Tyrosine phosphorylation of focal adhesion kinase and paxillin regulates the signaling mechanism of the rapid nongenomic action of dexamethasone on actin cytoskeleton.
We have previously shown that dexamethasone (DEX) stimulates rapid polymerization of actin and stabilization of microfilaments in human endometrial adenocarcinoma cells. As the content of total cellular actin and the concentration of the actin transcript did not change, we concluded that polymerization of actin by glucocorticoids involves nongenomic mechanisms. However, the signaling events by which the latter is achieved remain unknown. In the present study we evaluated whether tyrosine phosphorylation is required for the rapid, nongenomic DEX effect on actin assembly. In cells preincubated with the tyrosine kinase inhibitors, genistein or erbstatin analogue (EA), before adding DEX the G-/total actin ratio remained unchanged, whereas DEX in the absence of both inhibitors reduced the ratio by 25%. In addition, when cells were preincubated with the protein tyrosine phosphatase inhibitor pervanadate and subsequently incubated with DEX, the G-/total actin ratio was dramatically reduced by 65%. Furthermore, DEX increased transiently the levels of tyrosine phosphorylation of focal adhesion kinase (FAK) and paxillin within 2 to 15 min, without a change in their expression levels. Pervanadate mimicked this effect of DEX and enhanced tyrosine phosphorylation of both proteins. In addition, when cells were exposed to the anticytoskeletal agent cytochalasin B, the basal levels of tyrosine phosphorylation of both proteins were reduced. This effect was reversed by DEX, indicating that actin cytoskeleton integrity is required for the effect of DEX on tyrosine phosphorylation of FAK and paxillin. Finally, we documented enhanced expression of the Ras-related GTP-binding protein Rho-B after long-term (12- and 24-hr) treatment with DEX, whereas Rho-B levels remained unchanged after short-term (3- and 6-hr) treatment. Our observations demonstrate a novel mechanism through which the rapid nongenomic effect of DEX on actin assembly requires tyrosine phosphorylation of the cytoskeleton-associated proteins FAK and paxillin. We also propose that the DEX-induced actin polymerization may constitute a mechanism for transduction of signals resulting in tyrosine phosphorylation of FAK and paxillin. Moreover, the enhanced Rho-B levels observed after long-term treatment with DEX imply a mechanism for the well-described, long-term effects of glucocorticoids on actin cytoskeleton.
Koukouritaki
S B
SB
Department of Biochemistry, School of Medicine, University of Crete, Heraklion, Greece.
Gravanis
A
A
Stournaras
C
C
D000199:Actins / Q000187:drug effects* / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000378:metabolism*; D003571:Cytochalasin B / Q000494:pharmacology; D003598:Cytoskeletal Proteins / Q000378:metabolism*; D003599:Cytoskeleton / Q000187:drug effects* / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D003907:Dexamethasone / Q000494:pharmacology*; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; D006801:Humans; D051419:Paxillin; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011108:Polymers / Q000378:metabolism; D017027:Protein Tyrosine Phosphatases / Q000037:antagonists & inhibitors / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism*; D015398:Signal Transduction; D013997:Time Factors; D014407:Tumor Cells, Cultured; D014443:Tyrosine / Q000378:metabolism*; D020743:rhoB GTP-Binding Protein / Q000187:drug effects / Q000378:metabolism
D000199:Actins; D015815:Cell Adhesion Molecules; D003598:Cytoskeletal Proteins; C496061:PXN protein, human; D051419:Paxillin; D010750:Phosphoproteins; D011108:Polymers; D003571:Cytochalasin B; D014443:Tyrosine; D003907:Dexamethasone; D011505:Protein-Tyrosine Kinases; D051417:Focal Adhesion Kinase 1; D051416:Focal Adhesion Protein-Tyrosine Kinases; C496050:PTK2 protein, human; D017027:Protein Tyrosine Phosphatases; D020743:rhoB GTP-Binding Protein