CYCLIC GMP-DEPENDENT PROTEIN-KINASE IS REQUIRED FOR THROMBOSPONDIN AND TENASCIN MEDIATED FOCAL ADHESION DISASSEMBLY

Focal adhesions are specialized regions of cell membranes that are foc i for the transmission of signals between the outside and the inside o f the cell, Intracellular signaling events are important in the organi zation and stability of these structures, In previous work, we showed that the counter-adhesive extracellular matrix proteins, thrombospondi n, tenascin, and SPARC, induce the disassembly of focal adhesion plaqu es and we identified the active regions of these proteins, In order to determine the mechanisms whereby the anti-adhesive matrix proteins mo dulate cytoskeletal organization and focal adhesion integrity, we exam ined the role of protein kinases in mediating the loss of focal adhesi ons by these proteins, Data from these studies show that cGMP-dependen t protein kinase is necessary to mediate focal adhesion disassembly tr iggered by either thrombospondin or tenascin, but not by SPARC, In exp eriments using various protein kinase inhibitors, we observed that sel ective inhibitors of cyclic GMP-dependent protein kinase, KT5823 and R p-8-Br-cGMPS, blocked the effects of both the active sequence of throm bospondin 1 (hep I) and the alternatively - spliced segment (TNfnA-D) of tenascin-C on focal adhesion disassembly, Moreover, early passage r at aortic smooth muscle cells which have high levels of cGMP-dependent protein kinase were sensitive to hep I treatment, in contrast to pass aged cGMP-dependent protein kinase deficient cells which were refracto ry to hep I or TNfnA-D treatment, but were sensitive to SPARC, Transfe ction of passaged smooth muscle cells with the catalytic domain of PKG I alpha restored responsiveness to hep I and TNfnA-D. While these stu dies show that cGMP-dependent protein kinase activity is necessary for thrombospondin and tenascin-mediated focal adhesion disassembly, kina se activity alone is not sufficient to induce disassembly as transfect ion of the catalytic domain of the kinase in the absence of additional stimuli does not result in loss of focal adhesions.