Inhibition of the Motility and Growth of B16F10 Mouse Melanoma Cells by Dominant Negative Mutants of Dok-1

Abstract

Dok-1 (p62Dok ) is a multiple-site docking protein that acts downstream of receptor and nonreceptor tyrosine kinases. Although it has been proposed to contribute to the control of cell growth and migration through association with the Ras GTPase-activating protein and the adapter protein Nck, the role of Dok-1 remains largely unknown. The functions of Dok-1 have now been investigated by the generation of two different COOHterminal truncation mutants of this protein: one (DokPH؉PTB) containing the pleckstrin homology and phosphotyrosine-binding domains, and the other (DokPH) composed only of the pleckstrin homology domain. Both of these mutant proteins were shown to act in a dominant negative manner. Overexpression of each of the mutants in highly metastatic B16F10 mouse melanoma cells thus both inhibited the tyrosine phosphorylation of endogenous Dok-1 induced by cell adhesion as well as reduced the association of the endogenous protein with cellular membranes and the cytoskeleton. Overexpression of DokPH؉PTB in these cells also markedly reduced both the rates of cell spreading, migration, and growth as well as the extent of Ras activation. The effects of DokPH on these processes were less pronounced than were those of DokPH؉PTB, indicating the importance of the phosphotyrosine-binding domain. These results suggest that at least in B16F10 cells, Dok-1 positively regulates not only cell spreading and migration but also cell growth and Ras activity.The abilities to metastasize and to invade tissue are important characteristics of transformed cells, often complicating cancer therapy and becoming critical determinants of patient prognosis. Although metastasis and tissue invasion are complex, an enhanced motility of cancer cells is thought to contribute to both processes. Cell motility is itself controlled by a complex mechanism; however, reorganization of the actin cytoskeleton and adhesion to extracellular matrix (ECM) proteins have been shown to play crucial roles (20,26,35).The p62 Dok (Dok-1) protein was first identified as a common substrate for activated protein tyrosine kinases (PTKs) such as v- Abl,. Subsequent studies revealed that Dok-1 is constitutively phosphorylated on tyrosine residues in chronic myelogenous leukemia cells expressing the oncoprotein p210 bcr-abl (2, 44). The extent of tyrosine phosphorylation of Dok-1 in cells also correlates with the transforming activity of activated PTKs (1, 5, 25, 27). These observations suggest a causal role for Dok-1 in the acquired features of transformed cells, such as the aberrant growth and multiple abnormalities in cytoskeletal function (13). However, the physiological significance of tyrosine phosphorylation of Dok-1 has remained unclear.The NH 2 -terminal region of Dok-1 contains a pleckstrin homology (PH) domain and a putative phosphotyrosine-binding (PTB) domain, which are thought to mediate the association of this protein with the cell membrane and with phosphotyrosine-containing NPXpY motifs, respectively (2, 45). Dok-1 also contains several consensus ...

Mobility and integration sites of a murine C57BL/6 melanoma endogenous retrovirus involved in tumor progression in vivo

“…19 Concerning the other targeted gene, the dok5 gene, it has been previously shown that another member of the dok family, dok1, positively regulates B16F10 cell mobility ex vivo. 43 Futhermore, as illustrated in Figure 5, we were able to show that transfection of the B16 melanoma cells with a dok5 expression vector led to a significant enhancement (2-to 3-fold) of the metastatic potential of the cells in an in vivo metastasis assay. 19 This is consistent with the notion that dok5 activation upon MelARV insertion is indeed contributing to the enhanced metastatic potential of B16F10 cells.…”

Frameshift mutation in the Dok1 gene in chronic lymphocytic leukemia

“…In fact, this region has been shown to be important for Dok1 to suppress cell proliferation and cell transformation (Hosooka et al, 2001;Songyang et al, 2001;Shah and Shokat, 2002). In addition, tyrosine residues 296 and 315, which are necessary for Dok1 to interact with Ras-GAP in order to downregulate MAP kinase activity (Songyang et al, 2001;Shah and Shokat, 2002), are lost in Dok1-CLL13.…”

“…Dok1 acts as a negative regulator of MAP kinase activation and inhibits cell proliferation Hosooka et al, 2001). To examine the effect of Dok1-CLL13 on MAPK activation, 293 cells stably expressing F-Dok1 and F-Dok1-CLL13, and the control cell line containing empty vector, were stimulated by PDGF at different time points.…”

“…Dok1 downregulates several cell signaling processes: for example, it inhibits MAP kinase activity, downregulates cell proliferation, affects T-and B-cell signaling and plays a role in activin-induced apoptosis (Tamir et al, 2000;Yamanashi et al, 2000;Di Cristofano et al, 2001;Martelli et al, 2001;Nemorin et al, 2001;Songyang et al, 2001;Wick et al, 2001;Zhao et al, 2001;Kato et al, 2002;Yamakawa et al, 2002). Dok1 can also affect cell adhesion, spreading and migration (Noguchi et al, 1999;Hosooka et al, 2001). Tyrosine phosphorylation and PH domain-mediated plasma membrane translocation are necessary for Dok1 function (Liang et al, 2002;Zhao et al, 2001).…”

Interaction of SAP-1, a Transmembrane-type Protein-tyrosine Phosphatase, with the Tyrosine Kinase Lck

“…dok is thought to contribute to the positive regulation of cell migration in fibroblasts, melanoma cells, and leukemia cells, and this regulation also appears to require either the Ras GTPaseactivating protein or the adapter protein Nck (30,46,47). Previous studies also indicate that Lck and the activation of MAP kinase are important for the positive regulation of T cell migration (37,48,49).…”