GSKIP- and GSK3-mediated anchoring strengthens cAMP/PKA/Drp1 axis signaling in the regulation of mitochondrial elongation.

GSK3ß binding of GSKIP affects neurite outgrowth, but the physiological significance of PKA binding to GSKIP remains to be determined. We hypothesized that GSKIP and GSK3ß mediate cAMP/PKA/Drp1 axis signaling and modulate mitochondrial morphology by forming a working complex comprising PKA/GSKIP/GSK3ß/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 GSK3ß 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 GSK3ß but not GSK3α resulted in a dramatic decrease in Drp1 S637 phosphorylation, revealing that both GSKIP and GSK3ß are required in this novel PKA/GSKIP/GSK3ß/Drp1 complex. Moreover, overexpressed kinase-dead GSK3ß-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 GSK3ß 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 GSK3ß function as anchoring proteins in the cAMP/PKA/Drp1 signaling axis modulating Drp1 phosphorylation.