Honda Shigenori, Shirotani-Ikejima Hiroko, Tadokoro Seiji, Tomiyama Yoshiaki, Miyata Toshiyuki
Department of Molecular Pathogenesis, National Cerebral and Cardiovascular Center, Suita, Japan.
Department of Hematology and Oncology, Osaka University Graduate School of Medicine, Suita, Osaka, Japan.
PLoS One. 2013 Dec 23;8(12):e85498. doi: 10.1371/journal.pone.0085498. eCollection 2013.
Integrin-linked kinase (ILK) is an important signaling regulator that assembles into the heteroternary complex with adaptor proteins PINCH and parvin (termed the IPP complex). We recently reported that ILK is important for integrin activation in a Chinese hamster ovary (CHO) cell system. We previously established parental CHO cells expressing a constitutively active chimeric integrin (αIIbα6Bβ3) and mutant CHO cells expressing inactive αIIbα6Bβ3 due to ILK deficiency. In this study, we further investigated the underlying mechanisms for ILK-dependent integrin activation. ILK-deficient mutant cells had trace levels of PINCH and α-parvin, and transfection of ILK cDNA into the mutant cells increased not only ILK but also PINCH and α-parvin, resulting in the restoration of αIIbα6Bβ3 activation. In the parental cells expressing active αIIbα6Bβ3, ILK, PINCH, and α-parvin were co-immunoprecipitated, indicating the formation of the IPP complex. Moreover, short interfering RNA (siRNA) experiments targeting PINCH-1 or both α- and β-parvin mRNA in the parent cells impaired the αIIbα6Bβ3 activation as well as the expression of the other components of the IPP complex. In addition, ILK mutants possessing defects in either PINCH or parvin binding failed to restore αIIbα6Bβ3 activation in the mutant cells. Kindlin-2 siRNA in the parental cells impaired αIIbα6Bβ3 activation without disturbing the expression of ILK. For CHO cells stably expressing wild-type αIIbβ3 that is an inactive form, overexpression of a talin head domain (THD) induced αIIbβ3 activation and the THD-induced αIIbβ3 activation was impaired by ILK siRNA through a significant reduction in the expression of the IPP complex. In contrast, overexpression of all IPP components in the αIIbβ3-expressing CHO cells further augmented THD-induced αIIbβ3 activation, whereas they did not induce αIIbβ3 activation without THD. These data suggest that the IPP complex rather than ILK plays an important role and supports integrin activation probably through stabilization of the active conformation.
整合素连接激酶（ILK）是一种重要的信号调节因子，它与衔接蛋白PINCH和桩蛋白组装成异源三聚体复合物（称为IPP复合物）。我们最近报道，在中华仓鼠卵巢（CHO）细胞系统中，ILK对整合素激活很重要。我们之前建立了表达组成型活性嵌合整合素（αIIbα6Bβ3）的亲本CHO细胞，以及由于ILK缺陷而表达无活性αIIbα6Bβ3的突变CHO细胞。在本研究中，我们进一步研究了ILK依赖性整合素激活的潜在机制。ILK缺陷型突变细胞中PINCH和α-桩蛋白水平极低，将ILK cDNA转染到突变细胞中不仅增加了ILK，还增加了PINCH和α-桩蛋白，从而恢复了αIIbα6Bβ3的激活。在表达活性αIIbα6Bβ3的亲本细胞中，ILK、PINCH和α-桩蛋白共免疫沉淀，表明形成了IPP复合物。此外，在亲本细胞中靶向PINCH-1或α-和β-桩蛋白mRNA的短发夹RNA（siRNA）实验损害了αIIbα6Bβ3的激活以及IPP复合物其他成分的表达。此外，在PINCH或桩蛋白结合方面存在缺陷的ILK突变体无法恢复突变细胞中αIIbα6Bβ3的激活。亲本细胞中的Kindlin-2 siRNA损害了αIIbα6Bβ3的激活，而不干扰ILK的表达。对于稳定表达无活性形式野生型αIIbβ3的CHO细胞，踝蛋白头部结构域（THD）的过表达诱导了αIIbβ3的激活，并且ILK siRNA通过显著降低IPP复合物的表达损害了THD诱导的αIIbβ3激活。相反，在表达αIIbβ3的CHO细胞中过表达所有IPP成分进一步增强了THD诱导的αIIbβ3激活，而在没有THD的情况下它们不会诱导αIIbβ3激活。这些数据表明，IPP复合物而非ILK起着重要作用，并且可能通过稳定活性构象来支持整合素激活。
