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.
Keywords: AKAP; Anchoring; Drp1; GSK3; GSKIP; Mitochondrial morphology.
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