Role of integrin-linked kinaae in neurite outgrowth and axon guidance on neuronal repair

在层粘连蛋白基质上生长的小鼠N1 E-115细胞对血清剥夺反应表现出神经突生长。用针对β1整联蛋白的抗体处理细胞抑制神经突生长。因此，β1整联蛋白参与层粘连蛋白基质上的N1 E-115细胞的轴突发生。整合素连接激酶（integrin linked kinase，ILK）是近年来发现的一种与β1整合素胞浆区结合的丝氨酸/苏氨酸蛋白激酶，在整合素介导的跨膜信号转导中起重要作用。我们报告，ILK在N1 E-115细胞中表达，其表达水平在正常和分化条件下均恒定。ILK（DN-ILK）的激酶缺陷突变体的稳定转染导致在层粘连蛋白上生长的血清饥饿的N1 E-115细胞中神经突生长的抑制。另一方面，野生型ILK的瞬时表达刺激神经突生长。在层粘连蛋白基质上接种后，亲代细胞中ILK活性被瞬时激活， ...更多信息 而DN-ILK转染细胞则无此现象。这些结果表明，ILK的瞬时激活所需的血清饥饿的N1 E-115细胞层粘连蛋白上的神经突生长。在相同条件下，N1 E-115细胞粘附层粘连蛋白后，p38丝裂原活化蛋白（MAP）激酶被瞬时激活，但MAP激酶/细胞外信号调节激酶（MEK）和细胞外信号调节激酶（ERK）均未被激活，而DN-ILK表达的细胞则未被激活。p38 MAP激酶激活的调控过程与ILK激活的调控过程非常相似。此外，p38 MAP激酶抑制剂，SB 203580，显着阻止神经突生长。因此，p38 MAP激酶的激活参与ILK介导的信号转导，导致N1 E-115细胞中的整合素依赖性神经突生长。此外，ILK还通过调节N1 E-115细胞中糖原合成酶激酶-3 β（GSK-3β）的活性来控制tau蛋白的磷酸化。DN-ILK的稳定转染导致N1 E-115细胞中Tau-1抗体识别位点的异常tau磷酸化，其与阿尔茨海默病患者脑中成对螺旋丝（PHF）-tau中的一些磷酸化位点相同。表达DN-ILK的细胞中的tau磷酸化水平在正常和分化条件下是恒定的。另一方面，在亲本对照细胞中未观察到异常tau磷酸化。ILK失活导致GSK-3β的活性形式增加，但非活性形式减少，GSK-3β是参与PHF-tau形成的候选激酶。此外，用锂抑制GSK-3β可防止DN-ILK表达细胞中异常的tau磷酸化。这些结果表明，ILK失活通过持续激活N1 E-115细胞中的GSK-3β导致异常tau磷酸化。ILK直接磷酸化GSK-3β并抑制其活性。因此，内源性ILK通过抑制N1 E-115细胞中的GSK-3β活性来防止GSK-3β诱导的异常tau磷酸化。少

Mouse N1E-115 cells grown on a laminin matrix exhibit neurite outgrowth in response to serum deprivation. Treatment of cells with an antibody against β1 integrin inhibits neurite outgrowth. Thus, β1 integrin is involved in the neuritogenesis of N1E-115 cells on a laminin matrix. Integrin-linked kinase (ILK), a recently identified cytoplasmic serine/threonine protein kinase that binds to the cytoplasmic domain of β1 integrin, has an important role in transmembrane signal transduction via integrins. We report that ILK is expressed in N1E-115 cells, the expression levels of which are constant under both normal and differentiating conditions. A stable transfection of a kinase-deficient mutant of ILK (DN-ILK) results in inhibition of neurite outgrowth in serum-starved N1E-115 cells grown on laminin. On the other hand, a transient expression of wild-type ILK stimulated neurite outgrowth. The ILK activity in the parental cells was transiently activated after seeding on the laminin matrix, whe … More reas that in the DN-ILK transfected cells was not. These results suggest that transient activation of ILK is required for neurite outgrowth in serum-starved N1E-115 cells on laminin. Under the same conditions, p38 mitogen-activated protein (MAP) kinase, but neither MAP kinase/extracellular signal-regulated kinases kinase (MEK) nor extracellular signal-regulated kinases (ERK), was transiently activated after N1E-115 cell attachment to laminin, but not in the DN-ILK expressed cells. The tune course of p38 MAP kinase activation was very similar to that of ILK activation. Furthermore, a p38 MAP kinase inhibitor, SB203580, significantly blocked neurite outgrowth. Thus, activation of p38 MAP kinase is involved in ILK-mediated signal transduction leading to integrin-dependent neurite outgrowth in N1E-115 cells. Moreover, ILK also controls tau phosphorylation via regulation of glycogen synthase kinase-3β (GSK-3β) activity in N1E-115 cells. Stable transfection of DN-ILK resulted in aberrant tau phosphorylation in N1E-115 cells at sites recognized by the Tau-1 antibody, which are identical to some of the phosphorylation sites in paired helical filaments, (PHF)-tau, in brains of patients with Alzheimer's disease. The tau phosphorylation levels in the DN-ILK-expressing cells are constant under normal and differentiating conditions. On the other hand, aberrant tau phosphorylation was not observed in the parental control cells. ILK inactivation resulted in an increase in the active form but a decrease in the inactive form of GSK-3β, which is a candidate kinase involved in PHF-tau formation. Moreover, inhibition of GSK-3β with lithium prevented aberrant tau phosphorylation in the DN-ILK-expressing cells. These results suggest that ILK inactivation results in aberrant tau phosphorylation via sustained activation of GSK-3β in N1E-115 Cells. ILK directly phosphorylates GSK-3β and inhibits its activity. Therefore, endogenous ILK protects against GSK-3β-induced aberrant tau phosphorylation via inhibition of GSK-3β activity in N1E-115 cells. Less