Laboratory of Bioimaging and Cell Signaling, Graduate School of Biostudies, Kyoto University, Sakyo-ku, Kyoto, Japan.
Mol Biol Cell. 2011 May 15;22(10):1780-90. doi: 10.1091/mbc.E10-09-0783. Epub 2011 Apr 1.
The second messenger cAMP plays a pivotal role in neurite/axon growth and guidance, but its downstream pathways leading to the regulation of Rho GTPases, centrally implicated in neuronal morphogenesis, remain elusive. We examined spatiotemporal changes in Rac1 and Cdc42 activity and phosphatidylinositol 3,4,5-triphosphate (PIP(3)) concentration in dibutyryl cAMP (dbcAMP)-treated PC12D cells using Förster resonance energy transfer-based biosensors. During a 30-min incubation with dbcAMP, Rac1 activity gradually increased throughout the cells and remained at its maximal level. There was no change in PIP(3) concentration. After a 5-h incubation with dbcAMP, Rac1 and Cdc42 were activated at the protruding tips of neurites without PIP(3) accumulation. dbcAMP-induced Rac1 activation was principally mediated by protein kinase A (PKA) and Sif- and Tiam1-like exchange factor (STEF)/Tiam2. STEF depletion drastically reduced dbcAMP-induced neurite outgrowth. PKA phosphorylates STEF at three residues (Thr-749, Ser-782, Ser-1562); Thr-749 phosphorylation was critical for dbcAMP-induced Rac1 activation and neurite extension. During dbcAMP-induced neurite outgrowth, PKA activation at the plasma membrane became localized to neurite tips; this localization may contribute to local Rac1 activation at the same neurite tips. Considering the critical role of Rac1 in neuronal morphogenesis, the PKA-STEF-Rac1 pathway may play a crucial role in cytoskeletal regulation during neurite/axon outgrowth and guidance, which depend on cAMP signals.
第二信使 cAMP 在轴突/神经元生长和导向中起着关键作用，但其下游途径导致 Rho GTPases 的调节，这些 GTPases在神经元形态发生中起着核心作用，仍然难以捉摸。我们使用基于Förster 共振能量转移的生物传感器，研究了 dbcAMP（二丁酰环腺苷酸）处理的 PC12D 细胞中 Rac1 和 Cdc42 活性以及磷脂酰肌醇 3,4,5-三磷酸（PIP（3））浓度的时空变化。在与 dbcAMP 孵育 30 分钟期间，Rac1 活性逐渐在整个细胞中增加，并保持在最大水平。PIP（3）浓度没有变化。在与 dbcAMP 孵育 5 小时后，Rac1 和 Cdc42 在没有 PIP（3）积累的情况下在神经元突起的突出尖端被激活。dbcAMP 诱导的 Rac1 激活主要由蛋白激酶 A（PKA）和 Sif 和 Tiam1 样交换因子（STEF）/Tiam2 介导。STEF 耗尽极大地减少了 dbcAMP 诱导的神经突生长。PKA 在三个残基（Thr-749、Ser-782、Ser-1562）上磷酸化 STEF；Thr-749 磷酸化对于 dbcAMP 诱导的 Rac1 激活和神经突延伸至关重要。在 dbcAMP 诱导的神经突生长过程中，质膜上的 PKA 激活定位于神经突尖端；这种定位可能有助于在同一神经突尖端处进行局部 Rac1 激活。考虑到 Rac1 在神经元形态发生中的关键作用，PKA-STEF-Rac1 途径可能在依赖于 cAMP 信号的神经突/轴突生长和导向过程中的细胞骨架调节中发挥关键作用。
