Identification of the Serine 307 of LKB1 as a Novel Phosphorylation Site Essential for Its Nucleocytoplasmic Transport and Endothelial Cell Angiogenesis
Identification of the Serine 307 of LKB1 as a Novel Phosphorylation Site Essential for Its Nucleocytoplasmic Transport and Endothelial Cell Angiogenesis
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DOI：
10.1128/mcb.01417-08
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发表时间：
2009-07-01
影响因子：
5.3
通讯作者：
Zou, Ming-Hui
中科院分区：
文献类型：
--
作者：
Xie, Zhonglin;Dong, Yunzhou;Zou, Ming-Hui
LKB 1是一种主要存在于细胞核中的激酶，它控制至少13种下游蛋白激酶，包括AMP活化蛋白激酶（AMPK）。LKB 1激活的关键步骤是其从细胞核输出到细胞质。在这里，我们确定了LKB 1的S307作为一个推定的新的磷酸化位点，这是必不可少的核质运输。在无细胞系统中，重组PKC-zeta在S307磷酸化LKB 1。AMPK激活剂刺激内皮细胞、肝细胞、骨骼肌细胞和血管平滑肌细胞中的PKC-zeta活性和S307处的LKB 1磷酸化。与激酶死亡的LKB 1 D194 A突变体（Asp 194突变为Ala）一样，组成型核定位的LKB 1 SL 26突变体和LKB 1 S307 A突变体（Ser 307突变为Ala）与STRAD α的相关性降低。有趣的是，围绕LKB 1 S307的PKC-ζ共有序列在LKB 1 SL 26突变体中被破坏，从而为这种突变导致LKB 1功能障碍提供了可能的分子解释。此外，LKB 1核质运输和AMPK激活响应过氧亚硝酸盐显着减少的药理学抑制CRM 1，这通常有利于核输出LKB 1-STRAD复合物。与LKB 1野生型相比，S307 A突变体复合物显示与CRM 1的缔合减少。最后，腺病毒过表达野生型LKB 1抑制，而LKB 1 S307 A突变体增加，管形成和过氧化氢增强培养的内皮细胞凋亡。两者合计，我们的研究结果表明，在多种细胞类型的信号通路从事的几种生理刺激汇聚在PKC-zeta依赖的LKB 1磷酸化S307，指导核质运输LKB 1和随之而来的AMPK激活。
LKB1, a master kinase that controls at least 13 downstream protein kinases including the AMP-activated protein kinase (AMPK), resides mainly in the nucleus. A key step in LKB1 activation is its export from the nucleus to the cytoplasm. Here, we identified S307 of LKB1 as a putative novel phosphorylation site which is essential for its nucleocytoplasmic transport. In a cell-free system, recombinant PKC-zeta phosphorylates LKB1 at S307. AMPK-activating agents stimulate PKC-zeta activity and LKB1 phosphorylation at S307 in endothelial cells, hepatocytes, skeletal muscle cells, and vascular smooth muscle cells. Like the kinase-dead LKB1 D194A mutant (mutation of Asp194 to Ala), the constitutively nucleus-localized LKB1 SL26 mutant and the LKB1 S307A mutant (Ser307 to Ala) exhibit a decreased association with STRAD alpha. Interestingly, the PKC-zeta consensus sequence surrounding LKB1 S307 is disrupted in the LKB1 SL26 mutant, thus providing a likely molecular explanation for this mutation causing LKB1 dysfunction. In addition, LKB1 nucleocytoplasmic transport and AMPK activation in response to peroxynitrite are markedly reduced by pharmacological inhibition of CRM1, which normally facilitates nuclear export of LKB1-STRAD complexes. In comparison to the LKB1 wild type, the S307A mutant complexes show reduced association with CRM1. Finally, adenoviral overexpression of wild-type LKB1 suppresses, while the LKB1 S307A mutant increases, tube formation and hydrogen peroxide-enhanced apoptosis in cultured endothelial cells. Taken together, our results suggest that, in multiple cell types the signaling pathways engaged by several physiological stimuli converge upon PKC-zeta-dependent LKB1 phosphorylation at S307, which directs the nucleocytoplasmic transport of LKB1 and consequent AMPK activation.