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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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Expression of the C-terminus of RTKB2 in mammalian cells. SYF cells were transiently transfected with plasmids encoding GFP or GFP-RTKB2-C-terminus (alone or together with a plasmid encoding FLAG-tagged MbSrc1). Top panels: anti-GFP immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with anti-phosphotyrosine antibodies. The membrane was subsequently stripped and reprobed with anti-GFP antibodies. The bottom two panels show expression of GFP, GFP-RTKB2-CT, and FLAG-MbSrc1 in the SYF cell lysates.
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Figure 6: Expression of the C-terminus of RTKB2 in mammalian cells. SYF cells were transiently transfected with plasmids encoding GFP or GFP-RTKB2-C-terminus (alone or together with a plasmid encoding FLAG-tagged MbSrc1). Top panels: anti-GFP immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with anti-phosphotyrosine antibodies. The membrane was subsequently stripped and reprobed with anti-GFP antibodies. The bottom two panels show expression of GFP, GFP-RTKB2-CT, and FLAG-MbSrc1 in the SYF cell lysates.

Mentions: To study RM2 domain phosphorylation in a cellular context, we amplified a cDNA encoding the C-terminal tail of the RTK (all six RM2 domains), and cloned it into a mammalian expression vector to produce a fusion with green fluorescent protein (GFP). We expressed the protein in Src/Yes/Fyn triple knockout fibroblast cells (SYF cells), which lack all Src family kinases [21]. Fluorescence microscopy showed that the GFP-RTKB2-CT has a diffuse cytoplasmic localization (Additional file 3: Figure S3). Next, we expressed GFP-RTKB2-CT in SYF cells alone or in the presence of Flag-tagged MbSrc1 kinase (Figure 6). We isolated RTKB2 by anti-GFP immunoprecipitation and analyzed tyrosine phosphorylation by Western blotting. RTKB2-CT was weakly tyrosine-phosphorylated in these experiments, and the addition of MbSrc1 gave only a modest increase in phosphorylation (Figure 6). We were unable to detect a complex between RTKB2-CT and MbSrc1 by co-immunoprecipitation (data not shown). While these experiments confirm the ability of the RTKB2 C-tail to be phosphorylated, it is likely that ligand-stimulated RTKB2 kinase would produce much higher levels of tyrosine phosphorylation. Furthermore, the lack of membrane-anchoring motifs in the RTKB2-CT and MbSrc1 constructs likely reduces the efficiency of their interaction.


Metazoan-like signaling in a unicellular receptor tyrosine kinase.

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

Expression of the C-terminus of RTKB2 in mammalian cells. SYF cells were transiently transfected with plasmids encoding GFP or GFP-RTKB2-C-terminus (alone or together with a plasmid encoding FLAG-tagged MbSrc1). Top panels: anti-GFP immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with anti-phosphotyrosine antibodies. The membrane was subsequently stripped and reprobed with anti-GFP antibodies. The bottom two panels show expression of GFP, GFP-RTKB2-CT, and FLAG-MbSrc1 in the SYF cell lysates.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Expression of the C-terminus of RTKB2 in mammalian cells. SYF cells were transiently transfected with plasmids encoding GFP or GFP-RTKB2-C-terminus (alone or together with a plasmid encoding FLAG-tagged MbSrc1). Top panels: anti-GFP immunoprecipitates were separated by SDS-PAGE and analyzed by Western blotting with anti-phosphotyrosine antibodies. The membrane was subsequently stripped and reprobed with anti-GFP antibodies. The bottom two panels show expression of GFP, GFP-RTKB2-CT, and FLAG-MbSrc1 in the SYF cell lysates.
Mentions: To study RM2 domain phosphorylation in a cellular context, we amplified a cDNA encoding the C-terminal tail of the RTK (all six RM2 domains), and cloned it into a mammalian expression vector to produce a fusion with green fluorescent protein (GFP). We expressed the protein in Src/Yes/Fyn triple knockout fibroblast cells (SYF cells), which lack all Src family kinases [21]. Fluorescence microscopy showed that the GFP-RTKB2-CT has a diffuse cytoplasmic localization (Additional file 3: Figure S3). Next, we expressed GFP-RTKB2-CT in SYF cells alone or in the presence of Flag-tagged MbSrc1 kinase (Figure 6). We isolated RTKB2 by anti-GFP immunoprecipitation and analyzed tyrosine phosphorylation by Western blotting. RTKB2-CT was weakly tyrosine-phosphorylated in these experiments, and the addition of MbSrc1 gave only a modest increase in phosphorylation (Figure 6). We were unable to detect a complex between RTKB2-CT and MbSrc1 by co-immunoprecipitation (data not shown). While these experiments confirm the ability of the RTKB2 C-tail to be phosphorylated, it is likely that ligand-stimulated RTKB2 kinase would produce much higher levels of tyrosine phosphorylation. Furthermore, the lack of membrane-anchoring motifs in the RTKB2-CT and MbSrc1 constructs likely reduces the efficiency of their interaction.

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