Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially mediated by phosphorylation of RhoA
Inhibition of sustained smooth muscle contraction by PKA and PKG preferentially mediated by phosphorylation of RhoA
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DOI：
10.1152/ajpgi.00465.2002
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发表时间：
2003-06-01
影响因子：
4.5
通讯作者：
Makhlouf, GM
中科院分区：
文献类型：
--
作者：
Murthy, KS;Zhou, HP;Makhlouf, GM
在新鲜分散和培养的表达野生型RhoA、组成型活性Rho（V14）和磷酸化位点缺陷型Rho（A188）的平滑肌细胞中，研究了RhoA在PKA和PKG引起的肌球蛋白轻链（MLC）20去磷酸化和平滑肌松弛中的作用。PKA激活剂（5，6-二氯-1-β-呋喃核糖基苯并咪唑3 '，5'-环状单硫代磷酸酯，Sp-异构体; cBIMPS）或PKG [8-（4-氯苯硫基）鸟苷3 '，5'-环一磷酸（8-pCPT-cGMP），硝普钠（SNP）]或PKA和PKG（VIP）诱导组成型活性Rho（V14）和激动剂（ACh）-或GTP γ S-刺激的野生型RhoA型，而不是RhoA 188型。磷酸化伴随着膜结合的野生型RhoA和Rho（V14）易位到胞质溶胶和ACh刺激的Rho激酶和磷脂酶D的活动，RhoA/ Rho激酶协会，MLC 20磷酸化和持续的肌肉收缩的完全抑制。这些事件中的每一个都被PKG抑制剂KT 5823或PKA抑制剂肉豆蔻酰化PKI阻断，这取决于所使用的药剂。以先前通过直接测量激酶活性所示的浓度（1 μ M）使用抑制剂以选择性地抑制相应的激酶。在过表达活性磷酸化位点缺陷突变体Rho（A188）的肌细胞中，MLC 20磷酸化被SNP、VIP、cBIMPS和8- pCPT-cGMP部分抑制，表明RhoA下游存在独立的抑制机制。结果表明，MLC 20的去磷酸化和平滑肌松弛优先介导PKG和PKA依赖的磷酸化和RhoA的失活。
The role of RhoA in myosin light-chain (MLC) 20 dephosphorylation and smooth muscle relaxation by PKA and PKG was examined in freshly dispersed and cultured smooth muscle cells expressing wild-type RhoA, constitutively active Rho(V14), and phosphorylation site-deficient Rho(A188). Activators of PKA (5,6-dichloro-1-beta-ribofuranosyl benzimidazole 3', 5'-cyclic monophosphothionate, Sp-isomer; cBIMPS) or PKG [8-(4-chlorophenylthio) guanosine 3', 5'-cyclic monophosphate (8-pCPT-cGMP), sodium nitroprusside (SNP)] or both PKA and PKG ( VIP) induced phosphorylation of constitutively active Rho(V14) and agonist (ACh)- or GTPgammaS-stimulated wild-type RhoA but not RhoA188. Phosphorylation was accompanied by translocation of membrane-bound wild-type RhoA and Rho(V14) to the cytosol and complete inhibition of ACh-stimulated Rho kinase and phospholipase D activities, RhoA/ Rho kinase association, MLC20 phosphorylation, and sustained muscle contraction. Each of these events was blocked depending on the agent used, by the PKG inhibitor KT5823 or the PKA inhibitor myristoylated PKI. Inhibitors were used at a concentration ( 1 muM) previously shown by direct measurement of kinase activity to selectively inhibit the corresponding kinase. In muscle cells overexpressing the active phosphorylation site-deficient mutant Rho(A188), MLC20 phosphorylation was partly inhibited by SNP, VIP, cBIMPS, and 8- pCPT-cGMP, suggesting the existence of an independent inhibitory mechanism downstream of RhoA. Results demonstrate that dephosphorylation of MLC20 and smooth muscle relaxation are preferentially mediated by PKG- and PKA-dependent phosphorylation and inactivation of RhoA.