Luo Ruibai, Chen Pei-Wen, Kuo Jean-Cheng, Jenkins Lisa, Jian Xiaoying, Waterman Clare M, Randazzo Paul A
Laboratory of Cellular and Molecular Biology, National Cancer Institute, Bethesda, MD, 20892, USA.
Department of Biology, Williams College, Williamstown, MA, 01267, USA.
Biol Cell. 2018 Dec;110(12):257-270. doi: 10.1111/boc.201800044. Epub 2018 Sep 10.
ARAP2, an Arf GTPase-activating protein (Arf GAP) that binds to adaptor protein with PH domain, PTB domain and leucine zipper motifs 1 (APPL1), regulates focal adhesions (FAs). APPL1 affects FA dynamics by regulating Akt. Here, we tested the hypothesis that ARAP2 affects FAs in part by regulating Akt through APPL1.
We found that ARAP2 controlled FA dynamics dependent on its enzymatic Arf GAP activity. In some cells, ARAP2 also regulated phosphoAkt (pAkt) levels. However, ARAP2 control of FAs did not require Akt and conversely, the effects on pAkt were independent of FAs. Reducing ARAP2 expression reduced the size and number of FAs in U118, HeLa and MDA-MB-231 cells. Decreasing ARAP2 expression increased pAkt in U118 cells and HeLa cells and overexpressing ARAP2 decreased pAkt in U118 cells; in contrast, ARAP2 had no effect on pAkt in MDA-MB-231 cells. An Akt inhibitor did not block the effect of reduced ARAP2 on FAs in U118. Furthermore, the effect of ARAP2 on Akt did not require Arf GAP activity, which is necessary for effects on FAs and integrin traffic. Altering FAs by other means did not induce the same changes in pAkt as those seen by reducing ARAP2 in U118 cells. In addition, we discovered that ARAP2 and APPL1 had co-ordinated effects on pAkt in U118 cells. Reduced APPL1 expression, as for ARAP2, increased pAkt in U118 and the effect of reduced APPL1 expression was reversed by overexpressing ARAP2. Conversely, the effect of reduced ARAP2 expression was reversed by overexpressing APPL1. ARAP2 is an Arf GAP that has previously been reported to affect FAs by regulating Arf6 and integrin trafficking and to bind to the adaptor proteins APPL1. Here, we report that ARAP2 suppresses pAkt levels in cells co-ordinately with APPL1 and independently of GAP activity and its effect on the dynamic behaviour of FAs.
We conclude that ARAP2 affects Akt signalling in some cells by a mechanism independent of FAs or membrane traffic.
Our results highlight an Arf GAP-independent function of ARAP2 in regulating Akt activity and distinguish the effect of ARAP2 on Akt from that on FAs and integrin trafficking, which requires regulation of Arf6.
ARAP2是一种Arf GTP酶激活蛋白（Arf GAP），它与具有PH结构域、PTB结构域和亮氨酸拉链基序1（APPL1）的衔接蛋白结合，调节粘着斑（FAs）。APPL1通过调节Akt影响粘着斑动态变化。在此，我们检验了ARAP2部分通过APPL1调节Akt来影响粘着斑的假说。
我们发现ARAP2依赖其酶促Arf GAP活性控制粘着斑动态变化。在某些细胞中，ARAP2也调节磷酸化Akt（pAkt）水平。然而，ARAP2对粘着斑的控制并不需要Akt，相反，对pAkt的影响独立于粘着斑。降低ARAP2表达可减少U118、HeLa和MDA - MB - 231细胞中粘着斑的大小和数量。降低ARAP2表达可增加U118细胞和HeLa细胞中的pAkt，而过表达ARAP2可降低U118细胞中的pAkt；相比之下，ARAP2对MDA - MB - 231细胞中的pAkt无影响。Akt抑制剂不能阻断降低ARAP2对U118细胞中粘着斑的影响。此外，ARAP2对Akt的影响不需要Arf GAP活性，而Arf GAP活性对粘着斑和整合素运输的影响是必需的。通过其他方式改变粘着斑不会像在U118细胞中降低ARAP2那样引起pAkt的相同变化。此外，我们发现ARAP2和APPL1对U118细胞中的pAkt具有协同作用。与ARAP2一样，降低APPL1表达可增加U118细胞中的pAkt，而过表达ARAP2可逆转降低APPL1表达的影响。相反，过表达APPL1可逆转降低ARAP2表达的影响。ARAP2是一种Arf GAP，此前有报道称其通过调节Arf6和整合素运输来影响粘着斑，并与衔接蛋白APPL1结合。在此，我们报道ARAP2与APPL1协同抑制细胞中的pAkt水平，且独立于GAP活性及其对粘着斑动态行为的影响。
我们得出结论，ARAP2通过一种独立于粘着斑或膜运输的机制影响某些细胞中的Akt信号传导。
我们的结果突出了ARAP2在调节Akt活性方面的一种不依赖Arf GAP的功能，并区分了ARAP2对Akt的影响与对粘着斑和整合素运输的影响，后者需要调节Arf6。
