lack an ordered structure when it is removed from the context of the surrounding protein (Additional file 4: Figure S4). These binding sites at the C-terminus of RTKB2 may serve a similar recruitment function as the tyrosine motifs found in the short, unstructured cytoplasmic tails of the thrombopoietin or erythropoietin receptors [30, 31] or the phosphorylation sites in the C-termini of RTKs such as the epidermal growth factor receptor.
Binding of the M. brevicollis MbSrc1 kinase to the phosphorylated RM2 domain raises the possibility that receptor and nonreceptor tyrosine kinase signaling are linked, as in metazoans. A classic example of the linkage in mammalian cells is observed in the role for Src in relaying the signal through the platelet-derived growth factor (PDGF) family of receptors. Src stably associates with the cytoplasmic portion of the PDGF receptor, leading to enhanced Src activity [32–34]. For PDGFRa, Src is required for the phosphorylation of the adaptor protein Shc . After binding to PDGFRb, Src phosphorylates a tyrosine residue on the receptor; this inhibits a signaling pathway leading to motility, but increases mitogenic signaling . MbSrc1 phosphorylates the C-tail of RTKB2 weakly in the absence of an activating signal (Figure 6). Our results are consistent with a model in which RTKB2 activation (by an unknown signal) stimulates receptor autophosphorylation within the RM2 domains. This leads to MbSrc1 recruitment and further phosphorylation, which may serve to increase or dampen specific downstream signals. Identifying the nature of these signals will require the development of methodology to manipulate gene function in choanoflagellates.
We conducted the first biochemical study of a unicellular receptor tyrosine kinase. We cloned, expressed, and purified the RTKB2 kinase, and showed that it is enzymatically active. The activity of RTKB2 is regulated by autophosphorylation. The receptor possesses six copies of a unique domain (designated RM2) in its C-terminal tail. An isolated RM2 domain was a substrate for RTKB2 kinase, and the phosphorylated RM2 domain bound to the SH2 domain of a Src family kinase. Thus, this unicellular signaling system contains many of the features found in metazoan RTK pathways.
The protein sequence of RTKB2 was predicted from release 1.0 of the Monosiga brevicollis genome (http://genome.jgi-psf.org/Monbr1/Monbr1.home.html) . The kinase domain (residues 1450–1724) or C-terminal tail (residues 1722–2200) were amplified by PCR from an M. brevicollis cDNA library . For baculovirus expression, the kinase cDNA was cloned into the BamHI and XbaI sites of pFastbac-Htb (Invitrogen). For mammalian cell expression, the C-terminal tail cDNA was cloned into the EcoRI and BamHI sites of plasmid pEGFP-C1 (Clontech). For bacterial expression of RM2-6, the gene was synthesized with a sequence optimized for E. coli (GenScript) and cloned into plasmid pET-15b.
His-tagged RTKB2 kinase was produced in Spodoptera frugiperda (Sf9) insect cells using the Bac-to-Bac system (Invitrogen), using methods developed for other tyrosine kinases [17, 20]. The enzyme was purified by column chromatography with nickel-nitrilotriacetic acid (NiNTA) resin (Qiagen) and stored in 40% glycerol at −20°C. His-tagged RM2-6 was expressed in 1-liter E. coli cultures and purified by Ni-NTA chromatography.
The following conditions were used to produce labeled RM2-6 for NMR: the RM2-6 domain cDNA was cloned into pSKB3-His10 (customized pET28 vector) as a fusion featuring an N-terminal His10 tag and TEV protease cleavage site. For 15N labeled RM2-6 expression, pSKB3-His10-RM2 was transformed into E.coli BL21 (DE3) and selected for with Kanamycin (50 μg/μL), subsequently 2L of 15N enriched M9 minimal media containing 15NH4Cl as the sole source of nitrogen was inoculated with the cells and grown at 37°C. At O.D600nm ~ 0.4, the cultures were cooled to 16°C and induced with 0.5 mM IPTG at O.D600nm ~ 0.7-0.8. Expression was allowed to proceed overnight.
Cells were harvested by centrifugation at 3000 g for 10 minutes at 4°C, resuspended in lysis buffer (20 mM Tris pH 8, 500 mM NaCl, 5% glycerol), lysed by sonication on ice, centrifuged for 30 min at 18000g and then purified by Ni2+ NTA affinity chromatography. RM2-6 was eluted using a linear 0–100% imidazole gradient (lysis buffer plus 500 mM imidazole). Fractions containing RM2-6 were pooled and diluted 2-fold with 20 mM Tris pH 8, then purified further by anion exchange on a Q column. RM2-6 was eluted over a linear 0–40% NaCl gradient (QA buffer: 20 mM Tris pH 8, 5% glycerol, 1 mM DTT, QB buffer: same as QA buffer plus 1 M NaCl). Subsequent RM2-6 containing fractions were pooled and buffer exchanged into 50 mM NaH2PO4/Na2HPO4 pH 7, 150 mM NaCl, 1 mM DTT, then concentrated to 800 μM. A 500 μL RM2-6 sample at 720 μM was prepared with 10% 2H2O for NMR analysis.
RTKB2 synthetic peptide assays were performed with [γ-32P]-ATP using the phosphocellulose paper binding assay [36, 37]. Reaction mixtures contained 20 mM Tris–HCl (pH 7.4), 10 mM MgCl2, 0.1 mM Na2VO4, 0.5 mM DTT, 0.25 mM ATP, varying concentrations of peptide substrate, and [γ-32P]-ATP (200–400 cpm/pmol). The sequences of the peptides tested were: Src peptide, AEEEIYGEFEAKKKKG ; RTKB2-1, SEEVYGAVVDKKK; RTKB2-2, AEEVYEAIADKKK; insulin receptor substrate (E4YM4), KKEEEEYMMMMG. RTKB2 autophosphorylation was measured after treatment with a glutathione S-transferase (GST) fusion protein containing Yersinia YOP phosphatase. Reaction mixtures contained 10 mM MgCl2 and 0.5 mM unlabeled ATP (for Western blotting experiments) or 0.5 mM [γ-32P]-ATP (for analysis by autoradiography).
A 1H-15N HSQC spectrum of 15N labeled RM2-6 was acquired on a Bruker 700 MHz spectrometer at 25°C using 16 scans, data points TD2 = 2048, TD1 = 128, with spectral widths of 11160.71 Hz (1H) and 2483.23 Hz (15N) respectively. The spectrum was processed with Topspin and the spectrum figure was created using CCPNMR analysis 2.1.
SYF cells were cultured in Dulbecco’s modified Eagle’s medium plus 10% fetal bovine serum at 37°C in 5% CO2. Cells were transfected using TransIT polyamine transfection reagent (Mirus) according to the manufacturer’s instructions. Cells were lysed in buffer containing 10 mM Tris–HCl, pH 7.4, 50 mM NaCl, 5 mM EDTA, 1% TritonX-100, 50 mM NaF, 2 mM Na3VO4, 1mMPMSF, 1mg/ml aprotinin, and 1mg/ml leupeptin. After centrifugation, protein concentrations were determined using a Bio-Rad protein assay. For immunoprecipitation experiments, lysates (1 mg total protein) were precleared by mixing with 50 ml of protein A-agarose in lysis buffer for one hour at 4°C. After pre-clearing, 2 μg of anti-GFP antibody was added to the lysate and incubated for one hour at 4°C with rocking. Antibody protein complexes were collected with 50 μl protein A beads. The beads were washed 5 times in lysis buffer and boiled in 40 μl gel loading buffer. After separation by SDS-PAGE, proteins were transferred to PVDF membrane and analyzed by Western blotting. Western blotting experiments were carried out with the following antibodies: anti-phosphotyrosine antibody (4G10, Upstate), anti-Flag antibody (M2, Sigma) and anti-GFP antibody (Santa Cruz). Detection was by enhanced chemiluminescence (GE Healthcare).
For immunofluorescence miscroscopy, cells expressing GFP-RTKB2 were grown on 35mm glass bottom dishes (In Vitro Scientific). The cells were first washed with 1x PBS, then fixed with dilute 3.7% formaldehyde in 1x PBS for 15 minutes at room temperature. The fixed cells were washed several times with 1x PBS then mounted on coverslips using VECTASHIELD medium (Vector Laboratories). The GFP expressing cells were visualized by epifluorescence microscopy using a Zeiss Axiovert 200M inverted microscope and a Plan Apochromat 63x/1.40 oil objective. Images were captured using a GFP-Chroma Filter Set, AxioVision software and an AxioCam MRm CCD camera.
Src homology 2
Ethylenediamine tetraacetic acid
Epidermal growth factor
Green fluorescent protein
Heteronuclear single quantum coherence
Polymerase chain reaction
Platelet derived growth factor
Receptor tyrosine kinase
Src/Yes/Fyn deficient cells
Tobacco etch virus protease.
We thank Chris Gordon for assistance with fluorescence microscopy. This work was supported by NIH grant CA58530 to W.T.M.
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