Abstract
Vasodilator-stimulated phosphoprotein (VASP) associates with cytoskeletal structures and promotes F-actin formation. RhoA, a member of the Ras superfamily of proteins, activates serum response element (SRE)-dependent transcription through changes in actin dynamics. We now show that the F-actin binding region of VASP is required for VASP stimulation of SRE-dependent transcription, and that VASP is downstream of RhoA in stimulating SRE-dependent transcription. The isolated carboxyl-terminal coiled-coil region of VASP mediates protein tetramerization and has been used as a dominant negative form of VASP; we found that it forms complexes with endogenous VASP in vivo and inhibits in a dose-dependent fashion serum-, RhoA-, and VASP-stimulated SRE-dependent transcription. Cyclic GMP-dependent protein kinase (G-kinase) inhibits RhoA activation of SRE-dependent transcription (Gudi, T., Chen, J. C., Casteel, D. E., Seasholtz, T. M., Boss, G. R., and Pilz, R. B. (2002) J. Biol. Chem. 277, 37382-37393). We now show that the G-kinase inhibition that occurs downstream of RhoA can be explained, at least in part, by G-kinase phosphorylation of VASP on Ser(239) at the carboxyl-terminal end of the G-actin binding site, with some contribution by phosphorylation of Ser(157), which is proximal to the profilin binding site. A phosphorylation-deficient VASP mutant can partly prevent cGMP/G-kinase inhibition of serum- and RhoA-induced SRE-dependent transcription. These studies show that VASP, an important component of the cellular microfilament system, plays a major role in regulating SRE-dependent transcription, and that G-kinase regulates VASP activity.
Highlights
Serum response factor (SRF)1 is a ubiquitous transcription factor that regulates immediate early genes such as c-fos, mus
We found that transfection of full-length Vasodilator-stimulated phosphoprotein (VASP) into serum-starved rat C6 glioma cells stimulated SREdependent transcription: as little as two to three times the amount of endogenous VASP increased serum response element (SRE)-dependent transcription with further increases in transcriptional rates occurring at higher amounts of transfected VASP (Fig. 1, A and B)
The VASP constructs were expressed at relatively similar levels (Fig. 1C), and their differential effects on SRE-dependent transcription could not be attributed to variable expression; the isolated G- and F-actin binding regions consisting of amino acids 225–278 did not express sufficiently well to be used in these studies
Summary
Serum response factor (SRF)1 is a ubiquitous transcription factor that regulates immediate early genes such as c-fos, mus-. These results demonstrate that the dominant negative effect of the EVH2-(278 –380) construct depends on the presence of VASP; they show that RhoA can stimulate SRE-dependent transcription in the absence of VASP, i.e. through a VASP-independent pathway, but that VASP clearly enhances RhoA-induced SRF activation.
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