Dok1 Inhibits Dorsal Ruffle Formation In Response To EGF In Cos-7 Cells

Objective: The Dok proteins (downstream of tyrosine kinases) belongto a family of adaptor proteins and are related with tyrosine kinases.The p62Dok (Dokl) protein is a common substrate for activated protein tyrosine kinases(PTKs) such as v-Ab1 v-Src Tec and the p210bcr-abl fusion oncoprotein and is also rapidly tyrosine- phosphorylated in response to receptor tyrosine kinases(RTKs) such as EGFR PDGFR IR and Eph. As a docking molecule, Dokl harbors several characteristic motifs and domains,like the insulin receptor substrate(IRS)protein. At its N terminus, Dokl contains a pleckstrin homology (PH) domain followed by a phosphotyrosine -binding (PTB) domain, while the COOH-terminal tail of Dokl harbors several potential tyrosine phosphorylation sites and PxxP motifs. The PH domain is necessary for targeting Dok to the membrane, because PH domains preferentially bind phospholipids. The multiple tyrosine residues in the Dok carboxyl-terminal tail are candidate sites for tyrosine kinases. When phosphorylated, they become potential docking sites for Src homology 2-containing proteins such as p120 rasGAP. Nck and Csk. The putative PTB domain is most homologous to the IRS-1 PTB domains, which recognizephosphotyrosine (pY)-containing sequences.lt remains to be determined whether through the PTB domain Dokl can bind phosphopeptides of EGFR(RTK) , and the functional significance of the PTB domain has yet to be addressed.The physiollgical role of tyrosine phosphorylation of Dokl is not clear.The result has identified Dokl as a signal transducer that potentially links,through its interaction with NCK or rasGAP,cell adhesion and insulin receptors to the machinery that controls cell motility. Tyrosine phosphorylation of Dokl correlates well with the transforming activity of such viral oncogene-encoded activated tyrosine kinases,suggesting that this protein plays an important role in cellular transformation.There are some alter in almost all transformed cell cytoskeletons.Epidermal growth factor (EGF) specifically binds to and activates the EGF receptor (EGFR), resulting in stimulation of cellular responses such as membrane ruffle formation. Ruffle is one of the cytoskeleton forms rich in polymerized actin and is related with cell migration. To gain further insight into the functional significance of the PTB domain,we have now generated mutants of important domains and sites-Yellow Fluorescent Protein chimera.We introduce these mutant proteins into Cos-7 cells by transefection and find how can Dokl affects the signal pathway in response to epidermal growth factor,and in turn influences its biological functions, including cytoskeleton transform.These studies can not only reveal the function of the PTB domain in tyrosine phosphorylation of Dokl in response to EGF stimulation from cytology,but also provide support for the relation of Dokl and cellular transformation.Furthermore, elucidation of the mechanism of downstream of tyrosine kinases can light up to the therapy of metastasis of cancer. Both theorical and practical significance are important and profound.Methods: Using DNA reconstruction technology, such as PCR, restrict enzyme digestion, T4 DNA ligase ligation, et al, we insert the cDNA fragmentof Dokl wide type and mutants(lacking PH or PTB domains,R207A,R222A) into clone vector pSK and expression vector pEYFP-Nl; select positive clone by LacZ blue/white blot, restrict enzyme digestion, and DNA sequencing. Transient transfections are used for all experiments with Cos-7 cells.Cells are transfected with YFP-Dokl or YFP-mutants using the Lipofectamine method. Filamentous actin is stained with Texas Red-phalloidin. Fluorescence imaging reveal a change in cell morphology posterior to the transfection of Dokl-WT.To explore the potential role of the critical domains and sites mediating the biological functions in signaling pathways of Dokl,we contrast Dokl-WT to Dokl-mutants affecting dorsal ruffle formation in response to EGF in Cos-7 cells.To identify the particular signaling pathway of Dokl, we carried out pat...