Higuchi O, Amano T, Yang N, Mizuno K
Department of Biology, Faculty of Science, Kyushu University, Hakozaki, Fukuoka, Japan.
Oncogene. 1997 Apr 17;14(15):1819-25. doi: 10.1038/sj.onc.1201020.
LIM-kinase 1 and 2 (LIMK1 and LIMK2) are members of a novel class of protein kinases with structures composed of two LIM motifs at the N-terminus and an unusual protein kinase domain at the C-terminus. The cellular functions of the LIMK family proteins have remained unknown. In the present study, we examined effects of LIMKs on neuronal differentiation of PC12 pheochromocytoma cells. Transient expression analyses revealed that LIMK1, in itself, had no apparent effect on PC12 cells, but the oncogenic Ras-induced differentiation of PC12 cells was notably inhibited by co-expression with LIMK1 or LIMK2. A mutant of LIMK1 lacking a protein kinase domain (delta K) similarly inhibited Ras-induced differentiation of PC12 cells, but a mutant lacking a LIM domain (delta LIM) failed to do so, indicating that a LIM domain but not a protein kinase domain is required for the inhibitory activity. This notion was further supported by the finding that mutation, changing conserved cysteines involved in zinc coordination to glycines in both of two LIM motifs, abolished the inhibitory activity of delta K. Additionally, we also found that the constitutively activated MAP kinase kinase (MAPKK)-induced differentiation of PC12 cells was inhibited by co-expression with delta K. Furthermore, AK did not inhibit the kinase activity of MAP kinase (MAPK) stimulated by MAPKK, when co-expressed in COS7 cells. These findings suggest that LIMK1 inhibits neuronal differentiation of PC12 cells, through its LIM domain and by interfering with events downstream of MAPK activation.
LIM激酶1和2（LIMK1和LIMK2）是一类新型蛋白激酶的成员，其结构在N端由两个LIM基序组成，在C端有一个不同寻常的蛋白激酶结构域。LIMK家族蛋白的细胞功能一直未知。在本研究中，我们检测了LIMK对PC12嗜铬细胞瘤细胞神经元分化的影响。瞬时表达分析显示，LIMK1本身对PC12细胞没有明显影响，但与LIMK1或LIMK2共表达时，致癌性Ras诱导的PC12细胞分化受到显著抑制。缺乏蛋白激酶结构域的LIMK1突变体（delta K）同样抑制了Ras诱导的PC12细胞分化，但缺乏LIM结构域的突变体（delta LIM）则没有，这表明抑制活性需要LIM结构域而非蛋白激酶结构域。这一观点进一步得到以下发现的支持：将两个LIM基序中参与锌配位的保守半胱氨酸突变为甘氨酸，消除了delta K的抑制活性。此外，我们还发现，组成型激活的丝裂原活化蛋白激酶激酶（MAPKK）诱导的PC12细胞分化在与delta K共表达时受到抑制。此外，当在COS7细胞中共表达时，AK并不抑制由MAPKK刺激的丝裂原活化蛋白激酶（MAPK）的激酶活性。这些发现表明，LIMK1通过其LIM结构域并通过干扰MAPK激活下游的事件来抑制PC12细胞的神经元分化。
