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Tyrosine phosphorylation of p62(Dok) induced by cell adhesion and insulin: possible role in cell migration
T Noguchi1, T Matozaki, K Inagaki
1Second Department of Internal Medicine, Kobe University School of Medicine, Kusunoki-cho, Chuo-ku, Kobe 650-0017, USA.
Abstract:
Dok, a 62-kDa Ras GTPase-activating protein (rasGAP)-associated phosphotyrosyl protein, is thought to act as a multiple docking protein downstream of receptor or non-receptor tyrosine kinases. Cell adhesion to extracellular matrix proteins induced marked tyrosine phosphorylation of Dok. This adhesion-dependent phosphorylation of Dok was mediated, at least in part, by Src family tyrosine kinases. The maximal insulin-induced tyrosine phosphorylation of Dok required a Src family kinase. A mutant Dok (DokDeltaPH) that lacked its pleckstrin homology domain failed to undergo tyrosine phosphorylation in response to cell adhesion or insulin. Furthermore, unlike the wild-type protein, DokDeltaPH did not localize to subcellular membrane components. Insulin promoted the association of tyrosine-phosphorylated Dok with the adapter protein NCK and rasGAP. In contrast, a mutant Dok (DokY361F), in which Tyr361 was replaced by phenylalanine, failed to bind NCK but partially retained the ability to bind rasGAP in response to insulin. Overexpression of wild-type Dok, but not that of DokDeltaPH or DokY361F, enhanced the cell migratory response to insulin without affecting insulin activation of mitogen-activated protein kinase. These results identify Dok 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.
Insights
Dok, a docking protein, is phosphorylated by cell adhesion and insulin, mediating cell migration. Its interactions with NCK and rasGAP link these signals to cell motility machinery.
Area of Science:
- Cellular signaling
- Molecular biology
- Signal transduction
Background:
- Dok is a 62-kDa Ras GTPase-activating protein (rasGAP)-associated phosphotyrosyl protein.
- Dok functions as a docking protein downstream of tyrosine kinases.
- Cell adhesion and insulin signaling involve complex molecular pathways.
Purpose of the Study:
- To investigate the role of Dok in cellular responses to cell adhesion and insulin.
- To elucidate the mechanisms of Dok phosphorylation and its downstream interactions.
- To determine Dok's contribution to cell migration.
Main Methods:
- Investigated Dok tyrosine phosphorylation induced by cell adhesion and insulin.
- Utilized Dok mutants (DokDeltaPH, DokY361F) to study domain functions.
- Examined protein-protein interactions using co-immunoprecipitation.
- Assessed the effect of Dok overexpression on cell migration and MAPK activation.
Main Results:
- Cell adhesion and insulin induced Dok tyrosine phosphorylation, mediated by Src family kinases.
- Dok's pleckstrin homology domain is crucial for its phosphorylation and membrane localization.
- Insulin promoted Dok association with NCK and rasGAP; Tyr361 is critical for NCK binding.
- Overexpression of wild-type Dok enhanced insulin-induced cell migration, independent of MAPK activation.
Conclusions:
- Dok acts as a signal transducer linking cell adhesion and insulin receptors to cell motility.
- Dok's interactions with NCK and rasGAP are key to its function in cell migration.
- Dok phosphorylation and localization are essential for its role in insulin signaling and cell movement.