Targeting of the Akt/PKB kinase to the actin skeleton
Targeting of the Akt/PKB kinase to the actin skeleton
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DOI：
10.1007/s00018-003-3349-4
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发表时间：
2003-12-01
影响因子：
8
通讯作者：
Marmiroli, S
中科院分区：
文献类型：
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作者：
Cenni, V;Sirri, A;Marmiroli, S
丝氨酸/苏氨酸激酶Akt/PKB细胞内分布在激动剂如血小板衍生生长因子（PDGF）或胰岛素样生长因子（IGF）的作用下发生快速变化。最近出现的概念是Akt亚细胞运动是通过与非底物配体的相互作用促进的。本研究表明，在pdgf处理的Saos2细胞原位细胞骨架基质制备中，Akt与肌动蛋白骨架结合，表明这两种蛋白之间存在相互作用。事实上，通过免疫沉淀和亚细胞分离，我们证明了内源性Akt和肌动蛋白的物理相互作用。通过重组蛋白的体外结合和覆盖实验，我们进一步证明Akt与肌动蛋白直接相互作用。Akt突变体的表达强烈表明Akt的n端PH结构域介导了这种相互作用。更重要的是，我们发现肌动蛋白结合和未结合的Akt之间的划分不是恒定的，而是受生长因子刺激调节的。事实上，PDGF处理血清饥饿细胞引发与肌动蛋白骨架相关的Akt数量增加，同时Akt磷酸化增加。相反，表达Ser473和Thr308突变为丙氨酸的Akt突变体完全消除了pdgf诱导的结合。小gtpase Rac1和Cdc42似乎促进肌动蛋白结合，可能增加Akt磷酸化。
Serine/threonine kinase Akt/PKB intracellular distribution undergoes rapid changes in response to agonists such as Platelet-derived growth factor (PDGF) or Insulin-like growth factor (IGF). The concept has recently emerged that Akt subcellular movements are facilitated by interaction with nonsubstrate ligands. Here we show that Akt is bound to the actin skeleton in in situ cytoskeletal matrix preparations from PDGF-treated Saos2 cells, suggesting an interaction between the two proteins. Indeed, by immunoprecipitation and subcellular fractioning, we demonstrate that endogenous Akt and actin physically interact. Using recombinant proteins in in vitro binding and overlay assays, we further demonstrate that Akt interacts with actin directly. Expression of Akt mutants strongly indicates that the N-terminal PH domain of Akt mediates this interaction. More important, we show that the partition between actin bound and unbound Akt is not constant, but is modulated by growth factor stimulation. In fact, PDGF treatment of serum-starved cells triggers an increase in the amount of Akt associated with the actin skeleton, concomitant with an increase in Akt phosphorylation. Conversely, expression of an Akt mutant in which both Ser473 and Thr308 have been mutated to alanine completely abrogates PDGF-induced binding. The small GTPases Rac1 and Cdc42 seem to facilitate actin binding, possibly increasing Akt phosphorylation.