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

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 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.

Mitochondrial elongation-mediated glucose metabolism reprogramming is essential for tumour cell survival during energy stress

“…23 Similar results have also been observed in non-transformed cells, including MEF and HEK293 cells. 6,24 Consistent with these observations, we have shown that activation of PKA induced by energy stress clearly promotes DRP1 phosphorylation at Ser637 and subsequent mitochondrial elongation in HCC cells. However, we still cannot rule out the possibility that other kinases may be involved in this process.…”

Drp1-dependent remodeling of mitochondrial morphology triggered by EBV-LMP1 increases cisplatin resistance

“…However, Ser637 phosphorylation reverses this process. 5 Drp1 is phosphorylated by specific kinases and signaling pathways during mitochondrial fission, including phosphoglycerate mutase family member 5, 6 AMPK, [7][8][9] MAPK, [10][11][12] , and cyclin-dependent kinase 1/cyclin B1 (Cdk1/cyclin B1). 13 AMPK is not only an important detector of energy levels but is also a negative moderator of glycolysis.…”

TGR5 signalling promotes mitochondrial fission and beige remodelling of white adipose tissue

“…One of the main proteins implicated in mitochondrial fission is the GTPase dynamin-1-like protein (DRP1) that can be activated through phosphorylation by ERK at Serine 616 in mouse and human cells 37 , 38 . Conversely, phosphorylation of DRP1 at Serine 637 by PKA inactivates the protein leading to mitochondrial fusion 39 – 41 . Of interest, TGR5 stimulation in Tgr5 +/+ adipocytes increased DRP1 phosphorylation specifically at Serine 616, but not at Serine 637 (Fig.…”