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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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Related in: MedlinePlus

Alignment of the amino acid sequences of the six RM2 domains [10]. Sequences were aligned using the ClustalW program [15] and formatted with BOXSHADE (version 3.21, written by K. Hofmann and M. Baron). The conserved tyrosine residue is indicated by a red asterisk. The sixth RM2 domain, shown in red, was chosen for further analysis by bacterial expression.
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Figure 4: Alignment of the amino acid sequences of the six RM2 domains [10]. Sequences were aligned using the ClustalW program [15] and formatted with BOXSHADE (version 3.21, written by K. Hofmann and M. Baron). The conserved tyrosine residue is indicated by a red asterisk. The sixth RM2 domain, shown in red, was chosen for further analysis by bacterial expression.

Mentions: The six RM2 domains in the C-tail of RTKB2 contain tyrosines near their N-termini that are predicted to serve as phosphorylation and SH2-binding sites (Figure 4) [10]. As shown above in Figure 2, synthetic peptides derived from two of the domains are indeed substrates for RTKB2 kinase. To study an intact RM2 domain, we synthesized the gene corresponding to the sixth RM2 domain (the sequence of RM2-6 is presented in Figure 4). The sixth RM2 domain was chosen because it contains a predicted Src phosphorylation/SH2-binding site that conforms well to the consensus (EEVYEAI), and because the RTKB2-2 peptide (Figure 2) corresponds to this site. We cloned the cDNA into a bacterial expression vector, expressed the protein in E. coli cells, and purified it by metal affinity chromatography (Additional file 2: Figure S2A). Despite a predicted molecular weight of 10,089 daltons, purified RM2-6 migrated with an apparent molecular weight of approximately 20,000 daltons on SDS-PAGE (Additional file 2: Figure S2A). The aberrant mobility of RM2-6 may be due to the highly acidic amino acid composition (pI = 3.9). We confirmed the molecular weight of the bacterially-expressed RM2 domain by matrix-assisted laser desorption/ionization mass spectrometry (Additional file 2: Figure S2B).


Metazoan-like signaling in a unicellular receptor tyrosine kinase.

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

Alignment of the amino acid sequences of the six RM2 domains [10]. Sequences were aligned using the ClustalW program [15] and formatted with BOXSHADE (version 3.21, written by K. Hofmann and M. Baron). The conserved tyrosine residue is indicated by a red asterisk. The sixth RM2 domain, shown in red, was chosen for further analysis by bacterial expression.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Alignment of the amino acid sequences of the six RM2 domains [10]. Sequences were aligned using the ClustalW program [15] and formatted with BOXSHADE (version 3.21, written by K. Hofmann and M. Baron). The conserved tyrosine residue is indicated by a red asterisk. The sixth RM2 domain, shown in red, was chosen for further analysis by bacterial expression.
Mentions: The six RM2 domains in the C-tail of RTKB2 contain tyrosines near their N-termini that are predicted to serve as phosphorylation and SH2-binding sites (Figure 4) [10]. As shown above in Figure 2, synthetic peptides derived from two of the domains are indeed substrates for RTKB2 kinase. To study an intact RM2 domain, we synthesized the gene corresponding to the sixth RM2 domain (the sequence of RM2-6 is presented in Figure 4). The sixth RM2 domain was chosen because it contains a predicted Src phosphorylation/SH2-binding site that conforms well to the consensus (EEVYEAI), and because the RTKB2-2 peptide (Figure 2) corresponds to this site. We cloned the cDNA into a bacterial expression vector, expressed the protein in E. coli cells, and purified it by metal affinity chromatography (Additional file 2: Figure S2A). Despite a predicted molecular weight of 10,089 daltons, purified RM2-6 migrated with an apparent molecular weight of approximately 20,000 daltons on SDS-PAGE (Additional file 2: Figure S2A). The aberrant mobility of RM2-6 may be due to the highly acidic amino acid composition (pI = 3.9). We confirmed the molecular weight of the bacterially-expressed RM2 domain by matrix-assisted laser desorption/ionization mass spectrometry (Additional file 2: Figure S2B).

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