GSKIP- and GSK3-mediated anchoring strengthens cAMP/PKA/Drp1 axis signaling in the regulation of mitochondrial elongation.
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.
Department of Neurosurgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan; Department of Surgery, Kaohsiung Municipal Hsiao-Kang Hospital, Kaohsiung Medical University, Kaohsiung, Taiwan.
Joon-Khim
JK
Loh
Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Ching-Chih
CC
Lin
Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan; Laboratory of Medical Research, Center of Education and Faculty Development, Kaohsiung Armed Forces General Hospital, Kaohsiung, Taiwan.
Ming-Chang
MC
Yang
Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.
Chia-Hua
CH
Chou
Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan.
Wan-Shia
WS
Chen
Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and College of Medicine, Chang Gung University, Kaohsiung, Taiwan.
Ming-Chang
MC
Hong
Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.
Chung-Lung
CL
Cho
Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.
Ching-Mei
CM
Hsu
Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan.
Jiin-Tsuey
JT
Cheng
Department of Anesthesiology, Kaohsiung Chang Gung Memorial Hospital and College of Medicine, Chang Gung University, Kaohsiung, Taiwan.
An-Kuo
AK
Chou
Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Dermatology, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
Chung-Hsing
CH
Chang
Graduate Institute of Natural Products, Kaohsiung Medical University, Kaohsiung, Taiwan.
Chao-Neng
CN
Tseng
Department of Biotechnology, Kaohsiung Medical University, Kaohsiung, Taiwan.
Chi-Huei
CH
Wang
Department of Neurosurgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
Ann-Shung
AS
Lieu
Department of Neurosurgery, Kaohsiung Medical University Hospital, Kaohsiung, Taiwan.
Shen-Long
SL
Howng
Department of Biochemistry, Faculty of Medicine, College of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan; Department of Biological Sciences, National Sun Yat-Sen University, Kaohsiung, Taiwan; Graduate Institute of Medicine, Kaohsiung Medical University, Kaohsiung, Taiwan. Electronic address: m835016@cc.kmu.edu.tw.
Yi-Ren
YR
Hong
D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism*; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism*; D034281:Dynamins; D020558:GTP Phosphohydrolases / Q000235:genetics / Q000378:metabolism*; D038362:Glycogen Synthase Kinase 3 / Q000378:metabolism / Q000502:physiology*; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D008869:Microtubule-Associated Proteins / Q000235:genetics / Q000378:metabolism*; D008928:Mitochondria / Q000235:genetics / Q000378:metabolism*; D063154:Mitochondrial Dynamics / Q000502:physiology; D024101:Mitochondrial Proteins / Q000235:genetics / Q000378:metabolism*; D010766:Phosphorylation; D012097:Repressor Proteins / Q000378:metabolism / Q000502:physiology*; D015398:Signal Transduction / Q000235:genetics
C514503:GSKIP protein, human; D008869:Microtubule-Associated Proteins; D024101:Mitochondrial Proteins; D012097:Repressor Proteins; D000242:Cyclic AMP; D017868:Cyclic AMP-Dependent Protein Kinases; D038362:Glycogen Synthase Kinase 3; D020558:GTP Phosphohydrolases; C110768:DNM1L protein, human; D034281:Dynamins
AKAP; Anchoring; Drp1; GSK3; GSKIP; Mitochondrial morphology