Identification of a novel, putative Rho-specific GDP/GTP exchange factor and a RhoA-binding protein: control of neuronal morphology.

The small GTP-binding protein Rho has been implicated in the control of neuronal morphology. In N1E-115 neuronal cells, the Rho-inactivating C3 toxin stimulates neurite outgrowth and prevents actomyosin-based neurite retraction and cell rounding induced by lysophosphatidic acid (LPA), sphingosine-1-phosphate, or thrombin acting on their cognate G protein-coupled receptors. We have identified a novel putative GDP/GTP exchange factor, RhoGEF (190 kD), that interacts with both wild-type and activated RhoA, but not with Rac or Cdc42. RhoGEF, like activated RhoA, mimics receptor stimulation in inducing cell rounding and in preventing neurite outgrowth. Furthermore, we have identified a 116-kD protein, p116(Rip), that interacts with both the GDP- and GTP-bound forms of RhoA in N1E-115 cells. Overexpression of p116(Rip) stimulates cell flattening and neurite outgrowth in a similar way to dominant-negative RhoA and C3 toxin. Cells overexpressing p116(Rip) fail to change their shape in response to LPA, as is observed after Rho inactivation. Our results indicate that (a) RhoGEF may link G protein-coupled receptors to RhoA activation and ensuing neurite retraction and cell rounding; and (b) p116(Rip) inhibits RhoA-stimulated contractility and promotes neurite outgrowth.

Properties and functions of diacylglycerol kinases.

Diacylglycerol kinases (DGKs) phosphorylate the second-messenger diacylglycerol (DAG) to phosphatidic acid (PA). The family of DGKs is well conserved among most species. Nine mammalian isotypes have been identified, and are classified into five subgroups based on their primary structure. DGKs contain a conserved catalytic domain and an array of other conserved motifs that are likely to play a role in lipid-protein and protein-protein interactions in various signalling pathways dependent on DAG and/or PA production. DGK is therefore believed to be activated at the (plasma) membrane where DAG is generated. Some isotypes are found associated with and/or regulated by small GTPases of the Rho family, presumably acting in cytoskeletal rearrangements. Others are (also) found in the nucleus, in association with other regulatory enzymes of the phosphoinositide cycle, and have an effect on cell cycle progression. Most DGK isotypes show high expression in the brain, often in distinct brain regions, suggesting that each individual isotype has a unique function.

Diacylglycerol kinases in signal transduction.

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol (DAG) to phosphatidic acid. A family of nine mammalian isotypes have been identified. Their primary structure shows a diverse array of conserved domains, such as a catalytic domain, zinc fingers, pleckstrin homology domains and EF-hand structures, known to interact with other proteins, lipids or Ca2+, in signal transduction processes. DGK is believed to act in the phosphoinositide cycle in which DAG is enriched with arachidonoyl moieties, but the majority of DGK isotypes do not show specificity for this DAG species in vitro. This could imply that DGKs may also have other functions in the cell. DGK activity is not only found in membranes, but also in the nucleus and at the cytoskeleton. Agonist-induced translocations of DGK to or from these subcellular sites are known to occur. Some isotypes are contained in signaling complexes in specific association with members of the Rho family of small GTP binding proteins, suggesting that they are involved in Rho-mediated processes such as cytoskeletal reorganization.

Diacylglycerol kinase theta binds to and is negatively regulated by active RhoA.

Diacylglycerol kinase (DGK) phosphorylates the second messenger diacylglycerol to yield phosphatidic acid. To date, very little is known about the regulation of DGK activity. We have previously identified the DGKtheta isotype, which is predominantly expressed in brain (Houssa, B., Schaap, D., van der Wal, J., Goto, K., Kondo, H., Yamakawa, A., Shibata, M., Takenawa, T., and Van Blitterswijk, W. J. (1997) J. Biol. Chem. 272, 10422-10428). We now report that DGKtheta binds specifically to activated RhoA in transfected COS cells as well as in nontransfected neuronal N1E-115 cells. Binding is abolished by a point mutation (Y34N) in the effector loop of RhoA. DGKtheta does not bind to inactive RhoA, nor to the other Rho-family GTPases, Rac or Cdc42. Like active RhoA, DGKtheta localizes to the plasma membrane. Strikingly, the binding of activated RhoA to DGKtheta completely inhibits DGK catalytic activity. Our results suggest that DGKtheta is a downstream effector of RhoA and that its activity is negatively regulated by RhoA. Through accumulation of newly produced diacylglycerol, RhoA-mediated inhibition of DGKtheta may lead to enhanced PKC activity in response to external stimuli.

Diacylglycerol kinase theta is translocated and phosphoinositide 3-kinase-dependently activated by noradrenaline but not angiotensin II in intact small arteries.

Diacylglycerol (DG) kinase (DGK) phosphorylates the lipid second messenger DG to phosphatidic acid. We reported previously that noradrenaline (NA), but not angiotensin II (AII), increases membrane-associated DGK activity in rat small arteries [Ohanian and Heagerty (1994) Biochem. J. 300, 51-56]. Here, we have identified this DGK activity as DGKtheta, present in both smooth muscle and endothelial cells of these small vessels. Subcellular fractionation of artery homogenates revealed that DGKtheta was present in nuclear, plasma membrane (and/or Golgi) and cytosolic fractions. Upon NA stimulation, DGKtheta translocated towards the membrane and cytosol (155 and 153% increases relative to the control, respectively) at 30 s, followed by a return to near-basal levels at 5 min; AII was without effect. Translocation to the membrane was to both Triton-soluble and -insoluble fractions. NA, but not AII, transiently increased DGKtheta activity in immunoprecipitates (126% at 60 s). Membrane translocation and DGKtheta activation were regulated differently: NA-induced DGKtheta activation, but not translocation, was dependent on transient activation of phosphoinositide 3-kinase (PI 3-K). In addition, DGK activity co-immunoprecipitated with protein kinase B, a downstream effector of PI 3-K, and was increased greatly by NA stimulation. The rapid and agonist-specific activation of DGKtheta suggests that this pathway may have a physiological role in vascular smooth-muscle responses.

Cloning of a novel human diacylglycerol kinase (DGKtheta) containing three cysteine-rich domains, a proline-rich region, and a pleckstrin homology domain with an overlapping Ras-associating domain.

Diacylglycerol kinase (DGK) attenuates levels of second messenger diacylglycerol in cells and produces another (putative) messenger, phosphatidic acid. We have previously purified a 110-kDa DGK from rat brain (Kato, M., and Takenawa, T. (1990) J. Biol. Chem. 265, 794-800). Here we report the cDNA cloning from human brain and retina cDNA libraries. The cDNA encodes a novel DGK isotype, termed DGKtheta, of 941 amino acids with an apparent molecular mass of 110 kDa. DGKtheta contains a C-terminal putative catalytic domain, which is present in all eukaryotic DGKs. In contrast to other DGK isotypes, DGKtheta contains three cysteine-rich domains instead of two. The third cysteine-rich domain is most homologous to the second one in other DGK isotypes. This particular sequence homology extends C-terminally beyond the typical cysteine/histidine core structure and is DGK-specific. DGKtheta furthermore contains various domains for protein-protein interaction, such as a proline- and glycine-rich domain with a putative SH3 domain-binding site and a pleckstrin homology domain with an overlapping Ras-associating domain. DGKtheta is expressed in the brain and, to a lesser extent, in the small intestine, duodenum, and liver. In situ hybridization of DGKtheta mRNA in adult rat brain reveals high expression in the cerebellar cortex and hippocampus. DGKtheta activity in COS cell lysates is optimal toward diacylglycerols containing an unsaturated fatty acid at the sn-2 position.