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The deleted in brachydactyly B domain of ROR2 is required for receptor activation by recruitment of Src.

Akbarzadeh S, Wheldon LM, Sweet SM, Talma S, Mardakheh FK, Heath JK - PLoS ONE (2008)

Bottom Line: Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity.Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes.These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src.

View Article: PubMed Central - PubMed

Affiliation: CR-UK Growth Factor Group, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

ABSTRACT
The transmembrane receptor 'ROR2' resembles members of the receptor tyrosine kinase family of signalling receptors in sequence but its' signal transduction mechanisms remain enigmatic. This problem has particular importance because mutations in ROR2 are associated with two human skeletal dysmorphology syndromes, recessive Robinow Syndrome (RS) and dominant acting Brachydactyly type B (BDB). Here we show, using a constitutive dimerisation approach, that ROR2 exhibits dimerisation-induced tyrosine kinase activity and the ROR2 C-terminal domain, which is deleted in BDB, is required for recruitment and activation of the non-receptor tyrosine kinase Src. Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity. Eight sites of Src-mediated ROR2 phosphorylation have been identified by mass spectrometry. Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes. These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src.

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ROR2 receptor is a substrate for Src.Chondrocytes were co-transfected with myc-tagged ROR2 WT, ROR2 KD and myc-tagged deletion mutants ROR2 P860X and ROR2 L747X and either vector alone (V), constitutively active Src (Src A) or inactive Src (Src MF). Lanes 5 and 6 are controls for Src A and Src MF, respectively. The receptor constructs were immunoprecipitated with anti-myc (IP-myc) and probed with anti-phosphotyrosine antibodies (WB-pY, upper panel) then stripped and reprobed with anti-myc (loading control, WB-myc). Src A and MF expression was confirmed on whole cell lysate prior to immunoprecipitation using anti-chicken Src antibody (WB-EC10, lower panel).
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pone-0001873-g004: ROR2 receptor is a substrate for Src.Chondrocytes were co-transfected with myc-tagged ROR2 WT, ROR2 KD and myc-tagged deletion mutants ROR2 P860X and ROR2 L747X and either vector alone (V), constitutively active Src (Src A) or inactive Src (Src MF). Lanes 5 and 6 are controls for Src A and Src MF, respectively. The receptor constructs were immunoprecipitated with anti-myc (IP-myc) and probed with anti-phosphotyrosine antibodies (WB-pY, upper panel) then stripped and reprobed with anti-myc (loading control, WB-myc). Src A and MF expression was confirmed on whole cell lysate prior to immunoprecipitation using anti-chicken Src antibody (WB-EC10, lower panel).

Mentions: We next sought to identify the ROR2 cytoplasmic domain(s) that are essential for Src kinase binding. Full length ROR2-myc WT, KD, P860X and L747X were co-expressed in chondrocytes with either a constitutively active Src Y527F (Src A) or a kinase dead Src K295M/Y527F (Src MF). ROR2-myc WT, KD, and P860X receptors were tyrosine phosphorylated in a ligand-independent manner when co-expressed with Src Y527F (Fig. 4). Phosphorylation of L747X mutant was almost ablated compared to the WT receptor, indicating that the C-terminal proline-rich and/or the first serine/threonine-rich regions are essential for Src binding. No receptor phosphorylation was observed when Src MF was co-expressed with any of the ROR2-myc constructs.


The deleted in brachydactyly B domain of ROR2 is required for receptor activation by recruitment of Src.

Akbarzadeh S, Wheldon LM, Sweet SM, Talma S, Mardakheh FK, Heath JK - PLoS ONE (2008)

ROR2 receptor is a substrate for Src.Chondrocytes were co-transfected with myc-tagged ROR2 WT, ROR2 KD and myc-tagged deletion mutants ROR2 P860X and ROR2 L747X and either vector alone (V), constitutively active Src (Src A) or inactive Src (Src MF). Lanes 5 and 6 are controls for Src A and Src MF, respectively. The receptor constructs were immunoprecipitated with anti-myc (IP-myc) and probed with anti-phosphotyrosine antibodies (WB-pY, upper panel) then stripped and reprobed with anti-myc (loading control, WB-myc). Src A and MF expression was confirmed on whole cell lysate prior to immunoprecipitation using anti-chicken Src antibody (WB-EC10, lower panel).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2268744&req=5

pone-0001873-g004: ROR2 receptor is a substrate for Src.Chondrocytes were co-transfected with myc-tagged ROR2 WT, ROR2 KD and myc-tagged deletion mutants ROR2 P860X and ROR2 L747X and either vector alone (V), constitutively active Src (Src A) or inactive Src (Src MF). Lanes 5 and 6 are controls for Src A and Src MF, respectively. The receptor constructs were immunoprecipitated with anti-myc (IP-myc) and probed with anti-phosphotyrosine antibodies (WB-pY, upper panel) then stripped and reprobed with anti-myc (loading control, WB-myc). Src A and MF expression was confirmed on whole cell lysate prior to immunoprecipitation using anti-chicken Src antibody (WB-EC10, lower panel).
Mentions: We next sought to identify the ROR2 cytoplasmic domain(s) that are essential for Src kinase binding. Full length ROR2-myc WT, KD, P860X and L747X were co-expressed in chondrocytes with either a constitutively active Src Y527F (Src A) or a kinase dead Src K295M/Y527F (Src MF). ROR2-myc WT, KD, and P860X receptors were tyrosine phosphorylated in a ligand-independent manner when co-expressed with Src Y527F (Fig. 4). Phosphorylation of L747X mutant was almost ablated compared to the WT receptor, indicating that the C-terminal proline-rich and/or the first serine/threonine-rich regions are essential for Src binding. No receptor phosphorylation was observed when Src MF was co-expressed with any of the ROR2-myc constructs.

Bottom Line: Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity.Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes.These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src.

View Article: PubMed Central - PubMed

Affiliation: CR-UK Growth Factor Group, School of Biosciences, University of Birmingham, Edgbaston, Birmingham, United Kingdom.

ABSTRACT
The transmembrane receptor 'ROR2' resembles members of the receptor tyrosine kinase family of signalling receptors in sequence but its' signal transduction mechanisms remain enigmatic. This problem has particular importance because mutations in ROR2 are associated with two human skeletal dysmorphology syndromes, recessive Robinow Syndrome (RS) and dominant acting Brachydactyly type B (BDB). Here we show, using a constitutive dimerisation approach, that ROR2 exhibits dimerisation-induced tyrosine kinase activity and the ROR2 C-terminal domain, which is deleted in BDB, is required for recruitment and activation of the non-receptor tyrosine kinase Src. Native ROR2 phosphorylation is induced by the ligand Wnt5a and is blocked by pharmacological inhibition of Src kinase activity. Eight sites of Src-mediated ROR2 phosphorylation have been identified by mass spectrometry. Activation via tyrosine phosphorylation of ROR2 receptor leads to its internalisation into Rab5 positive endosomes. These findings show that BDB mutant receptors are defective in kinase activation as a result of failure to recruit Src.

Show MeSH
Related in: MedlinePlus