Barrett, A; (2014) The Role of the Adaptor Protein Downstream of Tyrosine Kinase 1 (DOK1) in Glioma Cell Motility. Doctoral thesis , UCL (University College London).

Abstract

The adaptor protein Downstream of tyrosine kinase 1 (DOK1) was identified as a 62kDa protein highly phosphorylated as a result tyrosine kinase activation. The role of DOK1 in cell motility had not been extensively explored prior to this thesis, despite reports of DOK1 associations with several proteins involved with cell motility, including p130Cas and the β3-integrin subunit. Results in this thesis show that DOK1 is expressed in glioma cell lines and biopsy samples. DOK1 is tyrosine phosphorylated following PDGF-BB stimulation in the malignant glioma cell line U87, requiring the activities of Src, Abl, and phosphoinositide 3-kinase. siRNA-mediated knockdown of DOK1, or expression of a DOK1 mutant (DOK1FF; Tyr362/398Phe), resulted in inhibition of PDGF-BB mediated p130Cas phosphorylation and Rap1 GTP loading. Furthermore, PDGF-BB directed U87 cell migration and invasion were significantly reduced. These data indicate a critical role for DOK1 in the regulation of PDGF-BB mediated U87 cell motility through a novel DOK1-p130Cas-Rap1 signalling pathway, with implications for the mechanisms underlying the pathogenesis and metastasis of glioma. PDGF-BB mediated DOK1 phosphorylation was found to be suppressed during integrin activation in U87 cells. Additionally, adhesion (to fibronectin or vitronectin) and PDGF-BB-stimulation were respectively found to induce opposing effects on the phosphorylation of the proximal NXXY motifs of β1- and β3-integrin subunits. Treatment of U87 cells with the Abl/PDGFR/c-Kit inhibitor imatinib appeared to activate integrin signalling, inducing p130Cas and FAK phosphorylation in the absence of PDGF-BB, whilst inhibiting PDGF-BB-stimulated PDGFRβ and DOK1 phosphorylation. Conversely, treatment with an RGD-peptide integrin inhibitor induced the opposite effects. Additionally, inhibition of FAK and PYK2 induced an increase in PDGF-BB-stimulated DOK1 phosphorylation. These findings support a novel model in which PDGFRβ and integrin subunits β1 and β3 compete for Src association, resulting in an antagonistic relationship between integrin-driven adhesive signalling and PDGFRβ-driven chemotactic signalling.

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