Akt/PKB localisation and 3′ phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction
Akt/PKB localisation and 3′ phosphoinositide generation at sites of epithelial cell-matrix and cell-cell interaction
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DOI：
10.1016/s0960-9822(99)80192-4
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发表时间：
1999-04-22
期刊：
影响因子：
9.2
通讯作者：
Downward, J
中科院分区：
文献类型：
--
作者：
Watton, SJ;Downward, J
蛋白激酶B（PKB或Akt）是一种丝裂原调节的蛋白激酶，参与保护细胞免于凋亡、促进细胞增殖和多种代谢反应[1]。其激活通过3'磷酸化肌醇脂质与其普列克底物蛋白同源性（PH）结构域的结合启动，导致通过上游激酶（如磷酸肌醇依赖性蛋白激酶-1（PDK 1））诱导残基Thr 308和Ser 473处的活化磷酸化[2]。上皮细胞与细胞外基质的粘附通过磷酸肌醇（PI）3-激酶和Akt/PKB的激活通过未知机制导致细胞凋亡保护[3，4]。在这里，我们使用Akt/PKB在细胞内的定位来探测PI 3-激酶活性诱导的位点。在成纤维细胞中，免疫荧光显微镜显示，内源性Akt/PKB定位于有丝分裂原处理后细胞外边缘的膜皱褶，绿色荧光蛋白（GFP）与全长Akt/PKB或其PH结构域单独融合也是如此。在上皮细胞中，PH结构域的Akt/PKB定位于细胞-细胞和细胞-基质接触的网站，不同于局灶性接触，即使在没有血清。由于这种定位被PI 3-激酶抑制药物和抑制与磷酸肌醇相互作用的突变破坏，因此它可能代表提供细胞存活信号的组成型3'磷酸肌醇产生的位点。我们提出，附着诱导的，PI-3激酶介导的上皮细胞的生存信号不仅是由细胞-基质相互作用，但也由细胞-细胞相互作用。
Protein kinase B (PKB or Akt) is a mitogen-regulated protein kinase involved in the protection of cells from apoptosis, the promotion of cell proliferation and diverse metabolic responses [1]. Its activation is initiated by the binding of 3' phosphorylated phosphoinositide lipids to its pleckstrin homology (PH) domain, resulting in the induction of activating phosphorylation at residues Thr308 and Ser473 by upstream kinases such as phosphoinositide-dependent protein kinase-1 (PDK1) [2]. Adhesion of epithelial cells to extracellular matrix leads to protection from apoptosis via the activation of phosphoinositide (PI) 3-kinase and Akt/PKB through an unknown mechanism [3,4]. Here, we use the localisation of Akt/PKB within the cell to probe the sites of induction of PI 3-kinase activity. In fibroblasts, immunofluorescence microscopy showed that endogenous Akt/PKB localised to membrane ruffles at the outer edge of the cell following mitogen treatment as did green fluorescent protein (GFP) fusions with full-length Akt/PKB or its PH domain alone. In epithelial cells, the PH domain of Akt/PKB localised to sites of cell-cell and cell-matrix contact, distinct from focal contacts, even in the absence of serum. As this localisation was disrupted by PI 3-kinase inhibitory drugs and by mutations that inhibit interaction with phosphoinositides, it is likely to represent the sites of constitutive 3' phosphoinositide generation that provide a cellular survival signal. We propose that the attachment-induced, PI-3-kinase-mediated survival signal in epithelial cells is generated not only by cell-matrix interaction but also by cell-cell interaction.