Diacylglycerol Kinase θ Binds to and Is Negatively Regulated by Active RhoA

Nine diacylglycerol kinase (DGK) isozymes have been identified. However, our knowledge of their individual functions is still limited. Here, we demonstrate the role of DGK␥ in regulating Rac1-governed cell morphology. We found that the expression of kinase-dead DGK␥, which acts as a dominant-negative mutant, and inhibition of endogenous DGK␥ activity with R59949 induced lamellipodium and membrane ruffle formation in NIH3T3 fibroblasts in the absence of growth factor stimulation. Reciprocally, lamellipodium formation induced by plateletderived growth factor was significantly inhibited upon expression of constitutively active DGK␥. Moreover, the constitutively active DGK␥ mutant suppressed integrinmediated cell spreading. These effects are isoform-specific because, in the same experiments, none of the corresponding mutants of DGK␣ and DGK␤, closely related isoforms, affected cell morphology. These results suggest that DGK␥ specifically participates in the Rac1-mediated signaling pathway leading to cytoskeletal reorganization. In support of this, DGK␥ co-localized with dominant-active Rac1 especially in lamellipodia. Moreover, we found that endogenous DGK␥ was physically associated with cellular Rac1. Dominant-negative Rac1 expression blocked the lamellipodium formation induced by kinase-dead DGK␥, indicating that DGK␥ acts upstream of Rac1. This model is supported by studies demonstrating that kinase-dead DGK␥ selectively activated Rac1, but not Cdc42. Taken together, these results strongly suggest that DGK␥ functions through its catalytic action as an upstream suppressor of Rac1 and, consequently, lamellipodium/ruffle formation.It is well recognized that a variety of lipid second messengers in low abundance carry out specific tasks for a wide range of biological processes in eukaryotic cells. The cellular concentrations of such signaling lipids must be strictly regulated by the action of metabolic enzymes. Diacylglycerol kinase (DGK) 1 phosphorylates diacylglycerol (DAG) to yield phosphatidic acid (PA) (1). DAG is an established activator of conventional and novel protein kinases C (PKCs), Unc-13, and Ras guanyl nucleotide-releasing protein (2-6). PA has also been reported to regulate a number of signaling proteins such as phosphatidylinositol-4-phosphate 5-kinase, Ras GTPase-activating protein, Raf-1 kinase, atypical PKC, and chimaerins (6 -8). Moreover, specific PA-binding sites have recently been identified in several important proteins such as Raf-1 (9), mTOR (mammalian target of rapamycin) (10), and p47 phox (11). Thus, DGK can potentially participate in a diverse range of cellular events through modulating the balance between two bioactive lipids, DAG and PA.Mammalian DGK is known to exist as a large protein family consisting of nine isozymes classified into five subtypes according to their structural features (12-15). These subfamilies can be characterized by the presence of a variety of regulatory domains of known and/or predicted functions, clearly indicating their distinct functions and regulatory mecha...

Section: Fig 9 Negative Regulation Of Rac1 Activity By Dgk␥mentioning

confidence: 99%

Diacylglycerol Kinase γ Serves as an Upstream Suppressor of Rac1 and Lamellipodium Formation

Tsushima

Kai

Yamada

et al. 2004

“…In this study we show that YpkAs RhoA binding and enzymatic activities appear to be independent of one another. YpkAs RhoA-independent kinase activity sets it apart from other RhoA-binding kinases in which RhoA binding appears to modulate, either positively or negatively, enzymatic activity (30,32,38). RhoA binding is also thought to function to localize RhoAbinding kinases to the plasma membrane apparently in order to place them near their substrates (39 -41).…”

Section: Fig 6 Yeast Two-hybrid Assaysmentioning

confidence: 99%

The Yersinia Protein Kinase A Is a Host Factor Inducible RhoA/Rac-binding Virulence Factor

Dukuzumuremyi

Rosqvist

Hållberg

et al. 2000

103

112

The pathogenic yersiniae inject proteins directly into eukaryotic cells that interfere with a number of cellular processes including phagocytosis and inflammatory-associated host responses. One of these injected proteins, the Yersinia protein kinase A (YpkA), has previously been shown to affect the morphology of cultured eukaryotic cells as well as to localize to the plasma membrane following its injection into HeLa cells. Here it is shown that these activities are mediated by separable domains of YpkA. The amino terminus, which contains the kinase domain, is sufficient to localize YpkA to the plasma membrane while the carboxyl terminus of YpkA is required for YpkAs morphological effects. YpkAs carboxyl-terminal region was found to affect the levels of actin-containing stress fibers as well as block the activation of the GTPase RhoA in Yersinia-infected cells. We show that the carboxyl-terminal region of YpkA, which contains sequences that bear similarity to the RhoAbinding domains of several eukaryotic RhoA-binding kinases, directly interacts with RhoA as well as Rac (but not Cdc42) and displays a slight but measurable binding preference for the GDP-bound form of RhoA. Surprisingly, YpkA binding to RhoA GDP affected neither the intrinsic nor guanine nucleotide exchange factor-mediated GDP/GTP exchange reaction suggesting that YpkA controls activated RhoA levels by a mechanism other than by simply blocking guanine nucleotide exchange factor activity. We go on to show that YpkAs kinase activity is neither dependent on nor promoted by its interaction with RhoA and Rac but is, however, entirely dependent on heat-sensitive eukaryotic factors present in HeLa cell extracts and fetal calf serum. Collectively, our data show that YpkA possesses both similarities and differences with the eukaryotic RhoA/Rac-binding kinases and suggest that the yersiniae utilize the Rho GTPases for unique activities during their interaction with eukaryotic cells.

“…The lipid kinase family of DGKs catalyzes the conversion of diacylglycerol to phosphatidic acid and thereby regulate events downstream of these two lipid signals in part by antagonizing PKC activation by diacylglycerol (44,45). DGKQ activity in turn is inhibited by GTP-bound RhoA (46). Although the connection between the knockdown of DGKQ and ␣-synuclein expression is presently unclear, it is tempting to speculate that DGKQ activity might repress RhoA-mediated transcriptional up-regulation of SNCA mRNA by GATA transcription factors (47,48), possibly through PKC inhibition.…”

Section: Volume 287 • Number 40 • September 28 2012mentioning

confidence: 99%

Novel One-step Immunoassays to Quantify α-Synuclein

Bidinosti

Shimshek

Mollenhauer

et al. 2012

Background: Robust assays for ␣-synuclein quantification are essential for Parkinson disease therapeutic development. Results: TR-FRET immunoassays were validated for total and oligomeric ␣-synuclein and used to screen small molecules and kinases that regulate ␣-synuclein expression. Conclusion: TR-FRET immunoassays are suitable for biomarker development and high-throughput screening. Significance: This is the first platform for large-scale drug discovery and for neuronal pathway analysis of ␣-synuclein expression regulation.