| Author |
Yang, Ming-chang Cho, Chung-lung Howng, Shen-long Chou, An-kuo Wang, Chi-huei Cheng, Jiin-tsuey Chang, Chung-hsing Chen, Wan-shia Hsu, Ching-mei Chou, Chia-hua Lieu, Ann-shung Hong, Ming-chang Lin, Ching-chih Hong, Yi-ren Loh, Joon-khim Tseng, Chao-neng |
| Description |
Author Affiliation: Loh JK ( Department of Neurosurgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan); Lin CC ( Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.); Yang MC ( Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan); Chou CH ( Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan); Chen WS ( Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.); Hong MC ( Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan); Cho CL ( Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.); Hsu CM ( Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.); Cheng JT ( Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.); Chou AK ( Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and College of Medicine, Chang Gung University, Kaohsiung, Taiwan.); Chang CH ( Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan); Tseng CN ( Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan.); Wang CH ( Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan.); Lieu AS ( Department of Neurosurgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.); Howng SL ( Department of Neurosurgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.); Hong YR ( Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan) |
| Abstract |
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 H $_{2}$ O $_{2}$ 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. |
