Lee Seunghyung, Wurtzel Jeremy G T, Goldfinger Lawrence E
Department of Anatomy & Cell Biology and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA.
Department of Anatomy & Cell Biology and The Sol Sherry Thrombosis Research Center, Temple University School of Medicine, Philadelphia, PA 19140, USA; Cancer Biology Program, Fox Chase Cancer Center, Philadelphia, PA 19111, USA.
Biochem Biophys Res Commun. 2014 Nov 28;454(4):560-5. doi: 10.1016/j.bbrc.2014.10.114. Epub 2014 Oct 30.
RLIP76 is a multifunctional protein involved in tumor growth and angiogenesis, and a promising therapeutic target in many cancers. RLIP76 harbors docking sites for many proteins, and we have found that it interacts with ARNO, a guanine nucleotide exchange factor for Arf6, and that RLIP76 regulates activation of Rac1 via Arf6, and regulates cell spreading and migration in an ARNO and Arf6-dependent manner. Here we show that ARNO interacts with the RLIP76 N-terminal domain, and this domain was required for RLIP76-dependent cell spreading and migration. We identified two sites in the RLIP76 N-terminus with differential effects on ARNO binding and downstream signaling: Ser29/Ser30 and Ser62. Ser29/30 mutation to Alanine inhibited ARNO interaction and was sufficient to block RLIP76-dependent cell spreading and migration, as well as RLIP76-dependent Arf6 activation. In contrast, RLIP76(S62A) interacted with ARNO and supported Arf6 activation. However, both sets of mutations blocked Rac1 activation. RLIP76-mediated Rac and Arf6 activation required PI3K activity. S29/30A mutations inhibited RLIP76-dependent PI3K activation, but S62A mutation did not. Together these results show that ARNO interaction with the RLIP76 N-terminus regulates cell spreading and motility via PI3K and Arf6, independent of RLIP76 control of Rac.
RLIP76是一种参与肿瘤生长和血管生成的多功能蛋白，是许多癌症中一个很有前景的治疗靶点。RLIP76含有许多蛋白质的对接位点，我们发现它与ARNO相互作用，ARNO是Arf6的鸟嘌呤核苷酸交换因子，并且RLIP76通过Arf6调节Rac1的激活，并以ARNO和Arf6依赖的方式调节细胞铺展和迁移。在这里，我们表明ARNO与RLIP76的N端结构域相互作用，并且该结构域是RLIP76依赖的细胞铺展和迁移所必需的。我们在RLIP76的N端鉴定出两个对ARNO结合和下游信号有不同影响的位点：Ser29/Ser30和Ser62。将Ser29/30突变为丙氨酸会抑制ARNO相互作用，并且足以阻断RLIP76依赖的细胞铺展和迁移，以及RLIP76依赖的Arf6激活。相比之下，RLIP76(S62A)与ARNO相互作用并支持Arf6激活。然而，这两组突变都阻断了Rac1激活。RLIP76介导的Rac和Arf6激活需要PI3K活性。S29/30A突变抑制了RLIP76依赖的PI3K激活，但S62A突变没有。这些结果共同表明，ARNO与RLIP76 N端的相互作用通过PI3K和Arf6调节细胞铺展和运动，独立于RLIP76对Rac的控制。
