Loh Joon-Khim, Lin Ching-Chih, Yang Ming-Chang, Chou Chia-Hua, Chen Wan-Shia, Hong Ming-Chang, Cho Chung-Lung, Hsu Ching-Mei, Cheng Jiin-Tsuey, Chou An-Kuo, Chang Chung-Hsing, Tseng Chao-Neng, Wang Chi-Huei, Lieu Ann-Shung, Howng Shen-Long, Hong Yi-Ren
Department of Neurosurgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; Department of Surgery, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan.
Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Biochim Biophys Acta. 2015 Aug;1853(8):1796-807. doi: 10.1016/j.bbamcr.2015.04.013. Epub 2015 Apr 25.
GSK3β binding of GSKIP affects neurite outgrowth, but the physiological significance of PKA binding to GSKIP remains to be determined. We hypothesized that GSKIP and GSK3β mediate cAMP/PKA/Drp1 axis signaling and modulate mitochondrial morphology by forming a working complex comprising PKA/GSKIP/GSK3β/Drp1. We demonstrated that GSKIP wild-type overexpression increased phosphorylation of Drp1 S637 by 7-8-fold compared to PKA kinase-inactive mutants (V41/L45) and a GSK3β binding-defective mutant (L130) under H2O2 and forskolin challenge in HEK293 cells, indicating that not only V41/L45, but also L130 may be involved in Drp1-associated protection of GSKIP. Interestingly, silencing either GSKIP or GSK3β but not GSK3α resulted in a dramatic decrease in Drp1 S637 phosphorylation, revealing that both GSKIP and GSK3β are required in this novel PKA/GSKIP/GSK3β/Drp1 complex. Moreover, overexpressed kinase-dead GSK3β-K85R, which retains the capacity to bind GSKIP, but not K85M which shows total loss of GSKIP-binding, has a higher Drp1 S637 phosphorylation similar to the GSKIP wt overexpression group, indicating that GSK3β recruits Drp1 by anchoring rather than in a kinase role. With further overexpression of either V41/L45P or the L130P GSKIP mutant, the elongated mitochondrial phenotype was lost; however, ectopically expressed Drp1 S637D, a phosphomimetic mutant, but not S637A, a non-phosphorylated mutant, restored the elongated mitochondrial morphology, indicating that Drp1 is a downstream effector of direct PKA signaling and possibly has an indirect GSKIP function involved in the cAMP/PKA/Drp1 signaling axis. Collectively, our data revealed that both GSKIP and GSK3β function as anchoring proteins in the cAMP/PKA/Drp1 signaling axis modulating Drp1 phosphorylation.
GSKIP与GSK3β的结合会影响神经突生长，但PKA与GSKIP结合的生理意义仍有待确定。我们推测，GSKIP和GSK3β通过形成包含PKA/GSKIP/GSK3β/Drp1的工作复合物来介导cAMP/PKA/Drp1轴信号传导并调节线粒体形态。我们证明，在HEK293细胞中，与PKA激酶失活突变体（V41/L45）和GSK3β结合缺陷突变体（L130）相比，在H2O2和福斯可林刺激下，GSKIP野生型过表达使Drp1 S637的磷酸化增加了7-8倍，这表明不仅V41/L45，而且L130可能参与了GSKIP对Drp1的相关保护。有趣的是，沉默GSKIP或GSK3β而不是GSK3α会导致Drp1 S637磷酸化显著降低，这表明在这种新型的PKA/GSKIP/GSK3β/Drp1复合物中，GSKIP和GSK3β都是必需的。此外，过表达的激酶失活型GSK3β-K85R（保留与GSKIP结合的能力），而不是完全丧失与GSKIP结合能力的K85M突变体，具有与GSKIP野生型过表达组相似的更高的Drp1 S637磷酸化水平，这表明GSK3β通过锚定而非激酶作用来招募Drp1。随着V41/L45P或L130P GSKIP突变体的进一步过表达，线粒体的伸长表型消失；然而，异位表达的Drp1 S637D（一种磷酸模拟突变体），而不是非磷酸化突变体S637A，恢复了线粒体的伸长形态，这表明Drp1是PKA直接信号传导的下游效应物，并且可能在cAMP/PKA/Drp1信号轴中具有间接的GSKIP功能。总体而言，我们的数据表明，GSKIP和GSK3β在调节Drp1磷酸化的cAMP/PKA/Drp1信号轴中均作为锚定蛋白发挥作用。
