Defawe Olivier D, Kim Sarah, Chen Lihua, Huang Daming, Kenagy Richard D, Renné Thomas, Walter Ulrich, Daum Günter, Clowes Alexander W
Department of Surgery, University of Washington, Seattle, Washington 98109, USA.
J Cell Physiol. 2010 Jan;222(1):230-7. doi: 10.1002/jcp.21942.
Nitric oxide triggers cGMP-dependent kinase-mediated phosphorylation of the actin regulator vasodilator-stimulated phosphoprotein (VASP) at residue serine239. The function of this phosphorylation for smooth muscle cell (SMC) adhesion, spreading, matrix contraction, and invasion is not well understood. We reconstituted VASP deficient SMC with wild-type VASP (wt-VASP) or VASP mutants that mimic "locked" serine239 phosphorylation (S239D-VASP) or "blocked" serine239 phosphorylation (S239A-VASP). Collagen gel contraction was reduced in S239D-VASP compared to S239A-VASP and wt-VASP expressing cells and nitric oxide (NO) stimulation decreased gel contraction of wt-VASP reconstituted SMC. Invasion of collagen was enhanced in S239D-VASP and NO-stimulated wild-type SMCs compared to S239A-VASP expressing cells. Expression of S239D-VASP impaired SMC attachment to collagen, reduced the number of membrane protrusions, and caused cell rounding compared to expression of S239A-VASP. Treatment of wt-VASP reconstituted SMCs with NO exerted similar effects as expression of S239D-VASP. As unstimulated cells were spreading on collagen S239A-VASP and wt-VASP localized to actin fibers whereas S239D-VASP was enriched in the cytosol. NO interferes with SMC invasion and contraction of collagen matrices. This requires phosphorylation of VASP on serine239, which reduces VASP binding to actin fibers. These findings support the conclusion that VASP phosphorylation at serine239 regulates cytoskeleton remodeling.
一氧化氮触发肌动蛋白调节因子血管舒张刺激磷蛋白（VASP）在丝氨酸239位点的环磷酸鸟苷依赖性激酶介导的磷酸化。这种磷酸化对平滑肌细胞（SMC）黏附、铺展、基质收缩和侵袭的功能尚不清楚。我们用野生型VASP（wt-VASP）或模拟丝氨酸239“锁定”磷酸化（S239D-VASP）或丝氨酸239“阻断”磷酸化（S239A-VASP）的VASP突变体重构了VASP缺陷型SMC。与表达S239A-VASP和wt-VASP的细胞相比，S239D-VASP的胶原蛋白凝胶收缩减少，一氧化氮（NO）刺激降低了wt-VASP重构SMC的凝胶收缩。与表达S239A-VASP的细胞相比，S239D-VASP和NO刺激的野生型SMC对胶原蛋白的侵袭增强。与S239A-VASP的表达相比，S239D-VASP的表达损害了SMC与胶原蛋白的附着，减少了膜突起的数量，并导致细胞变圆。用NO处理wt-VASP重构的SMC产生了与S239D-VASP表达类似的效果。由于未刺激的细胞在胶原蛋白上铺展，S239A-VASP和wt-VASP定位于肌动蛋白纤维，而S239D-VASP富集于细胞质中。NO干扰SMC对胶原蛋白基质的侵袭和收缩。这需要VASP在丝氨酸239位点的磷酸化，从而减少VASP与肌动蛋白纤维的结合。这些发现支持丝氨酸239位点的VASP磷酸化调节细胞骨架重塑的结论。
