| Dematin is a widely expressed actin binding and bundling protein that was first characterized in the erythrocyte membrane. In the present study, I have utilized a combination of cell biology and biochemistry techniques to understand the function of dematin in non-erythroid cells. Using isolated mouse embryonic fibroblasts from the dematin headpiece-null (HPKO) model to examine the actin morphology, a time-dependent defect in stress fiber formation, membrane protrusions, cell motility, and adhesion was observed. Furthermore, biochemical analysis of the HPKO fibroblasts revealed a sustained hyper-phosphorylation of focal adhesion kinase (FAK) at tyrosine 397 as well as a two-fold increase in RhoA activation. Inhibition of both RhoA and FAK signaling revealed that dematin acts upstream of RhoA. To further explore the role of dematin in non-erythroid cells, I used primary HPKO and 3T3-L1 adipocytes. Recently, we identified a novel interaction between dematin and glucose transporter-1 (GLUT1) in the erythrocyte. Since homologues of dematin and GLUT1 exist in adipocytes we proposed that a conserved mechanism might couple related sugar transporters, such as the insulin-responsive glucose transporter-4 (GLUT4), to the actin cytoskeleton via dematin. Immunocytochemistry established the presence of dematin in 3T3-L1 adipocytes, and a small pool of dematin and GLUT4-containing vesicles co-localized in 3T3-L1 cells under both basal and insulin-stimulated conditions. Importantly, glucose transport was reduced in both 3T3-L1 adipocytes depleted of dematin, and in the HPKO primary adipocytes. Biochemical interaction between dematin and GLUT4 could not be verified using immunoprecipitation and transfection assays. However, dematin binds to the adaptor protein, 14-3-3zeta in an Akt-dependent manner at Serine-85. Disrupting the dematin and 14-3-3zeta interaction results in a decrease in glucose transport in 3T3-L1 cells. Furthermore, using pharmacological inhibitors as well as an in vitro kinase assay, Akt was identified as the protein kinase that phosphorylates dematin to mediate the biochemical interactions between dematin and 14-3-3zeta. Together, these results identify non-erythroid dematin as a regulator of RhoA signaling in mouse embryonic fibroblasts and glucose transporter trafficking in adipocytes with functional implications for glucose homeostasis and diabetes, as well as normal and pathogenic signaling pathways. |
