Serine phosphorylation negatively regulates RhoA in vivo
Serine phosphorylation negatively regulates RhoA in vivo
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DOI：
10.1074/jbc.m213066200
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发表时间：
2003-05-23
影响因子：
4.8
通讯作者：
Burridge, K
中科院分区：
文献类型：
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作者：
Ellerbroek, SM;Wennerberg, K;Burridge, K
以往的工作表明，cAMP或cGMP依赖的激酶使Ser(188)上的RhoA磷酸化抑制其活性。然而，这些研究缺乏直接研究体内磷酸化RhoA活性的可能性。因此，我们创建了含有拟磷脂残基的RhoA蛋白，取代了cAMP/cGMP依赖的激酶磷酸化位点。在体外，RhoA的磷酸化或仿磷取代不影响Rho鸟嘌呤核苷酸交换因子、GTP酶激活蛋白或香叶基香叶基转移酶的活性，但通过交换因子竞争实验，促进与Rho鸟嘌呤解离抑制剂的结合。RhoA模拟磷酸化蛋白与磷酸化的RhoA在体外的相似性使我们能够在体内研究磷酸化的RhoA的功能。当在NIH3T3细胞中瞬时表达时，RhoA磷酸化模拟蛋白显示GTP负载量降低。稳定表达的RhoA和RhoA(S188A)克隆的传播速度明显慢于模拟转染组或RhoA(S188E)克隆。RhoA(S188A)克隆不受cAMP激动剂的形态影响，而仿磷克隆表现出与对照细胞相似的应力纤维分解。总之，这些数据提供了体内证据，证明在磷酸化时在Ser(188)上添加一个带电基团对RhoA活性进行负面调节，并表明这是通过增强Rho鸟嘌呤解离抑制剂相互作用而发生的，而不是直接干扰鸟嘌呤核苷酸交换因子、GTPase激活蛋白或香叶基转移酶的活性。
Previous work indicates that RhoA phosphorylation on Ser(188) by cAMP or cGMP-dependent kinases inhibits its activity. However, these studies lacked the possibility to directly study phosphorylated RhoA activity in vivo. Therefore, we created RhoA proteins containing phosphomimetic residues in place of the cAMP/cGMP-dependent kinase phosphorylation site. RhoA phosphorylation or phosphomimetic substitution did not affect Rho guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity in vitro but promoted binding to the Rho guanine-dissociation inhibitor as measured by exchange factor competition assays. The in vitro similarities between RhoA phosphomimetic proteins and phosphorylated RhoA allowed us to study function of phosphorylated RhoA in vivo. RhoA phosphomimetic proteins display depressed GTP loading when transiently expressed in NIH 3T3 cells. Stable-expressing RhoA and RhoA(S188A) clones spread significantly slower than mock-transfected or RhoA(S188E) clones. RhoA(S188A) clones were protected from the morphological effects of a cAMP agonist, whereas phosphomimetic clones exhibit stress fiber disassembly similar to control cells. Together, these data provide in vivo evidence that addition of a charged group to Ser(188) upon phosphorylation negatively regulates RhoA activity and indicates that this occurs through enhanced Rho guanine-dissociation inhibitor interaction rather than direct perturbation of guanine nucleotide exchange factor, GTPase activating protein, or geranylgeranyl transferase activity.