Van Eyk J E, Arrell D K, Foster D B, Strauss J D, Heinonen T Y, Furmaniak-Kazmierczak E, Côté G P, Mak A S
Department of Biochemistry, Queen's University, Kingston, Ontario K7L 3N6, Canada.
J Biol Chem. 1998 Sep 4;273(36):23433-9. doi: 10.1074/jbc.273.36.23433.
Abnormal smooth muscle contraction may contribute to diseases such as asthma and hypertension. Alterations to myosin light chain kinase or phosphatase change the phosphorylation level of the 20-kDa myosin regulatory light chain (MRLC), increasing Ca2+ sensitivity and basal tone. One Rho family GTPase-dependent kinase, Rho-associated kinase (ROK or p160(ROCK)) can induce Ca2+-independent contraction of Triton-skinned smooth muscle by phosphorylating MRLC and/or myosin light chain phosphatase. We show that another Rho family GTPase-dependent kinase, p21-activated protein kinase (PAK), induces Triton-skinned smooth muscle contracts independently of calcium to 62 +/- 12% (n = 10) of the value observed in presence of calcium. Remarkably, PAK and ROK use different molecular mechanisms to achieve the Ca2+-independent contraction. Like ROK and myosin light chain kinase, PAK phosphorylates MRLC at serine 19 in vitro. However, PAK-induced contraction correlates with enhanced phosphorylation of caldesmon and desmin but not MRLC. The level of MRLC phosphorylation remains similar to that in relaxed muscle fibers (absence of GST-mPAK3 and calcium) even as the force induced by GST-mPAK3 increases from 26 to 70%. Thus, PAK uncouples force generation from MRLC phosphorylation. These data support a model of PAK-induced contraction in which myosin phosphorylation is at least complemented through regulation of thin filament proteins. Because ROK and PAK homologues are present in smooth muscle, they may work in parallel to regulate smooth muscle contraction.
异常的平滑肌收缩可能导致诸如哮喘和高血压等疾病。肌球蛋白轻链激酶或磷酸酶的改变会改变20 kDa肌球蛋白调节轻链（MRLC）的磷酸化水平，增加钙离子敏感性和基础张力。一种Rho家族GTP酶依赖性激酶，即Rho相关激酶（ROK或p160(ROCK)），可通过使MRLC和/或肌球蛋白轻链磷酸酶磷酸化，诱导经曲拉通处理的平滑肌发生不依赖钙离子的收缩。我们发现，另一种Rho家族GTP酶依赖性激酶，即p21活化蛋白激酶（PAK），可诱导经曲拉通处理的平滑肌发生不依赖钙离子的收缩，收缩程度达到在有钙离子存在时所观察到值的62±12%（n = 10）。值得注意的是，PAK和ROK采用不同的分子机制来实现不依赖钙离子的收缩。与ROK和肌球蛋白轻链激酶一样，PAK在体外可使MRLC的丝氨酸19位点磷酸化。然而，PAK诱导的收缩与钙调蛋白和结蛋白磷酸化增强相关，而与MRLC无关。即使GST-mPAK3诱导的张力从26%增加到70%，MRLC的磷酸化水平仍与松弛肌纤维（不存在GST-mPAK3和钙离子）中的水平相似。因此，PAK使力的产生与MRLC磷酸化脱钩。这些数据支持了一种PAK诱导收缩的模型，即肌球蛋白磷酸化至少通过细肌丝蛋白的调节得到补充。由于ROK和PAK的同源物存在于平滑肌中，它们可能协同作用来调节平滑肌收缩。
