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Updated: Nov 15, 2025
RhoC GTPase Activation Assay
Published on: August 22, 2010
PKCα-mediated phosphorylation of the diacylglycerol kinase ζ MARCKS domain switches cell migration modes by
Ryan Ard1, Jean-Christian Maillet1, Elias Daher1
1Department of Cellular and Molecular Medicine, University of Ottawa, Ottawa, Ontario, Canada; Centre for Neuromuscular Disease, University of Ottawa, Ottawa, Ontario, Canada.
Abstract:
Cells can switch between Rac1 (lamellipodia-based) and RhoA (blebbing-based) migration modes, but the molecular mechanisms regulating this shift are not fully understood. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, forms independent complexes with Rac1 and RhoA, selectively dissociating each from their common inhibitor RhoGDI. DGKζ catalytic activity is required for Rac1 dissociation but is dispensable for RhoA dissociation; instead, DGKζ stimulates RhoA release via a kinase-independent scaffolding mechanism. The molecular determinants that mediate the selective targeting of DGKζ to Rac1 or RhoA signaling complexes are unknown. Here, we show that protein kinase Cα (PKCα)-mediated phosphorylation of the DGKζ MARCKS domain increased DGKζ association with RhoA and decreased its interaction with Rac1. The same modification also enhanced DGKζ interaction with the scaffold protein syntrophin. Expression of a phosphomimetic DGKζ mutant stimulated membrane blebbing in mouse embryonic fibroblasts and C2C12 myoblasts, which was augmented by inhibition of endogenous Rac1. DGKζ expression in differentiated C2 myotubes, which have low endogenous Rac1 levels, also induced substantial membrane blebbing via the RhoA-ROCK pathway. These events were independent of DGKζ catalytic activity, but dependent upon a functional C-terminal PDZ-binding motif. Rescue of RhoA activity in DGKζ-null cells also required the PDZ-binding motif, suggesting that syntrophin interaction is necessary for optimal RhoA activation. Collectively, our results define a switch-like mechanism whereby DGKζ phosphorylation by PKCα plays a role in the interconversion between Rac1 and RhoA signaling pathways that underlie different cellular migration modes.
Insights
Diacylglycerol kinase ζ (DGKζ) phosphorylation by protein kinase Cα (PKCα) controls cell migration modes. This switch mechanism favors RhoA (blebbing) over Rac1 (lamellipodia) by altering DGKζ interactions and scaffolding.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Cells exhibit distinct migration modes, lamellipodia-based (Rac1) and blebbing-based (RhoA), regulated by complex molecular mechanisms.
- Diacylglycerol kinase ζ (DGKζ) interacts with both Rac1 and RhoA, influencing their dissociation from RhoGDI, a common inhibitor.
- The precise determinants governing DGKζ's selective targeting to Rac1 or RhoA signaling pathways remain largely unelucidated.
Purpose of the Study:
- To investigate the molecular mechanisms underlying the switch between Rac1 and RhoA signaling pathways in cellular migration.
- To determine the role of protein kinase Cα (PKCα)-mediated phosphorylation of DGKζ in regulating its interactions with Rac1 and RhoA.
Main Methods:
- Utilized phosphomimetic DGKζ mutants and examined protein interactions in mouse embryonic fibroblasts and C2C12 myoblasts.
- Assessed cellular migration modes, specifically membrane blebbing, under various experimental conditions including Rac1 inhibition and DGKζ expression.
- Investigated the dependence of DGKζ-mediated RhoA activation on its catalytic activity and C-terminal PDZ-binding motif.
Main Results:
- PKCα-mediated phosphorylation of DGKζ enhances its association with RhoA while decreasing interaction with Rac1, promoting a shift towards RhoA signaling.
- Phosphorylated DGKζ interacts with syntrophin, a scaffold protein, and this interaction is crucial for RhoA activation and membrane blebbing.
- DGKζ-induced membrane blebbing is independent of its catalytic activity but relies on its PDZ-binding motif, highlighting a scaffolding role.
Conclusions:
- DGKζ phosphorylation by PKCα acts as a critical switch, modulating the balance between Rac1 and RhoA signaling pathways.
- This phosphorylation-dependent switch mechanism dictates cellular migration modes, favoring RhoA-driven blebbing.
- The findings reveal a novel role for DGKζ scaffolding, mediated by syntrophin interaction, in regulating RhoA activity and cellular dynamics.
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