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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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Peptide phosphorylation by purified RTKB2 kinase. The enzymatic activity of RTKB2 (1 μM) towards four synthetic peptides (750 μM) was measured using the phosphocellulose paper binding assay. Reactions were carried out for 15 minutes at 30°C. Error bars indicate standard deviations.
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Figure 2: Peptide phosphorylation by purified RTKB2 kinase. The enzymatic activity of RTKB2 (1 μM) towards four synthetic peptides (750 μM) was measured using the phosphocellulose paper binding assay. Reactions were carried out for 15 minutes at 30°C. Error bars indicate standard deviations.

Mentions: To test whether the predicted RTKB2 protein is an active tyrosine kinase, we used PCR with kinase-specific primers to amplify the cDNA encoding the catalytic domain (residues 1450–1724) from a Monosiga brevicollis cDNA library. We cloned the RTKB2 kinase sequence into a baculovirus expression vector, infected Spodoptera frugiperda (Sf9) insect cells with the recombinant baculovirus, and purified the protein (Additional file 1: Figure S1). Using the phosphocellulose paper binding assay, we tested RTKB2 kinase activity towards several synthetic peptides (Figure 2). RTKB2 was highly active, with a specific activity similar to other purified RTK kinase domains (e.g., the kinase domain of insulin-like growth factor I receptor [17]). The highest activity was observed with a peptide (E4YM4) containing the EEEEYMMMM motif that was selected from a synthetic peptide library as a highly efficient insulin receptor substrate [18]. This activity (73 pmol/min/μg) is comparable to the activity of Monosiga brevicollis MbSrc1 toward its preferred substrates (145 pmol/min/μg; ref. 10). We also tested two peptides with sequences corresponding to predicted phosphorylation sites within RTKB2 RM2 domains. RTKB2-1 is derived from the fifth RM2 domain, while RTKB2-2 is derived from the sixth RM2. Both peptides were phosphorylated by RTKB2 kinase, consistent with the possibility that these sites on the RTKB2 C-tail serve as autophosphorylation sites. We previously showed that these peptides are also phosphorylated by the Monosiga Src-like kinase MbSrc1 [10].


Metazoan-like signaling in a unicellular receptor tyrosine kinase.

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

Peptide phosphorylation by purified RTKB2 kinase. The enzymatic activity of RTKB2 (1 μM) towards four synthetic peptides (750 μM) was measured using the phosphocellulose paper binding assay. Reactions were carried out for 15 minutes at 30°C. Error bars indicate standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Peptide phosphorylation by purified RTKB2 kinase. The enzymatic activity of RTKB2 (1 μM) towards four synthetic peptides (750 μM) was measured using the phosphocellulose paper binding assay. Reactions were carried out for 15 minutes at 30°C. Error bars indicate standard deviations.
Mentions: To test whether the predicted RTKB2 protein is an active tyrosine kinase, we used PCR with kinase-specific primers to amplify the cDNA encoding the catalytic domain (residues 1450–1724) from a Monosiga brevicollis cDNA library. We cloned the RTKB2 kinase sequence into a baculovirus expression vector, infected Spodoptera frugiperda (Sf9) insect cells with the recombinant baculovirus, and purified the protein (Additional file 1: Figure S1). Using the phosphocellulose paper binding assay, we tested RTKB2 kinase activity towards several synthetic peptides (Figure 2). RTKB2 was highly active, with a specific activity similar to other purified RTK kinase domains (e.g., the kinase domain of insulin-like growth factor I receptor [17]). The highest activity was observed with a peptide (E4YM4) containing the EEEEYMMMM motif that was selected from a synthetic peptide library as a highly efficient insulin receptor substrate [18]. This activity (73 pmol/min/μg) is comparable to the activity of Monosiga brevicollis MbSrc1 toward its preferred substrates (145 pmol/min/μg; ref. 10). We also tested two peptides with sequences corresponding to predicted phosphorylation sites within RTKB2 RM2 domains. RTKB2-1 is derived from the fifth RM2 domain, while RTKB2-2 is derived from the sixth RM2. Both peptides were phosphorylated by RTKB2 kinase, consistent with the possibility that these sites on the RTKB2 C-tail serve as autophosphorylation sites. We previously showed that these peptides are also phosphorylated by the Monosiga Src-like kinase MbSrc1 [10].

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