TYROSINE PHOSPHORYLATION OF FOCAL ADHESION KINASE (P125(FAK)) - REGULATION BY CAMP AND THROMBIN IN MESANGIAL CELLS

Stress fibers, composed of actin filaments, converge upon and associat e with a number of proteins, including focal adhesion kinase (p125(FAK )), and integrin receptors to form areas of close contact between cell s and the extracellular matrix referred to as focal adhesions. Treatme nt of mesangial cells with cAMP-elevating agents causes a loss of foca l adhesions, fragmentation of stress fibers, and decreased tyrosine ph osphorylation of p125(FAK). Thrombin reverses these effects of cAMP, a nd this model can be used to address some of the cellular mechanisms i nvolved in regulating the loss and formation of focal adhesions. This study reports the effects of cAMP and thrombin on mesangial cell shape , distribution of actin, formation of stress fibers, and tyrosine phos phorylation of p125(FAK). cAMP-treated cells display a condensed cell body with slender processes that traverse the area formerly covered by the cell. Addition of thrombin to these cells restores actin filament s (stress fibers) and increases tyrosine phosphorylation of p125(FAK), and the cells resume a flattened morphology, even In the continued pr esence of cAMP-elevating agents. Peptides that mimic the tethered liga nd portion of the thrombin receptor have the same effects on cell morp hology and stress fiber formation as thrombin. In selected experiments , agents that disrupt either stress fibers (cytochalasin D) or microtu bules (nocodazole; Sigma Chemical, St. Louis, MO) were used to examine the role of these cytoskeletal elements in thrombin-induced restorati on of focal adhesions. Cytochalasin D blocked the ability of thrombin to restore focal adhesions and phosphorylate p125(FAK). The effects of nocodazole, an agent that destabilizes microtubules (but which has no known receptor), are very similar to those of thrombin. The findings discussed in this study indicate that thrombin can modulate the format ion of focal adhesions. The organization of stress fibers and microtub ules is apparently intimately related to the phosphorylation of p125(F AK) and can be modulated by soluble receptor agonists such as thrombin or via altered polymerization of microtubules.