• Joon-Khim Loh, Ching-Chih Lin, Ming-Chang Yang, Chia-Hua Chou, Wan-Shia Chen, Ming-Chang Hong, Chung-Lung Cho, Ching-Mei Hsu, Jiin-Tsuey Cheng, An-Kuo Chou, Chung-Hsing Chang, Chao-Neng Tseng, Chi-Huei Wang, Ann-Shung Lieu, Shen-Long Howng, and Yi-Ren Hong.
• Department of Neurosurgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; Department of Surgery, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan.
• Biochim. Biophys. Acta. 2015 Aug 1; 1853 (8): 1796-807.

AbstractGSK3β 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.Copyright © 2015 Elsevier B.V. All rights reserved.

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