TY - JOUR

T1 - Diacylglycerol kinase ζ regulates RhoA activation via a kinase-independent scaffolding mechanism

AU - Ard, Ryan

AU - Mulatz, Kirk

AU - Abramovici, Hanan

AU - Maillet, Jean-Christian

AU - Fottinger, Alexandra

AU - Foley, Tanya

AU - Byham, Michèle-Renée

AU - Iqbal, Tasfia Ahmed

AU - Yoneda, Atsuko

AU - Couchman, John R

AU - Parks, Robin J

AU - Gee, Stephen H

PY - 2012/10

Y1 - 2012/10

N2 - Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα) selectively releases RhoA. Here we show DGKζ is required for RhoA activation and Ser-34 phosphorylation, which were decreased in DGKζ-deficient fibroblasts and rescued by wild-type DGKζ or a catalytically inactive mutant. DGKζ bound directly to the C-terminus of RhoA and the regulatory arm of RhoGDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest DGKζ functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGKζ is a critical factor linking changes in lipid signaling to actin reorganization.

AB - Rho GTPases share a common inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI), which regulates their expression levels, membrane localization, and activation state. The selective dissociation of individual Rho GTPases from RhoGDI ensures appropriate responses to cellular signals, but the underlying mechanisms are unclear. Diacylglycerol kinase ζ (DGKζ), which phosphorylates diacylglycerol to yield phosphatidic acid, selectively dissociates Rac1 by stimulating PAK1-mediated phosphorylation of RhoGDI on Ser-101/174. Similarly, phosphorylation of RhoGDI on Ser-34 by protein kinase Cα (PKCα) selectively releases RhoA. Here we show DGKζ is required for RhoA activation and Ser-34 phosphorylation, which were decreased in DGKζ-deficient fibroblasts and rescued by wild-type DGKζ or a catalytically inactive mutant. DGKζ bound directly to the C-terminus of RhoA and the regulatory arm of RhoGDI and was required for efficient interaction of PKCα and RhoA. DGKζ-null fibroblasts had condensed F-actin bundles and altered focal adhesion distribution, indicative of aberrant RhoA signaling. Two targets of the RhoA effector ROCK showed reduced phosphorylation in DGKζ-null cells. Collectively our findings suggest DGKζ functions as a scaffold to assemble a signaling complex that functions as a RhoA-selective, GDI dissociation factor. As a regulator of Rac1 and RhoA activity, DGKζ is a critical factor linking changes in lipid signaling to actin reorganization.

KW - Animals

KW - Biocatalysis

KW - Diacylglycerol Kinase

KW - Embryo, Mammalian

KW - Enzyme Activation

KW - Fibroblasts

KW - Focal Adhesions

KW - Mice

KW - Models, Biological

KW - Multiprotein Complexes

KW - Phosphorylation

KW - Phosphoserine

KW - Protein Binding

KW - Protein Kinase C-alpha

KW - Protein Stability

KW - Protein Structure, Tertiary

KW - Signal Transduction

KW - Stress Fibers

KW - rho-Specific Guanine Nucleotide Dissociation Inhibitors

KW - rhoA GTP-Binding Protein

U2 - 10.1091/mbc.E12-01-0026

DO - 10.1091/mbc.E12-01-0026

M3 - Journal article

C2 - 22918940

VL - 23

SP - 4008

EP - 4019

JO - Molecular Biology of the Cell

JF - Molecular Biology of the Cell

SN - 1059-1524

IS - 20

ER -