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Metazoan-like signaling in a unicellular receptor tyrosine kinase.

Schultheiss KP, Craddock BP, Tong M, Seeliger M, Miller WT - BMC Biochem. (2013)

Bottom Line: 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.Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, Basic Science Tower, T-6, School of Medicine, Stony Brook University, Stony Brook, NY 11794-8661, USA.

ABSTRACT

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.

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The RM2 domain acts as a kinase substrate and SH2-binding site. (A) Left panel: purified RM2-6 (1 μM) was incubated with RTKB2 (250 nM) in kinase assay buffer containing 0.8 mM ATP at 30°C for the indicated times. Aliquots were removed, boiled with SDS-PAGE sample buffer, and analyzed by Western blotting with anti-pTyr antibody. Right panel: similar experiments were carried out with purified MbSrc1 kinase. (B) Purified RM2-6 was phosphorylated by MbSrc1 in the presence of [γ-32P]-ATP. A sample of the reaction mixture is shown in the left panel. Right panel: the reaction mixture containing phosphorylated RM2 domain was incubated for 30 minutes with 25 μl of immobilized GST or a GST-MbSrc1 SH2 domain fusion protein [20] in binding buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, total volume = 200 μl). In one reaction, an SH2-binding synthetic peptide (Glu-Pro-Gln-pTyr-Glu-Glu-Ile-Pro-Ile-Lys-Gln; labeled “pY” on the gel) was added at a concentration of 100 μM as a competitor. The glutathione-agarose beads were washed 4 times with 1 ml of binding buffer. Proteins were eluted by boiling with SDS-PAGE sample buffer and visualized by autoradiography.
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Figure 5: The RM2 domain acts as a kinase substrate and SH2-binding site. (A) Left panel: purified RM2-6 (1 μM) was incubated with RTKB2 (250 nM) in kinase assay buffer containing 0.8 mM ATP at 30°C for the indicated times. Aliquots were removed, boiled with SDS-PAGE sample buffer, and analyzed by Western blotting with anti-pTyr antibody. Right panel: similar experiments were carried out with purified MbSrc1 kinase. (B) Purified RM2-6 was phosphorylated by MbSrc1 in the presence of [γ-32P]-ATP. A sample of the reaction mixture is shown in the left panel. Right panel: the reaction mixture containing phosphorylated RM2 domain was incubated for 30 minutes with 25 μl of immobilized GST or a GST-MbSrc1 SH2 domain fusion protein [20] in binding buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, total volume = 200 μl). In one reaction, an SH2-binding synthetic peptide (Glu-Pro-Gln-pTyr-Glu-Glu-Ile-Pro-Ile-Lys-Gln; labeled “pY” on the gel) was added at a concentration of 100 μM as a competitor. The glutathione-agarose beads were washed 4 times with 1 ml of binding buffer. Proteins were eluted by boiling with SDS-PAGE sample buffer and visualized by autoradiography.

Mentions: To determine whether the isolated RM2 domain could serve as a substrate for RTKB2 kinase, we incubated the two purified proteins with Mg-ATP and followed the reaction by anti-pTyr Western blotting (Figure 5A, left panel). Tyrosine-phosphorylated RM2-6 was detected after 5–10 minutes in this reaction, consistent with the role of RM2 domains as autophosphorylation sites. The purified RM2 domain could also be phosphorylated by M. brevicollis MbSrc1 kinase (Figure 5A, right panel). We determined the stoichiometry of RM2-6 phosphorylation by MbSrc1 in a parallel reaction with [γ-32P]-ATP. After 5 minutes, the stoichiometry was 2.02 ± 0.13 moles of phosphate per mole of RM2-6 domain. RM2-6 has three potential tyrosine phosphorylation sites; these results raise the possibility of multisite phosphorylation in the six RM2 domains in the C-terminus of RTKB2. The Tyr-Glu-Ala-Ile sequence within RM2-6 fits the consensus sequence for binding to the SH2 domains of Src-family kinases [19]. The MbSrc1 SH2 domain has a similar binding preference as its metazoan counterparts [20]. To test for MbSrc1 SH2 binding, we first phosphorylated RM2-6 using RTKB2 and [γ-32P]-ATP. Next, we incubated the pY-RM2 with an immobilized protein consisting of glutathione S-transferase (GST) fused to the SH2 domain of MbSrc1 (Figure 5B). The phosphorylated RM2 domain bound to the MbSrc1 SH2 domain, as detected by autoradiography. When the binding reaction was carried out in the presence of a high-affinity SH2 binding peptide (pYEEI), binding between RM-6 and the MbSrc1 SH2 domains was drastically reduced (Figure 5B). These results are consistent with a model in which RTKB2 autophosphorylation initiates the formation of a signaling complex, potentially containing MbSrc1 (by analogy to metazoan RTK signaling) and additional SH2-containing Monosiga proteins.


Metazoan-like signaling in a unicellular receptor tyrosine kinase.

Schultheiss KP, Craddock BP, Tong M, Seeliger M, Miller WT - BMC Biochem. (2013)

The RM2 domain acts as a kinase substrate and SH2-binding site. (A) Left panel: purified RM2-6 (1 μM) was incubated with RTKB2 (250 nM) in kinase assay buffer containing 0.8 mM ATP at 30°C for the indicated times. Aliquots were removed, boiled with SDS-PAGE sample buffer, and analyzed by Western blotting with anti-pTyr antibody. Right panel: similar experiments were carried out with purified MbSrc1 kinase. (B) Purified RM2-6 was phosphorylated by MbSrc1 in the presence of [γ-32P]-ATP. A sample of the reaction mixture is shown in the left panel. Right panel: the reaction mixture containing phosphorylated RM2 domain was incubated for 30 minutes with 25 μl of immobilized GST or a GST-MbSrc1 SH2 domain fusion protein [20] in binding buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, total volume = 200 μl). In one reaction, an SH2-binding synthetic peptide (Glu-Pro-Gln-pTyr-Glu-Glu-Ile-Pro-Ile-Lys-Gln; labeled “pY” on the gel) was added at a concentration of 100 μM as a competitor. The glutathione-agarose beads were washed 4 times with 1 ml of binding buffer. Proteins were eluted by boiling with SDS-PAGE sample buffer and visualized by autoradiography.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC3584944&req=5

Figure 5: The RM2 domain acts as a kinase substrate and SH2-binding site. (A) Left panel: purified RM2-6 (1 μM) was incubated with RTKB2 (250 nM) in kinase assay buffer containing 0.8 mM ATP at 30°C for the indicated times. Aliquots were removed, boiled with SDS-PAGE sample buffer, and analyzed by Western blotting with anti-pTyr antibody. Right panel: similar experiments were carried out with purified MbSrc1 kinase. (B) Purified RM2-6 was phosphorylated by MbSrc1 in the presence of [γ-32P]-ATP. A sample of the reaction mixture is shown in the left panel. Right panel: the reaction mixture containing phosphorylated RM2 domain was incubated for 30 minutes with 25 μl of immobilized GST or a GST-MbSrc1 SH2 domain fusion protein [20] in binding buffer (50 mM Tris, pH 7.5, 250 mM NaCl, 5 mM EDTA, 0.1% Triton X-100, total volume = 200 μl). In one reaction, an SH2-binding synthetic peptide (Glu-Pro-Gln-pTyr-Glu-Glu-Ile-Pro-Ile-Lys-Gln; labeled “pY” on the gel) was added at a concentration of 100 μM as a competitor. The glutathione-agarose beads were washed 4 times with 1 ml of binding buffer. Proteins were eluted by boiling with SDS-PAGE sample buffer and visualized by autoradiography.
Mentions: To determine whether the isolated RM2 domain could serve as a substrate for RTKB2 kinase, we incubated the two purified proteins with Mg-ATP and followed the reaction by anti-pTyr Western blotting (Figure 5A, left panel). Tyrosine-phosphorylated RM2-6 was detected after 5–10 minutes in this reaction, consistent with the role of RM2 domains as autophosphorylation sites. The purified RM2 domain could also be phosphorylated by M. brevicollis MbSrc1 kinase (Figure 5A, right panel). We determined the stoichiometry of RM2-6 phosphorylation by MbSrc1 in a parallel reaction with [γ-32P]-ATP. After 5 minutes, the stoichiometry was 2.02 ± 0.13 moles of phosphate per mole of RM2-6 domain. RM2-6 has three potential tyrosine phosphorylation sites; these results raise the possibility of multisite phosphorylation in the six RM2 domains in the C-terminus of RTKB2. The Tyr-Glu-Ala-Ile sequence within RM2-6 fits the consensus sequence for binding to the SH2 domains of Src-family kinases [19]. The MbSrc1 SH2 domain has a similar binding preference as its metazoan counterparts [20]. To test for MbSrc1 SH2 binding, we first phosphorylated RM2-6 using RTKB2 and [γ-32P]-ATP. Next, we incubated the pY-RM2 with an immobilized protein consisting of glutathione S-transferase (GST) fused to the SH2 domain of MbSrc1 (Figure 5B). The phosphorylated RM2 domain bound to the MbSrc1 SH2 domain, as detected by autoradiography. When the binding reaction was carried out in the presence of a high-affinity SH2 binding peptide (pYEEI), binding between RM-6 and the MbSrc1 SH2 domains was drastically reduced (Figure 5B). These results are consistent with a model in which RTKB2 autophosphorylation initiates the formation of a signaling complex, potentially containing MbSrc1 (by analogy to metazoan RTK signaling) and additional SH2-containing Monosiga proteins.

Bottom Line: 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.Thus, crucial features of signal transduction circuitry were established prior to the evolution of metazoans from their unicellular ancestors.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Biophysics, Basic Science Tower, T-6, School of Medicine, Stony Brook University, Stony Brook, NY 11794-8661, USA.

ABSTRACT

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.

Show MeSH
Related in: MedlinePlus