Huang Jiean, Mahavadi Sunila, Sriwai Wimolpak, Hu Wenhui, Murthy Karnam S
Department of Medicine, Medical College of Virginia Campus, Virginia Commonwealth University, Richmond, VA 23298, USA.
Biochem J. 2006 May 15;396(1):193-200. doi: 10.1042/BJ20051772.
Sustained smooth-muscle contraction or its experimental counterpart, Ca2+ sensitization, by G(q/13)-coupled receptor agonists is mediated via RhoA-dependent inhibition of MLC (myosin light chain) phosphatase and MLC20 (20 kDa regulatory light chain of myosin II) phosphorylation by a Ca2+-independent MLCK (MLC kinase). The present study identified the corresponding pathways initiated by G(i)-coupled receptors. Somatostatin acting via G(i)1-coupled sstr3 receptor, DPDPE ([D-Pen2,D-Pen5]enkephalin; where Pen is penicillamine) acting via G(i)2-coupled delta-opioid receptors, and cyclopentyl adenosine acting via G(i)3-coupled adenosine A1 receptors preferentially activated PI3K (phosphoinositide 3-kinase) and ILK (integrin-linked kinase), whereas ACh (acetylcholine) acting via G(i)3-coupled M2 receptors preferentially activated PI3K, Cdc42 (cell division cycle 42)/Rac1, PAK1 (p21-activated kinase 1) and p38 MAPK (mitogen-activated protein kinase). Only agonists that activated ILK induced sustained CPI-17 (protein kinase C potentiated inhibitor 17 kDa protein) phosphorylation at Thr38, MLC20 phosphorylation at Ser19, and contraction, consistent with recent evidence that ILK can act as a Ca2+-independent MLCK capable of phosphorylating the MLC phosphatase inhibitor, CPI-17, at Thr38. ILK activity, and CPI-17 and MLC20 phosphorylation were inhibited by LY294002 and in muscle cells expressing ILK(R211A) or treated with siRNA (small interfering RNA) for ILK. ACh acting via M2 receptors activated ILK, and induced CPI-17 and MLC20 phosphorylation and muscle contraction, but only after inhibition of p38 MAPK; all these responses were inhibited in cells expressing ILK(R211A). Conversely, ACh activated PAK1, a step upstream of p38 MAPK, whereas the three other agonists did so only in cells transfected with ILK(R211A) or siRNA for ILK. The results demonstrate reciprocal inhibition between two pathways downstream of PI3K, with ILK inhibiting PAK1, and p38 MAPK inhibiting ILK. Sustained contraction via G(i)-coupled receptors is dependent on CPI-17 and MLC20 phosphorylation by ILK.
G(q/13)偶联受体激动剂引起的平滑肌持续收缩或其实验对应物Ca2+致敏，是通过RhoA依赖性抑制肌球蛋白轻链（MLC）磷酸酶以及由不依赖Ca2+的肌球蛋白轻链激酶（MLCK）使肌球蛋白轻链20（肌球蛋白II的20 kDa调节性轻链，MLC20）磷酸化来介导的。本研究确定了由G(i)偶联受体启动的相应途径。通过G(i)1偶联的生长抑素受体3（sstr3）起作用的生长抑素、通过G(i)2偶联的δ-阿片受体起作用的DPDPE（[D-青霉胺2，D-青霉胺5]脑啡肽；其中青霉胺为penicillamine）以及通过G(i)3偶联的腺苷A1受体起作用的环戊基腺苷优先激活磷脂酰肌醇3激酶（PI3K）和整合素连接激酶（ILK），而通过G(i)3偶联的M2受体起作用的乙酰胆碱（ACh）优先激活PI3K、细胞分裂周期蛋白42（Cdc42）/Rac1、p21激活激酶1（PAK1）和p38丝裂原活化蛋白激酶（MAPK）。只有激活ILK的激动剂能诱导CPI-17（蛋白激酶C增强抑制因子17 kDa蛋白）在苏氨酸38位点持续磷酸化、MLC20在丝氨酸19位点磷酸化以及收缩，这与最近的证据一致，即ILK可作为一种不依赖Ca2+的MLCK，能够在苏氨酸38位点使MLC磷酸酶抑制剂CPI-17磷酸化。LY294002以及在表达ILK（R211A）的肌肉细胞中或用ILK的小干扰RNA（siRNA）处理后，可抑制ILK活性以及CPI-17和MLC20的磷酸化。通过M2受体起作用的ACh激活ILK，并诱导CPI-17和MLC2磷酸化以及肌肉收缩，但仅在p38 MAPK被抑制后；在表达ILK（R211A）的细胞中，所有这些反应均被抑制。相反，ACh激活PAK1，这是p38 MAPK上游的一个步骤，而其他三种激动剂仅在转染了ILK（R211A）或ILK的siRNA的细胞中才激活PAK1。结果表明PI3K下游的两条途径之间存在相互抑制，即ILK抑制PAK1，而p38 MAPK抑制ILK。通过G(i)偶联受体引起的持续收缩依赖于ILK使CPI-17和MLC20磷酸化。
