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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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(A) Domain organization of the M. brevicollis RTKB2 kinase [10]. List of domain abbreviations: Cys, cysteine-rich region; HYR, repeats similar to hyalin domains; SCR, short consensus repeat/complement control protein domain; E, epidermal growth factor repeat; tm, transmembrane sequence; RM2, unique M. brevicollis domain. (B) Amino acid sequence of the RTKB2 kinase domain, aligned with human fibroblast growth factor receptor 1 (FGFR1) and epidermal growth factor receptor (EGFR). Sequences were aligned using the ClustalW program [15] and formatted with BOXSHADE (version 3.21, written by K. Hofmann and M. Baron). The position of the kinase-conserved DFG motif at the beginning of the activation loop is indicated with a red bracket, and an asterisk indicates the position of the potential autophosphorylation site.
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Figure 1: (A) Domain organization of the M. brevicollis RTKB2 kinase [10]. List of domain abbreviations: Cys, cysteine-rich region; HYR, repeats similar to hyalin domains; SCR, short consensus repeat/complement control protein domain; E, epidermal growth factor repeat; tm, transmembrane sequence; RM2, unique M. brevicollis domain. (B) Amino acid sequence of the RTKB2 kinase domain, aligned with human fibroblast growth factor receptor 1 (FGFR1) and epidermal growth factor receptor (EGFR). Sequences were aligned using the ClustalW program [15] and formatted with BOXSHADE (version 3.21, written by K. Hofmann and M. Baron). The position of the kinase-conserved DFG motif at the beginning of the activation loop is indicated with a red bracket, and an asterisk indicates the position of the potential autophosphorylation site.

Mentions: The RTKB family of tyrosine kinases from M. brevicollis consists of nine members. While the sequences of the RTKB kinase catalytic domains are related, the extracellular regions are quite diverse [10]. Three of the nine RTKB kinases appear to lack important catalytic residues and may function as pseudokinases or scaffolding proteins. Of the six remaining RTKB kinases, four possess a unique modular domain (designated RM2) in their cytoplasmic tails (Figure 1). The RM2 domains contain potential Src-family kinase phosphorylation sites and SH2-binding sites, suggesting that they may link RTK activation with downstream cytoplasmic signals [10]. In this paper, we characterized Monosiga brevicollis RTKB2, a kinase with six RM2 domains in its C-tail. We cloned, expressed, and purified the RTKB2 kinase, and conducted biochemical studies of its activity. These studies are the first of an RTK from a unicellular organism.


Metazoan-like signaling in a unicellular receptor tyrosine kinase.

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

(A) Domain organization of the M. brevicollis RTKB2 kinase [10]. List of domain abbreviations: Cys, cysteine-rich region; HYR, repeats similar to hyalin domains; SCR, short consensus repeat/complement control protein domain; E, epidermal growth factor repeat; tm, transmembrane sequence; RM2, unique M. brevicollis domain. (B) Amino acid sequence of the RTKB2 kinase domain, aligned with human fibroblast growth factor receptor 1 (FGFR1) and epidermal growth factor receptor (EGFR). Sequences were aligned using the ClustalW program [15] and formatted with BOXSHADE (version 3.21, written by K. Hofmann and M. Baron). The position of the kinase-conserved DFG motif at the beginning of the activation loop is indicated with a red bracket, and an asterisk indicates the position of the potential autophosphorylation site.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: (A) Domain organization of the M. brevicollis RTKB2 kinase [10]. List of domain abbreviations: Cys, cysteine-rich region; HYR, repeats similar to hyalin domains; SCR, short consensus repeat/complement control protein domain; E, epidermal growth factor repeat; tm, transmembrane sequence; RM2, unique M. brevicollis domain. (B) Amino acid sequence of the RTKB2 kinase domain, aligned with human fibroblast growth factor receptor 1 (FGFR1) and epidermal growth factor receptor (EGFR). Sequences were aligned using the ClustalW program [15] and formatted with BOXSHADE (version 3.21, written by K. Hofmann and M. Baron). The position of the kinase-conserved DFG motif at the beginning of the activation loop is indicated with a red bracket, and an asterisk indicates the position of the potential autophosphorylation site.
Mentions: The RTKB family of tyrosine kinases from M. brevicollis consists of nine members. While the sequences of the RTKB kinase catalytic domains are related, the extracellular regions are quite diverse [10]. Three of the nine RTKB kinases appear to lack important catalytic residues and may function as pseudokinases or scaffolding proteins. Of the six remaining RTKB kinases, four possess a unique modular domain (designated RM2) in their cytoplasmic tails (Figure 1). The RM2 domains contain potential Src-family kinase phosphorylation sites and SH2-binding sites, suggesting that they may link RTK activation with downstream cytoplasmic signals [10]. In this paper, we characterized Monosiga brevicollis RTKB2, a kinase with six RM2 domains in its C-tail. We cloned, expressed, and purified the RTKB2 kinase, and conducted biochemical studies of its activity. These studies are the first of an RTK from a unicellular organism.

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