ABSTRACT
DOK1 regulates platelet-derived growth factor (PDGF)-BB-stimulated glioma cell motility. Mechanisms regulating tumour cell motility are essential for invasion and metastasis. We report here that PDGF-BB-mediated glioma cell invasion and migration are dependent on the adaptor protein downstream of kinase 1 (DOK1). DOK1 is expressed in several glioma cell lines and in tumour biopsies from high-grade gliomas. DOK1 becomes tyrosine phosphorylated upon PDGF-BB stimulation of human glioma cells. Knockdown of DOK1 or expression of a DOK1 mutant (DOK1FF) containing Phe in place of Tyr at residues 362 and 398, resulted in inhibition of both the PDGF-BB-induced tyrosine phosphorylation of p130Cas (also known as BCAR1) and the activation of Rap1 (also known as TERF2IP). DOK1 colocalises with tyrosine phosphorylated p130Cas at the cell membrane of PDGF-BB-treated cells. Expression of a non-tyrosine-phosphorylatable substrate domain mutant of p130Cas (p130Cas15F) inhibited PDGF-BB-mediated Rap1 activation. Knockdown of DOK1 and Rap1 inhibited PDGF-BB-induced chemotactic cell migration, and knockdown of DOK1 and Rap1 and expression of DOK1FF inhibited PDGF-mediated three-dimensional (3D) spheroid invasion. These data show a crucial role for DOK1 in the regulation of PDGF-BB-mediated tumour cell motility through a p130Cas–Rap1 signalling pathway.
Author contributions
A.B., I.M.E. and P.F. designed the study, performed the experiments, analysed the data and wrote the paper. A.F., G.B., C.P‐M. and M.Y. performed the experiments. A.F. and G.B. contributed equally. V.M., R.B., N.L. and S.B. generated essential reagents. I.C.Z. designed the study, analysed the data and wrote the paper.
Funding
This work was supported by grants from the Biotechnology and Biological Sciences Research Council (BB/G017921/1 and BB/K013068/1) to P.F. (A.B. and A.F.), a British Heart Foundation programme grant RG/06/003 and project grant PG/12/65/29840 to I.C.Z. (I.E., M.Y., G.B. and C.P-M.), and the Brain Tumour Charity (NL; Grant 8-128). This work was undertaken at UCLH/UCL who received a proportion of funding from the UK Department of Health's National Institute for Health Research (NIHR) Biomedical Research Centre's funding scheme (S.B.).
