Importin-α mediates the regulated nuclear targeting of serum- and glucocorticoid-inducible, protein kinase (Sgk) by recognition of a nuclear localization signal in the kinase central domain
Importin-α mediates the regulated nuclear targeting of serum- and glucocorticoid-inducible, protein kinase (Sgk) by recognition of a nuclear localization signal in the kinase central domain
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DOI：
10.1091/mbc.e02-03-0170
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发表时间：
2003-03-01
影响因子：
3.3
通讯作者：
Firestone, GL
中科院分区：
文献类型：
--
作者：
Maiyar, AC;Leong, MLL;Firestone, GL
转录调节的血清和糖皮质激素诱导蛋白激酶（Sgk）以血清依赖的方式定位于细胞核，酵母双杂交遗传筛选发现Sgk与importin- α核输入受体之间存在特异性相互作用。体外GST拉下实验表明，importin-alpha与内源性Sgk和外源性表达ha标记的Sgk存在强烈而直接的关联，而这两种成分在血清刺激后会共同免疫沉淀并共定位到细胞核。与核定位的积极机制一致，HA-Sgk在渗透细胞中的核输入需要ATP、细胞质和功能性核孔复合物。异位添加含有Sgk结合区的importin-alpha的107个氨基酸羧基末端片段，竞争性地抑制了内源性importin-alpha在体外将Sgk导入细胞核的能力。通过丙氨酸替代的赖氨酸诱变在Sgk的131-141氨基酸之间的中心区域定义了一个KKAILKKKEEK序列，该序列作为核定位信号（NLS），是体外与importin- α相互作用和培养细胞中全长Sgk的核输入所必需的。血清诱导的Sgk核输入需要nls依赖的输入蛋白α对Sgk的识别，以及pi3激酶依赖的Sgk磷酸化。我们的研究结果确定了进口蛋白- α在核定位蛋白激酶信号转导的刺激依赖性控制中的新作用。
The transcriptionally regulated serum and glucocorticoid inducible protein kinase (Sgk) is localized to the nucleus in a serum-dependent manner, and a yeast two-hybrid genetic screen uncovered a specific interaction between Sgk and the importin-alpha nuclear import receptor. In vitro GST pull down assays demonstrated a strong and direct association of importin-alpha with endogenous Sgk and exogenously expressed HA-tagged Sgk, whereas both components coimmunoprecipitate and colocalize to the nucleus after serum stimulation. Consistent with an active mechanism of nuclear localization, the nuclear import of HA-Sgk in permeabilized cells required ATP, cytoplasm, and a functional nuclear pore complex. Ectopic addition of a 107 amino acid carboxyterminal fragment of importin-alpha, which contains the Sgk binding region, competitively inhibited the ability of endogenous importin-alpha to import Sgk into nuclei in vitro. Mutagenesis of lysines by alanine substitution defined a KKAILKKKEEK sequence within the central domain of Sgk between amino acids 131-141 that functions as a nuclear localization signal (NLS) required for the in vitro interaction with importin-alpha and for nuclear import of full-length Sgk in cultured cells. The serum-induced nuclear import of Sgk requires the NLS-dependent recognition of Sgk by importin-alpha as well as the PI3-kinase- dependent phosphorylation of Sgk. Our results define a new role importin-alpha in the stimulus-dependent control of signal transduction by nuclear localized protein kinases.