Vascular Biology Center, Medical College of Georgia, Augusta, 30912-2500, USA.
Am J Physiol Heart Circ Physiol. 2011 Apr;300(4):H1336-44. doi: 10.1152/ajpheart.01163.2010. Epub 2011 Feb 4.
The mechanisms controlling the activity of NADPH oxidase 5 (Nox5) are unique in that they are independent of the protein: protein interactions that coordinate the activation of other Nox isoforms. Instead, the primary driving force for Nox5 activity is calcium. However, in a previous study we reported that the protein kinase C (PKC)-agonist PMA could induce a sustained activation of Nox5 that was independent of calcium changes. This apparent calcium-independent activation was found to be mediated by the PKC-dependent phosphorylation of specific serine and threonine residues on Nox5 which increased the calcium sensitivity of the enzyme and enabled activation at resting levels of calcium. However, the specific kinase(s) mediating the phosphorylation and activation of Nox5 are not known. As PKC can activate the MEK/ERK1/2 signaling pathway, we hypothesized that Nox5 is activated by the coordinated phosphorylation of both MAPK and PKC pathways. The inhibition of MEK1 using PD-98059 and U-0126 significantly reduced the phosphorylation and activity of Nox5 in response to PMA but not to the calcium-mobilizing stimulus ionomycin. Dominant negative MEK1 and knockdown of endogenous MEK1/2 using a specific small interfering RNA also inhibited Nox5 activity in response to PMA. The mutation of S498 to a nonphosphorylatable residue and to a lesser degree T494 blocked the ability of ERK to stimulate Nox5 activity. However, a constitutively active form of MEK1 failed to increase Nox5 activity in the absence of PMA stimulation. These results suggest that the MEK/ERK1/2 pathway is necessary but not sufficient to regulate the PMA-dependent activation of Nox5.
NADPH 氧化酶 5（Nox5）的活性调控机制独具特色，与其他 Nox 同工型的激活所需的蛋白-蛋白相互作用无关。相反，Nox5 活性的主要驱动力是钙。然而，在之前的一项研究中，我们报道蛋白激酶 C（PKC）激动剂 PMA 可以诱导 Nox5 的持续激活，而这种激活与钙变化无关。这种明显的钙不依赖性激活是通过 PKC 依赖性磷酸化 Nox5 上的特定丝氨酸和苏氨酸残基介导的，这增加了酶对钙的敏感性，并使酶在钙的静息水平下被激活。然而，介导 Nox5 磷酸化和激活的特定激酶尚不清楚。由于 PKC 可以激活 MEK/ERK1/2 信号通路，我们假设 Nox5 通过 MAPK 和 PKC 途径的协调磷酸化而被激活。使用 PD-98059 和 U-0126 抑制 MEK1，显著降低了 PMA 刺激下 Nox5 的磷酸化和活性，但对钙动员刺激剂离子霉素没有影响。显性负性 MEK1 和使用特异性小干扰 RNA 敲低内源性 MEK1/2 也抑制了 PMA 刺激下 Nox5 的活性。将 S498 突变为非磷酸化残基，以及在较小程度上突变为 T494，阻断了 ERK 刺激 Nox5 活性的能力。然而，在没有 PMA 刺激的情况下，组成型激活的 MEK1 未能增加 Nox5 的活性。这些结果表明，MEK/ERK1/2 通路是调节 PMA 依赖性 Nox5 激活所必需的，但不是充分的。
