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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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RTKB2 autophosphorylation. (A) RTKB2 kinase was first treated with GST-YOP phosphatase, then incubated for the indicated times in the presence of 0.5 mM ATP. Top panel: the autophosphorylation reaction contained [γ-32P]-ATP, and the reaction was analyzed by SDS-PAGE and autoradiography. Incorporation of 32P into RTKB2 kinase was also measured by scintillation counting, and the stoichiometry (mol phosphate/mol RTKB2) is presented below the gel. Bottom panel: unlabeled ATP was used in the reaction, which was analyzed by SDS-PAGE and Western blotting with anti-pTyr antibody. Also analyzed were samples of RTKB2 kinase before YOP treatment (“pre”) and after YOP treatment but before autophosphorylation (0 min.). (B) The activity of Sf9-purified RTKB2 towards the E4YM4 synthetic peptide was measured either directly (RTKB2), after treatment with YOP tyrosine phosphatase (dephos. RTKB2), or after treatment with YOP followed by an autophosphorylation reaction (30 min at 30°C; dephos. RTKB2 + MgATP). Activity was measured using the phosphocellulose paper binding assay.
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Figure 3: RTKB2 autophosphorylation. (A) RTKB2 kinase was first treated with GST-YOP phosphatase, then incubated for the indicated times in the presence of 0.5 mM ATP. Top panel: the autophosphorylation reaction contained [γ-32P]-ATP, and the reaction was analyzed by SDS-PAGE and autoradiography. Incorporation of 32P into RTKB2 kinase was also measured by scintillation counting, and the stoichiometry (mol phosphate/mol RTKB2) is presented below the gel. Bottom panel: unlabeled ATP was used in the reaction, which was analyzed by SDS-PAGE and Western blotting with anti-pTyr antibody. Also analyzed were samples of RTKB2 kinase before YOP treatment (“pre”) and after YOP treatment but before autophosphorylation (0 min.). (B) The activity of Sf9-purified RTKB2 towards the E4YM4 synthetic peptide was measured either directly (RTKB2), after treatment with YOP tyrosine phosphatase (dephos. RTKB2), or after treatment with YOP followed by an autophosphorylation reaction (30 min at 30°C; dephos. RTKB2 + MgATP). Activity was measured using the phosphocellulose paper binding assay.

Mentions: Many RTKs are regulated by autophosphorylation at one or more tyrosines within the activation loop, a flexible segment between the two lobes of the kinase catalytic domain [2]. RTKB2 has a single tyrosine in the predicted activation loop (Figure 1B). To test for RTKB2 autophosphorylation, we first treated the purified enzyme with Yersinia tyrosine-specific YOP phosphatase. Next, we incubated the dephosphorylated RTKB2 with magnesium and [γ-32P]-ATP and followed the reaction by SDS-PAGE and autoradiography (Figrue 3A, top panel). Labeled phosphate was incorporated into RTKB2 after 5–15 minutes under these conditions. The stoichiometry of phosphorylation after 15 minutes was 0.93 mol phosphate/mol protein. In a parallel experiment, the time-course of autophosphorylation was followed by Western blotting with anti-phosphotyrosine antibody (Figure 3A, bottom panel). In this experiment, the signal for pre-YOP treated RTKB2 was stronger than the signal for autophosphorylated RTKB2, raising the possibility that multiple sites are phosphorylated in Sf9 cells. To determine whether autophosphorylation influences RTKB2 kinase activity, we carried out a peptide phosphorylation assay with purified RTKB2, YOP-dephosphorylated RTKB2, and enzyme that had been allowed to re-phosphorylate as described above. Dephosphorylated RTKB2 lost significant kinase activity as compared to the starting sample, but the activity was regained upon re-phosphorylation (Figure 3B). These results are consistent with a role for RTKB2 autophosphorylation in the control of enzymatic function.


Metazoan-like signaling in a unicellular receptor tyrosine kinase.

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

RTKB2 autophosphorylation. (A) RTKB2 kinase was first treated with GST-YOP phosphatase, then incubated for the indicated times in the presence of 0.5 mM ATP. Top panel: the autophosphorylation reaction contained [γ-32P]-ATP, and the reaction was analyzed by SDS-PAGE and autoradiography. Incorporation of 32P into RTKB2 kinase was also measured by scintillation counting, and the stoichiometry (mol phosphate/mol RTKB2) is presented below the gel. Bottom panel: unlabeled ATP was used in the reaction, which was analyzed by SDS-PAGE and Western blotting with anti-pTyr antibody. Also analyzed were samples of RTKB2 kinase before YOP treatment (“pre”) and after YOP treatment but before autophosphorylation (0 min.). (B) The activity of Sf9-purified RTKB2 towards the E4YM4 synthetic peptide was measured either directly (RTKB2), after treatment with YOP tyrosine phosphatase (dephos. RTKB2), or after treatment with YOP followed by an autophosphorylation reaction (30 min at 30°C; dephos. RTKB2 + MgATP). Activity was measured using the phosphocellulose paper binding assay.
© Copyright Policy - open-access
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3584944&req=5

Figure 3: RTKB2 autophosphorylation. (A) RTKB2 kinase was first treated with GST-YOP phosphatase, then incubated for the indicated times in the presence of 0.5 mM ATP. Top panel: the autophosphorylation reaction contained [γ-32P]-ATP, and the reaction was analyzed by SDS-PAGE and autoradiography. Incorporation of 32P into RTKB2 kinase was also measured by scintillation counting, and the stoichiometry (mol phosphate/mol RTKB2) is presented below the gel. Bottom panel: unlabeled ATP was used in the reaction, which was analyzed by SDS-PAGE and Western blotting with anti-pTyr antibody. Also analyzed were samples of RTKB2 kinase before YOP treatment (“pre”) and after YOP treatment but before autophosphorylation (0 min.). (B) The activity of Sf9-purified RTKB2 towards the E4YM4 synthetic peptide was measured either directly (RTKB2), after treatment with YOP tyrosine phosphatase (dephos. RTKB2), or after treatment with YOP followed by an autophosphorylation reaction (30 min at 30°C; dephos. RTKB2 + MgATP). Activity was measured using the phosphocellulose paper binding assay.
Mentions: Many RTKs are regulated by autophosphorylation at one or more tyrosines within the activation loop, a flexible segment between the two lobes of the kinase catalytic domain [2]. RTKB2 has a single tyrosine in the predicted activation loop (Figure 1B). To test for RTKB2 autophosphorylation, we first treated the purified enzyme with Yersinia tyrosine-specific YOP phosphatase. Next, we incubated the dephosphorylated RTKB2 with magnesium and [γ-32P]-ATP and followed the reaction by SDS-PAGE and autoradiography (Figrue 3A, top panel). Labeled phosphate was incorporated into RTKB2 after 5–15 minutes under these conditions. The stoichiometry of phosphorylation after 15 minutes was 0.93 mol phosphate/mol protein. In a parallel experiment, the time-course of autophosphorylation was followed by Western blotting with anti-phosphotyrosine antibody (Figure 3A, bottom panel). In this experiment, the signal for pre-YOP treated RTKB2 was stronger than the signal for autophosphorylated RTKB2, raising the possibility that multiple sites are phosphorylated in Sf9 cells. To determine whether autophosphorylation influences RTKB2 kinase activity, we carried out a peptide phosphorylation assay with purified RTKB2, YOP-dephosphorylated RTKB2, and enzyme that had been allowed to re-phosphorylate as described above. Dephosphorylated RTKB2 lost significant kinase activity as compared to the starting sample, but the activity was regained upon re-phosphorylation (Figure 3B). These results are consistent with a role for RTKB2 autophosphorylation in the control of enzymatic function.

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