Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids
ReviewPhosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases
Introduction
Phosphatidic acid (PA) has been long proposed as a pleiotropic bioactive lipid regulating cell growth and proliferation, membrane trafficking, and cytoskeletal reorganization [1], [2]. The cellular signaling PA is produced through two major pathways: the hydrolysis of phosphatidylcholine (PC) by phospholipase D (PLD), and the phosphorylation of diacylglycerol (DAG) by diacylglycerol kinase (DGK).
The mammalian PLD family consists of two related gene products, PLD1 and PLD2 [3], [4], [5], [6]. PLD1 is directly regulated by classic protein kinase C (PKC) and members of the ADP-ribosylation factor (Arf) and Rho family small GTPases in conjunction with phosphatidylinositol-4,5-bisphosphate (PI(4,5)P2) [7]. In contrast to PLD1, PLD2 is constitutively active in the presence of PI(4,5)P2 and localized to the plasma membrane in most cell types [8], [9]. Subsequent studies have shown that PLD2 can also be activated in intact cells by agonists and can be regulated by small GTPases and PKC isoforms [5], [10], [11].
DGK family is defined by a conserved catalytic domain (DAGKc) located at the C-terminal half of the protein, and comprises 10 distinct enzymes [12], [13]. Based on the distinct regulatory domains, DGKs can be grouped into five subtypes: type I DGK (α, β and γ), type II DGK (δ, η and κ), type III DGK (ɛ), type IV DGK (ζ and ι), and type V DGK (θ) [12], [14]. Whereas all DGK catalyzes the same reaction, the presence of non-conserved regulatory regions appears to confer specificity to the distinct DGK isoforms by restricting their subcellular site of action and/or defining their activation mechanism [12], [14]. By regulating the level of both DAG and PA in a reciprocal manner, DGK can potentially act as terminators of DAG-mediated signals as well as activators of PA-mediated signals [12], [14].
The activity of both PLD and DGK is tightly regulated by external stimuli. However, compared to phosphatidylinositides and DAG, the signaling role of PA has not been fully appreciated until recently. PA is generally believed to be less stable, and is metabolized to DAG by phosphatidic acid phosphatase (PAP). In the studies published in the 1980s and early 1990s, the major role assigned to PLD and DGK was the control of intracellular DAG level, and many biological effects of modulating PLD and DGK activities were interpreted as a result of PKC overstimulation [14]. Recent studies by us and other groups have demonstrated that PA directly regulates the activities of some key signaling molecules, such as mTOR, Ras, and myosin II [9], [15], [16], [17], suggesting that PA itself is an important signaling lipid in cells.
The Ras superfamily of small guanosine triphosphatases (GTPases) contains over 150 human members. Based on sequence and functional similarities, the Ras superfamily of small GTPases is divided into at least five distinct branches: Ras, Rho, Rab, Arf, and Ran [18], [19]. Upon extracellular stimulation, activation of Ras-GTPases controls a diversity of downstream cytoplasmic signaling cascades and cellular functions. These small GTPases share a common biochemical mechanism and act as binary molecular switches. They exhibit high-affinity binding for GDP and GTP, and possess low intrinsic GTP hydrolysis and GDP/GTP exchange activities. GDP/GTP binding is controlled by two main classes of regulatory proteins: guanine nucleotide exchange factors (GEFs) promote formation of the active, GTP-bound form, whereas GTPase activating proteins (GAPs) accelerate the intrinsic GTPase activity to promote formation of the inactive GDP-bound form [18], [19]. The fine regulation of Ras superfamily of small GTPases facilitates their key involvement in a widely diverse spectrum of biochemical and biological processes.
Earlier studies on PLD mainly focused on the regulation of PLD activity by small GTPases. Recent studies have shown that PLD and DGK also act upstream to regulate the activities of some small GTPases. We have recently reported that PA directly binds to the pleckstrin homology (PH) domain of Sos, a Ras GEF, and thus recruits Sos to the plasma membrane and converts Ras-GDP to Ras-GTP [17]. This and other similar findings in the literature have suggested that regulation of GEFs and GAPs by PA metabolism might be a common mechanism for controlling the activities of small GTPases in response to signal stimulation. The source of PA and its relative contribution to the activity of individual small GTPases in a particular cellular context, however, remain to be further investigated. We have summarized the regulation of some members of Ras superfamily by PA and PA-metabolizing enzymes in Table 1.
Section snippets
Ras activation
Ras activation is tightly regulated by guanine nucleotide exchange factors (GEFs). To date, four subfamilies of GEF molecules have been identified: Sos, RasGRF, RasGRP and CNRasGEF [18], [19]. The ubiquitously expressed Sos signals downstream of receptor tyrosine kinases (RTKs). RasGRF proteins are expressed predominantly in the central nervous system. The family of RasGRP proteins is expressed primarily in haematopoietic cell lineages and function downstream of non-receptor tyrosine kinases
PLD and DGK regulation of Rac1
Rho family GTPases are key regulators of cytoskeletal dynamics and affect many cellular processes, including cell polarity, migration, vesicle trafficking and cytokinesis [38], [39]. Most of the functional information on Rho family proteins has come from studies of RhoA, Rac1 and Cdc42. In addition to Ras, some recent publications have suggested that PA regulates the activity of Rac1 small GTPase, which is well known for its activity in promoting actin polymerization and the formation of
PA regulation of Arf small GTPases
The Arf proteins are best known for their role in membrane trafficking, and also contribute significantly to the regulation of actin cytoskeletal organization. The significance of PA regulation of Arf activity has not been tested in any cellular context yet. However, PA, in cooperation with phosphoinositides, is an extremely potent activator for the activity of several Arf GAP proteins in vitro. The activity of ASAP1, an Arf GAP protein, is stimulated about 10, 000 fold by PI(4,5)P2 and PA. PA
Future perspectives
Recent results have shown specific and direct interactions between PA and PA-binding proteins [1], [2]. These types of interactions involve binding of PA to a positively charged site on a PA-binding protein, such as Raf-1, SHP-1 and mTOR [1], [2]. However, the primary structures of these PA-binding regions reveal no significant homology. They are diverse and include sequences that had not previously been described as lipid binding domains. Future analysis of the tertiary structures of
Acknowledgements
The authors thank Dr. Aimalohi Esechie for her comments on the manuscript. This work was supported by a research grant from National Institutes of Health to GD (GM071475).
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