Nishimura Yukio, Yoshioka Kiyoko, Bernard Ora, Bereczky Biborka, Itoh Kazuyuki
Graduate School of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Fukuoka 812-8582, Japan.
Histochem Cell Biol. 2006 Nov;126(5):627-38. doi: 10.1007/s00418-006-0198-x. Epub 2006 Jun 9.
We have previously shown that overexpression of LIM kinase1 (LIMK1) resulted in a marked retardation of the internalization of the receptor-mediated endocytic tracer, Texas red-labeled epidermal growth factor (EGF) in low-invasive human breast cancer cell MCF-7. We thereby postulate that LIMK1 signaling plays an important role in the regulation of ligand-induced endocytosis of EGF receptor (EGFR) in tumor cells by reorganizing and influencing actin-filament dynamics. In the present study, we further assessed the effect of wild-type LIMK1, a kinase-deficient dominant negative mutant of LIMK1 (DN-LIMK1) and an active, unphosphorylatable cofilin mutant (S3A cofilin) on internalization of EGF-EGFR in MDA-MB-231, a highly invasive human breast cancer cell line. We demonstrate here that a marked delay in the receptor-mediated internalization of Texas red-labeled EGF was observed in the wild-type LIMK1 transfectants, and that most of the internalized EGF staining were accumulated within transferrin receptor-positive early endosomes even after 30 min internalization. In contrast, the expression of dominant-negative LIMK1 mutant rescued the efficient endocytosis of Texas red-EGF, and large amounts of Texas red-EGF staining already reached LIMPII-positive late endosomes/lysosomal vacuoles after 15 min internalization. We further analyzed the effect of S3A cofilin mutant on EGFR trafficking, and found an efficient delivery of Texas red-EGF into late endosomes/lysosomes at 15-30 min after internalization. Taken together, our novel findings presented in this paper implicate that LIMK1 signaling indeed plays a pivotal role in the regulation of EGFR trafficking through the endocytic pathway in invasive tumor cells.
我们之前已经表明，在低侵袭性人乳腺癌细胞MCF-7中，LIM激酶1（LIMK1）的过表达导致受体介导的内吞示踪剂——德克萨斯红标记的表皮生长因子（EGF）的内化显著延迟。因此我们推测，LIMK1信号传导通过重组和影响肌动蛋白丝动力学，在肿瘤细胞中表皮生长因子受体（EGFR）的配体诱导内吞作用调节中发挥重要作用。在本研究中，我们进一步评估了野生型LIMK1、LIMK1的激酶缺陷型显性负性突变体（DN-LIMK1）和活性、不可磷酸化的丝切蛋白突变体（S3A丝切蛋白）对高侵袭性人乳腺癌细胞系MDA-MB-231中EGF-EGFR内化的影响。我们在此证明，在野生型LIMK1转染细胞中观察到德克萨斯红标记的EGF的受体介导内化显著延迟，并且即使在30分钟内化后，大多数内化的EGF染色仍积聚在转铁蛋白受体阳性的早期内体中。相比之下，显性负性LIMK1突变体的表达挽救了德克萨斯红-EGF的有效内吞作用，并且在15分钟内化后，大量的德克萨斯红-EGF染色已经到达LIMPII阳性的晚期内体/溶酶体空泡。我们进一步分析了S3A丝切蛋白突变体对EGFR运输的影响，发现在内化后15 - 30分钟，德克萨斯红-EGF能有效递送至晚期内体/溶酶体。综上所述，我们在本文中提出的新发现表明，LIMK1信号传导确实在侵袭性肿瘤细胞中通过内吞途径调节EGFR运输中起关键作用。
