Yang N, Higuchi O, Mizuno K
Department of Biology, Faculty of Science, Kyushu University, Fukuoka, Japan.
Exp Cell Res. 1998 May 25;241(1):242-52. doi: 10.1006/excr.1998.4053.
LIM-containing protein kinase 1 (LIMK1) is a serine/threonine kinase with a structure composed of two LIM domains, a PDZ domain, and a protein kinase domain. We examined the subcellular localization of LIMK1 and its variously deleted mutants in HeLa cells by transfection with these cDNAs. Immunofluorescence analysis revealed that the full-length LIMK1 and its mutants deleted with LIM domain or protein kinase domain preferentially localized in the cytoplasm, while the mutants deleted with the PDZ domain or a 52 amino acid region (B region) within the PDZ domain localized mainly in the nucleus. When the normally nuclear cyclin A was fused with the PDZ domain or the B region of LIMK1, it was localized in the cytoplasm of transfected cells. The corresponding region of the PDZ domain of postsynaptic density protein (PSD)-95 had no such function. Additionally, the PDZ domain of LIMK1 had no potential to bind to the C-terminal S/TXV peptides, to which the PSD-95 PDZ domain can bind. Taken together these results suggest that the PDZ domain, particularly the B region, of LIMK1 has a specific function to localize the protein in the cytoplasm. When glutathione S-transferase (GST) fused with the PDZ domain of LIMK1 (GST-PDZ) or GST-PDZ deleted with the B region (GST-PDZ delta B) was microinjected into the nucleus of COS cells, GST-PDZ was almost completely excluded from the nucleus within 30 min, whereas GST-PDZ delta B remained in the nucleus. These findings suggest that the B region of LIMK1 probably has nuclear export signal activity.
含LIM结构域的蛋白激酶1（LIMK1）是一种丝氨酸/苏氨酸激酶，其结构由两个LIM结构域、一个PDZ结构域和一个蛋白激酶结构域组成。我们通过用这些cDNA转染来检测HeLa细胞中LIMK1及其各种缺失突变体的亚细胞定位。免疫荧光分析显示，全长LIMK1及其缺失LIM结构域或蛋白激酶结构域的突变体优先定位于细胞质中，而缺失PDZ结构域或PDZ结构域内一个52个氨基酸区域（B区域）的突变体主要定位于细胞核中。当正常定位于细胞核的细胞周期蛋白A与LIMK1的PDZ结构域或B区域融合时，它定位于转染细胞的细胞质中。突触后致密蛋白（PSD）-95的PDZ结构域的相应区域没有这种功能。此外，LIMK1的PDZ结构域没有与PSD-95 PDZ结构域可结合的C末端S/TXV肽结合的潜力。综合这些结果表明，LIMK1的PDZ结构域，特别是B区域，具有将蛋白质定位于细胞质中的特定功能。当与LIMK1的PDZ结构域融合的谷胱甘肽S-转移酶（GST）（GST-PDZ）或缺失B区域的GST-PDZ（GST-PDZ delta B）被显微注射到COS细胞的细胞核中时，GST-PDZ在30分钟内几乎完全被排除在细胞核外，而GST-PDZ delta B仍留在细胞核中。这些发现表明，LIMK1的B区域可能具有核输出信号活性。
