Miura Yuki, Hongu Tsunaki, Yamauchi Yohei, Funakoshi Yuji, Katagiri Naohiro, Ohbayashi Norihiko, Kanaho Yasunori
Department of Physiological Chemistry, Faculty of Medicine and Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan School of Integrative and Global Majors, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan.
Department of Physiological Chemistry, Faculty of Medicine and Graduate School of Comprehensive Human Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8575, Japan.
Biochem J. 2016 Sep 1;473(17):2591-602. doi: 10.1042/BCJ20160183. Epub 2016 Jun 21.
ACAP3 (ArfGAP with coiled-coil, ankyrin repeat and pleckstrin homology domains 3) belongs to the ACAP family of GAPs (GTPase-activating proteins) for the small GTPase Arf (ADP-ribosylation factor). However, its specificity to Arf isoforms and physiological functions remain unclear. In the present study, we demonstrate that ACAP3 plays an important role in neurite outgrowth of mouse hippocampal neurons through its GAP activity specific to Arf6. In primary cultured mouse hippocampal neurons, knockdown of ACAP3 abrogated neurite outgrowth, which was rescued by ectopically expressed wild-type ACAP3, but not by its GAP activity-deficient mutant. Ectopically expressed ACAP3 in HEK (human embryonic kidney)-293T cells showed the GAP activity specific to Arf6. In support of this observation, the level of GTP-bound Arf6 was significantly increased by knockdown of ACAP3 in hippocampal neurons. In addition, knockdown and knockout of Arf6 in mouse hippocampal neurons suppressed neurite outgrowth. These results demonstrate that ACAP3 positively regulates neurite outgrowth through its GAP activity specific to Arf6. Furthermore, neurite outgrowth suppressed by ACAP3 knockdown was rescued by expression of a fast cycle mutant of Arf6 that spontaneously exchanges guanine nucleotides on Arf6, but not by that of wild-type, GTP- or GDP-locked mutant Arf6. Thus cycling between active and inactive forms of Arf6, which is precisely regulated by ACAP3 in concert with a guanine-nucleotide-exchange factor(s), seems to be required for neurite outgrowth of hippocampal neurons.
ACAP3（含卷曲螺旋、锚蛋白重复序列和普列克底物蛋白同源结构域3的ArfGAP）属于小GTP酶Arf（ADP核糖基化因子）的GAP（GTP酶激活蛋白）家族成员ACAP家族。然而，其对Arf亚型的特异性和生理功能仍不清楚。在本研究中，我们证明ACAP3通过其对Arf6的特异性GAP活性在小鼠海马神经元的轴突生长中发挥重要作用。在原代培养的小鼠海马神经元中，敲低ACAP3可消除轴突生长，而异位表达的野生型ACAP3可挽救这一现象，但其GAP活性缺陷型突变体则不能。在HEK（人胚肾）-293T细胞中异位表达的ACAP3表现出对Arf6的特异性GAP活性。为支持这一观察结果，海马神经元中敲低ACAP3后，结合GTP的Arf6水平显著升高。此外，敲低和敲除小鼠海马神经元中的Arf6可抑制轴突生长。这些结果表明，ACAP3通过其对Arf6的特异性GAP活性正向调节轴突生长。此外，ACAP3敲低所抑制的轴突生长可通过表达Arf6的快速循环突变体来挽救，该突变体可自发地在Arf6上交换鸟嘌呤核苷酸，而野生型、GTP或GDP锁定的突变体Arf6则不能。因此，海马神经元的轴突生长似乎需要Arf6的活性形式和非活性形式之间的循环，这一循环由ACAP3与鸟嘌呤核苷酸交换因子协同精确调控。
