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

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.

SUBMITTER: Akbarzadeh S

PROVIDER: S-EPMC2268744 | biostudies-literature | 2008 Mar

REPOSITORIES: biostudies-literature

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

Akbarzadeh Shiva S Wheldon Lee M LM Sweet Steve M M SM Talma Sonia S Mardakheh Faraz Khosravi FK Heath John K JK

PloS one 20080326 3

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 ...[more]

PMID: 18365018

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Project description:BackgroundThe mucin MUC1, a type I transmembrane glycoprotein, is overexpressed in breast cancer and has been correlated with increased metastasis. We were the first to report binding between MUC1 and Intercellular adhesion molecule-1 (ICAM-1), which is expressed on stromal and endothelial cells throughout the migratory tract of a metastasizing breast cancer cell. Subsequently, we found that MUC1/ICAM-1 binding results in pro-migratory calcium oscillations, cytoskeletal reorganization, and simulated transendothelial migration. These events were found to involve Src kinase, a non-receptor tyrosine kinase also implicated in breast cancer initiation and progression. Here, we further investigated the mechanism of MUC1/ICAM-1 signalling, focusing on the role of MUC1 dimerization in Src recruitment and pro-metastatic signalling.MethodsTo assay MUC1 dimerization, we used a chemical crosslinker which allowed for the detection of dimers on SDS-PAGE. We then generated MUC1 constructs containing an engineered domain which allowed for manipulation of dimerization status through the addition of ligands to the engineered domain. Following manipulation of dimerization, we immunoprecipitated MUC1 to investigate recruitment of Src, or assayed for our previously observed ICAM-1 binding induced events. To investigate the nature of MUC1 dimers, we used both non-reducing SDS-PAGE and generated a mutant construct lacking cysteine residues.ResultsWe first demonstrate that the previously observed MUC1/ICAM-1 signalling events are dependent on the activity of Src kinase. We then report that MUC1 forms constitutive cytoplasmic domain dimers which are necessary for Src recruitment, ICAM-1 induced calcium oscillations and simulated transendothelial migration. The dimers are not covalently linked constitutively or following ICAM-1 binding. In contrast to previously published reports, we found that membrane proximal cysteine residues were not involved in dimerization or ICAM-1 induced signalling.ConclusionsOur data implicates non-cysteine linked MUC1 dimerization in cell signalling pathways required for cancer cell migration.

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

Publications

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

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