Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506, USA.
J Biol Chem. 2010 Sep 10;285(37):28643-50. doi: 10.1074/jbc.M110.107649. Epub 2010 Jul 15.
GIPC1/synectin, a single PDZ domain-containing protein, binds to numerous proteins and is involved in multiple biological processes, including cell migration. We reported previously that MyoGEF, a guanine nucleotide exchange factor, plays a role in regulating breast cancer cell polarization and invasion. Here, we identify GIPC1 as an interacting partner of MyoGEF. Both in vitro and in vivo binding assays show that the GIPC1 PDZ domain binds to the PDZ-binding motif at the C terminus of MyoGEF. Immunofluorescence analysis shows that GIPC1 and MyoGEF colocalize to the cell leading edge. Depletion of GIPC1 by RNAi in MDA-MB-231 cells causes cells to shift from a polarized to a rounded morphology. Matrigel invasion assays show that RNAi-mediated depletion of GIPC1 dramatically decreases MDA-MB-231 cell invasion. Notably, an anti-MyoGEF peptide antibody, whose epitope is located at the C terminus of MyoGEF, interferes with GIPC1-MyoGEF complex formation. Treatment of MDA-MB-231 cells with the anti-MyoGEF peptide antibody disrupts cell polarization and invasion. Thus, our results suggest that GIPC1-MyoGEF complex formation plays an important role in regulating MDA-MB-231 breast cancer cell polarization and invasion.
GIPC1/synectin 是一种含有单个 PDZ 结构域的蛋白质，可与许多蛋白质结合，并参与多种生物学过程，包括细胞迁移。我们之前报道过，MyoGEF 是一种鸟嘌呤核苷酸交换因子，在调节乳腺癌细胞极化和侵袭中发挥作用。在这里，我们鉴定出 GIPC1 是 MyoGEF 的相互作用伙伴。体外和体内结合实验表明，GIPC1 PDZ 结构域与 MyoGEF C 末端的 PDZ 结合基序结合。免疫荧光分析表明，GIPC1 和 MyoGEF 共定位于细胞前缘。在 MDA-MB-231 细胞中通过 RNAi 耗尽 GIPC1 会导致细胞从极化状态转变为圆形形态。Matrigel 侵袭实验表明，RNAi 介导的 GIPC1 耗竭显著降低了 MDA-MB-231 细胞的侵袭能力。值得注意的是，一种抗 MyoGEF 肽抗体，其表位位于 MyoGEF 的 C 末端，干扰了 GIPC1-MyoGEF 复合物的形成。用抗 MyoGEF 肽抗体处理 MDA-MB-231 细胞会破坏细胞极化和侵袭。因此，我们的结果表明，GIPC1-MyoGEF 复合物的形成在调节 MDA-MB-231 乳腺癌细胞极化和侵袭中起着重要作用。
