Protein Kinase Cδ-Mediated Phosphorylation of Phospholipase D Controls Integrin-Mediated Cell Spreading

Integrin signaling plays critical roles in cell adhesion, spreading, and migration, and it is generally accepted that to regulate these integrin functions accurately, localized actin remodeling is required. However, the molecular mechanisms that control the targeting of actin regulation molecules to the proper sites are unknown. We previously demonstrated that integrin-mediated cell spreading and migration on fibronectin are dependent on the localized activation of phospholipase D (PLD). However, the mechanism underlying PLD activation by integrin is largely unknown. Here we demonstrate that protein kinase Cδ (PKCδ) is required for integrin-mediated PLD signaling. After integrin stimulation, PKCδ is activated and translocated to the edges of lamellipodia, where it colocalizes with PLD2. The abrogation of PKCδ activity inhibited integrin-induced PLD activation and cell spreading. Finally, we show that Thr566 of PLD2 is directly phosphorylated by PKCδ and that PLD2 mutation in this region prevents PLD2 activation, PLD2 translocation to the edge of lamellipodia, Rac translocation, and cell spreading after integrin activation. Together, these results suggest that PKCδ is a primary regulator of integrin-mediated PLD activation via the direct phosphorylation of PLD, which is essential for directing integrin-induced cell spreading.Integrin-mediated cell adhesion, spreading, and migration, which are essential for cellular differentiation, proliferation, survival, chemotaxis, and wound healing, require cell polarization with an environmental stimulus (32). To regulate these integrin-mediated functions accurately, coordinated and spatial control of localized cytoskeletal rearrangement is required. The key downstream signaling molecules of integrin-mediated actin cytoskeletal rearrangements include small GTPases of the Rho family, such as Rho, Cdc42, and Rac (57, 58). Recently it was suggested that integrin indirectly regulates the recruitment of small G proteins and their localized activation at a specific plasma membrane region called the cholesterol-enriched membrane microdomain. Furthermore, the membrane targeting of these molecules appears to be required for the activation of downstream effectors that induce actin reorganization (8, 9, 48). However, in the absence of integrin signaling, despite the GTP loading status, activated Rac and Cdc42 remain in the cytosol and cannot activate downstream effectors, such as p21-activated kinase (PAK) (8). This regulation of the localization of small GTPases to a specific site is supported by the observation that Rac1 is localized and activated at the leading edges of migrating cells, while Cdc42 is also activated in cellular protrusions and in the peripheral region (33, 51). The differentially localized activation of small GTPases results in coordinated spatially confined signaling leading to cytoskeletal rearrangement, which is critical for the regulation of integrin-mediated cell spreading and directional cell migration.The hydrolysis of phosphatidylcholine by phospholipase D1 (PLD1) and PLD2 generates the messenger lipid phosphatidic acid (PA) in response to a variety of signals, which include hormones, neurotransmitters, and growth factors (17). It has been shown that PA affects actin cytoskeletal rearrangement and hence lamellipodium extension and integrin-mediated cell spreading as well as migration. PLD activity has been found in detergent-insoluble membrane fractions in which a wide variety of cytoskeletal proteins, such as F-actin, α-actinin, vinculin, paxillin, and talin, were enriched (34). Furthermore, the stimulation of PLD with physiologic and pharmacologic agonists results in its association with actin filaments (34). In addition, actin polymerization and stress fiber formation are tightly coupled to the activation of PLD (14). The formation of lamellipodium structures and membrane ruffles is blocked by PLD inhibition (53, 60), and PLD activity is critical for epithelial cell, leukocyte, and neutrophil adhesion and migration (41, 43, 52). Furthermore, we have previously shown that the activity of PLD is upregulated, and that the activated PLD is translocated to lamellipodia, after integrin activation (3). The PLD product PA acts as a lipid anchor for the membrane translocation of Rac, and this PA-mediated localized activation of Rac is critical for integrin-mediated cell spreading and migration through Rac downstream signaling activation and actin cytoskeleton rearrangement (3). However, the mechanisms that regulate the activation and localization of PLD, which induce the localized downstream activation of integrin signaling, have not been elucidated.Members of the protein kinase C (PKC) family of serine-threonine kinases are known to play important roles in the transduction of signals from the activation of integrin to cell adhesion and spreading, as well as in cell migration via actin reorganization (11, 25, 61, 66). Several studies have shown that the activities of several PKC isozymes are modulated and are crucially required for integrin-mediated cell spreading and migration. The PKCα, -δ, and -ɛ isotypes were activated and then translocated from the cytosol to the membrane after integrin activation, and inhibition of these PKC isozymes prevented cell spreading (10, 47, 66). In addition, the activation of PKCα, -δ, and -ɛ rescued the spreading of α5 integrin-deficient cells on fibronectin (10), and PKCβΙ mediated platelet cell spreading and migration on fibrinogen (2). It has also been demonstrated that PKCθ activity is involved in endothelial cell migration (65). These results suggest that the kinase activities of diverse members of PKC are involved in the integrin-mediated signaling pathway leading to the actin cytoskeletal rearrangement required for cell spreading and migration. Several PKC substrates are known to influence the actin cytoskeleton directly (42). However, the natures of the isoform-specific functions of PKC members and of their specific downstream effectors for actin cytoskeletal rearrangement induction by integrin signaling remain to be elucidated.In this study, we found that PKCδ is an upstream modulator of localized PLD activation in the integrin signaling pathway. We demonstrate for the first time that PKCδ activity (not PKCα or PKCɛ activity) is critical for integrin-mediated PLD activation, and we found that PLD2 is phosphorylated at Thr566 by PKCδ in the integrin signaling pathway. Furthermore, we show that this phosphorylation is critical for integrin-mediated targeting of PLD to membrane ruffles, Rac translocation to the membrane, and lamellipodium formation during cell spreading. These findings strongly suggest a bridge between PKCδ and the signaling of actin cytoskeletal rearrangement by the integrin signaling pathway via PLD activation, and they provide a novel molecular mechanism for localized PLD activation via PKCδ phosphorylation, which is critical for the actin cytoskeletal rearrangements required for integrin-mediated cell spreading.     

Targeting and activation of Rac1 are mediated by the exchange factor beta-Pix

Rho guanosine triphosphatases (GTPases) are critical regulators of cytoskeletal dynamics and control complex functions such as cell adhesion, spreading, migration, and cell division. It is generally accepted that localized GTPase activation is required for the proper initiation of downstream signaling events, although the molecular mechanisms that control targeting of Rho GTPases are unknown. In this study, we show that the Rho GTPase Rac1, via a proline stretch in its COOH terminus, binds directly to the SH3 domain of the Cdc42/Rac activator beta-Pix (p21-activated kinase [Pak]-interacting exchange factor). The interaction with beta-Pix is nucleotide independent and is necessary and sufficient for Rac1 recruitment to membrane ruffles and to focal adhesions. In addition, the Rac1-beta-Pix interaction is required for Rac1 activation by beta-Pix as well as for Rac1-mediated spreading. Finally, using cells deficient for the beta-Pix-binding kinase Pak1, we show that Pak1 regulates the Rac1-beta-Pix interaction and controls cell spreading and adhesion-induced Rac1 activation. These data provide a model for the intracellular targeting and localized activation of Rac1 through its exchange factor beta-Pix.     

Diacylglycerol Kinase ζ Regulates Actin Cytoskeleton Reorganization through Dissociation of Rac1 from RhoGDI

Activation of Rac1 GTPase signaling is stimulated by phosphorylation and release of RhoGDI by the effector p21-activated kinase 1 (PAK1), but it is unclear what initiates this potential feed-forward mechanism for regulation of Rac activity. Phosphatidic acid (PA), which is produced from the lipid second messenger diacylglycerol (DAG) by the action of DAG kinases (DGKs), is known to activate PAK1. Here, we investigated whether PA produced by DGKζ initiates RhoGDI release and Rac1 activation. In DGKζ-deficient fibroblasts PAK1 phosphorylation and Rac1–RhoGDI dissociation were attenuated, leading to reduced Rac1 activation after platelet-derived growth factor stimulation. The cells were defective in Rac1-regulated behaviors, including lamellipodia formation, membrane ruffling, migration, and spreading. Wild-type DGKζ, but not a kinase-dead mutant, or addition of exogenous PA rescued Rac activation. DGKζ stably associated with PAK1 and RhoGDI, suggesting these proteins form a complex that functions as a Rac1-selective RhoGDI dissociation factor. These results define a pathway that links diacylglycerol, DGKζ, and PA to the activation of Rac1: the PA generated by DGKζ activates PAK1, which dissociates RhoGDI from Rac1 leading to changes in actin dynamics that facilitate the changes necessary for cell motility.     

B cell antigen receptor-induced Rac1 activation and Rac1-dependent spreading are impaired in transitional immature B cells due to levels of membrane cholesterol

The BCR-triggered responses of mature and transitional immature B cells differ at both the biochemical and functional level. In this study, we show that in mature B cells, BCR signaling triggers Vav phosphorylation and Rac1 activation. Furthermore, we demonstrate that although downstream actin-dependent BCR capping is independent of Rac1 activation, actin-dependent membrane ruffling and cell spreading are Rac1-dependent processes. In contrast, BCR-induced Vav phosphorylation and Rac1 activation is impaired in transitional immature B cells, resulting in defects in actin polymerization-dependent spreading and membrane ruffling while Rac1-independent BCR capping remains intact. Because transitional immature murine B cells maintain lower steady-state levels of plasma membrane cholesterol, we augmented their levels to that of mature B cells and found that BCR-induced Rac1 activation and Rac1-dependent membrane ruffling and cell spreading were restored. These studies provide a direct link between B cell cholesterol levels and downstream cellular signaling processes.     

v-Crk induces Rac-dependent membrane ruffling and cell migration in CAS-deficient embryonic fibroblasts

Crk-associated substrate (CAS) is a focal adhesion protein that is involved in integrin signaling and cell migration. CAS deficiency reduces the migration and spreading of cells, both of which are processes mediated by Rac activation. We examined the functions of v-Crk, the oncogene product of the CT10 virus p47gag-crk, which affects cell migration and spreading, membrane ruffling, and Rac activation in CAS-deficient mouse embryonic fibroblasts (CAS-/- MEFs). CAS-/- MEFs showed less spreading than did CAS+/+ MEFs, but spreading was recovered in mutant cells that expressed v-Crk (CAS-/-v-Crk MEF). We observed that the reduction in spreading was linked to the formation of membrane ruffles, which were accompanied by Rac activation. In CAS-/- MEFs, Rac activity was significantly reduced, and Rac was not localized to the membrane. In contrast, Rac was active and localized to the membrane in CAS-/-v-Crk MEFs. Lamellipodia protrusion and ruffle retraction velocities were both reduced in CAS-/- MEFs, but not in CAS-/-v-Crk MEFs. We also found that microinjection of anti-gag antibodies inhibited the migration of CAS-/-v-Crk MEFs. These findings indicate that v-Crk controls cell migration and membrane dynamics by activating Rac in CAS-deficient MEFs.     

Tyrosine 221 in Crk regulates adhesion-dependent membrane localization of Crk and Rac and activation of Rac signaling

The adaptor protein CrkII plays a central role in signal transduction cascades downstream of a number of different stimuli. We and others have previously shown that CrkII mediates attachment-induced JNK activation, membrane ruffling and cell motility in a Rac-dependent manner. We report here that cell attachment leads to tyrosine phosphorylation of CrkII on Y221, and that CrkII-Y221F mutant demonstrates enhanced association with the Crk-binding partners C3G and paxillin. Despite this enhanced signaling complex formation, CrkII-Y221F fails to induce JNK and PAK activation, membrane ruffling and cell migration, suggesting that it is defective in activating Rac signaling. Wild-type CrkII has no effect on adhesion-induced GTP loading of Rac, but its expression results in enhanced membrane localization of Rac, which is known to be required for Rac signaling. In contrast, CrkII-Y221F is deficient in enhancing membrane localization of Rac. Mutations in Rac and CrkII-Y221F that force membrane targeting of these molecules restore Rac signaling in adherent cells. Together, these results indicate that the Y221 site in CrkII regulates Rac membrane translocation upon cell adhesion, which is necessary for activation of downstream Rac signaling pathways.     

In vivo dynamics of Rac-membrane interactions

The small GTPase Rac cycles between the membrane and the cytosol as it is activated by nucleotide exchange factors (GEFs) and inactivated by GTPase-activating proteins (GAPs). Solubility in the cytosol is conferred by binding of Rac to guanine-nucleotide dissociation inhibitors (GDIs). To analyze the in vivo dynamics of Rac, we developed a photobleaching method to measure the dissociation rate constant (k(off)) of membrane-bound GFP-Rac. We find that k(off) is 0.048 s(-1) for wtRac and approximately 10-fold less (0.004 s(-1)) for G12VRac. Thus, the major route for dissociation is conversion of membrane-bound GTP-Rac to GDP-Rac; however, dissociation of GTP-Rac occurs at a detectable rate. Overexpression of the GEF Tiam1 unexpectedly decreased k(off) for wtRac, most likely by converting membrane-bound GDP-Rac back to GTP-Rac. Both overexpression and small hairpin RNA-mediated suppression of RhoGDI strongly affected the amount of membrane-bound Rac but surprisingly had only slight effects on k(off). These results indicate that RhoGDI controls Rac function mainly through effects on activation and/or membrane association.     

Phosphatidic acid signaling regulation of Ras superfamily of small guanosine triphosphatases

Phosphatidic acid (PA) has been increasingly recognized as an important signaling lipid regulating cell growth and proliferation, membrane trafficking, and cytoskeletal reorganization. Recent studies indicate that the signaling PA generated from phospholipase D (PLD) and diacylglycerol kinase (DGK) plays critical roles in regulating the activity of some members of Ras superfamily of small guanosine triphosphatases (GTPases), such as Ras, Rac and Arf. Change of PA levels regulates the activity of small GTPases by modulating membrane localization and activity of small GTPase regulatory proteins, guanine nucleotide exchange factors (GEFs) and GTPase activating proteins (GAPs). In addition, PA also targets some small GTPases to membranes by direct binding. This review summarizes the roles of PLD and DGK in regulating the activity of several Ras superfamily members and cellular processes they control. Some future directions and the implication of PA regulation of Ras small GTPases in pathology are also discussed.     

Suppression of RhoG activity is mediated by a syndecan 4–synectin–RhoGDI1 complex and is reversed by PKCα in a Rac1 activation pathway

Fibroblast growth factor 2 (FGF2) is a major regulator of developmental, pathological, and therapeutic angiogenesis. Its activity is partially mediated by binding to syndecan 4 (S4), a proteoglycan receptor. Angiogenesis requires polarized activation of the small guanosine triphosphatase Rac1, which involves localized dissociation from RhoGDI1 and association with the plasma membrane. Previous work has shown that genetic deletion of S4 or its adapter, synectin, leads to depolarized Rac activation, decreased endothelial migration, and other physiological defects. In this study, we show that Rac1 activation downstream of S4 is mediated by the RhoG activation pathway. RhoG is maintained in an inactive state by RhoGDI1, which is found in a ternary complex with synectin and S4. Binding of S4 to synectin increases the latter''s binding to RhoGDI1, which in turn enhances RhoGDI1''s affinity for RhoG. S4 clustering activates PKCα, which phosphorylates RhoGDI1 at Ser⁹⁶. This phosphorylation triggers release of RhoG, leading to polarized activation of Rac1. Thus, FGF2-induced Rac1 activation depends on the suppression of RhoG by a previously uncharacterized ternary S4–synectin–RhoGDI1 protein complex and activation via PKCα.     

Diacylglycerol kinase-alpha mediates hepatocyte growth factor-induced epithelial cell scatter by regulating Rac activation and membrane ruffling

Diacylglycerol kinases (Dgk) phosphorylate diacylglycerol (DG) to phosphatidic acid (PA), thus turning off and on, respectively, DG-mediated and PA-mediated signaling pathways. We previously showed that hepatocyte growth factor (HGF), vascular endothelial growth factor, and anaplastic lymphoma kinase activate Dgkalpha in endothelial and leukemia cells through a Src-mediated mechanism and that activation of Dgkalpha is required for chemotactic, proliferative, and angiogenic signaling in vitro. Here, we investigate the downstream events and signaling pathways regulated by Dgkalpha, leading to cell scatter and migration upon HGF treatment and v-Src expression in epithelial cells. We report that specific inhibition of Dgkalpha, obtained either pharmacologically by R59949 treatment, or by expression of Dgkalpha dominant-negative mutant, or by small interfering RNA-mediated down-regulation of endogenous Dgkalpha, impairs 1) HGF- and v-Src-induced cell scatter and migration, without affecting the loss of intercellular adhesions; 2) HGF-induced cell spreading, lamellipodia formation, membrane ruffling, and focal adhesions remodeling; and 3) HGF-induced Rac activation and membrane targeting. In summary, we provide evidence that Dgkalpha, activated downstream of tyrosine kinase receptors and Src, regulates crucial steps directing Rac activation and Rac-dependent remodeling of actin cytoskeleton and focal contacts in migrating epithelial cells.     

Scaffold Proteins IRSp53 and Spinophilin Regulate Localized Rac Activation by T-lymphocyte Invasion and Metastasis Protein 1 (TIAM1)

The Rac exchange factor Tiam1 is involved in diverse cell functions and signaling pathways through multiple protein interactions, raising the question of how signaling and functional specificity are achieved. We have shown that Tiam1 interactions with different scaffold proteins activate different Rac-dependent pathways by recruiting specific Rac effector proteins, and reasoned that there must be regulatory mechanisms governing each interaction. Fibroblasts express at least two Tiam1-interacting proteins, insulin receptor substrate protein 53 kDa (IRSp53) and spinophilin. We used fluorescent resonance energy transfer (FRET) to measure localized Rac activation associated with IRSp53 and spinophilin complexes in individual fibroblasts to test this hypothesis. Pervanadate or platelet-derived growth factor induced localized Rac activation dependent on Tiam1 and IRSp53. Forskolin or epinephrine induced localized Rac activation dependent on Tiam1 and spinophilin. In spinophilin-deficient cells, Tiam1 co-localized with IRSp53 in response to pervanadate or platelet-derived growth factor. In IRSp53-deficient cells, Tiam1 co-localized with spinophilin in response to forskolin or epinephrine. Total cellular levels of activated Rac were affected only in cells with exogenous Tiam1, and were primarily increased in the membrane fraction. Downstream effects of Rac activation were also stimulus and scaffold-specific. Cell ruffling, spreading, and cell adhesion were dependent on IRSp53, but not spinophilin. Epinephrine decreased IRSp53-dependent adhesion and increased cell migration in a Rac and spinophilin-dependent fashion. These results support the idea that Tiam1 interactions with different scaffold proteins couple distinct upstream signals to localized Rac activation and specific downstream pathways, and suggest that manipulating Tiam1-scaffold interactions can modulate Rac-dependent cellular behaviors.     

Deficiencies of the Lipid-Signaling Enzymes Phospholipase D1 and D2 Alter Cytoskeletal Organization,Macrophage Phagocytosis,and Cytokine-Stimulated Neutrophil Recruitment

Cell migration and phagocytosis ensue from extracellular-initiated signaling cascades that orchestrate dynamic reorganization of the actin cytoskeleton. The reorganization is mediated by effector proteins recruited to the site of activity by locally-generated lipid second messengers. Phosphatidic acid (PA), a membrane phospholipid generated by multiple enzyme families including Phospholipase D (PLD), has been proposed to function in this role. Here, we show that macrophages prepared from mice lacking either of the classical PLD isoforms PLD1 or PLD2, or wild-type macrophages whose PLD activity has been pharmacologically inhibited, display isoform-specific actin cytoskeleton abnormalities that likely underlie decreases observed in phagocytic capacity. Unexpectedly, PA continued to be detected on the phagosome in the absence of either isoform and even when all PLD activity was eliminated. However, a disorganized phagocytic cup was observed as visualized by imaging PA, F-actin, Rac1, an organizer of the F-actin network, and DOCK2, a Rac1 activator, suggesting that PLD-mediated PA production during phagocytosis is specifically critical for the integrity of the process. The abnormal F-actin reorganization additionally impacted neutrophil migration and extravasation from the vasculature into interstitial tissues. Although both PLD1 and PLD2 were important in these processes, we also observed isoform-specific functions. PLD1-driven processes in particular were observed to be critical in transmigration of macrophages exiting the vasculature during immune responses such as those seen in acute pancreatitis or irritant-induced skin vascularization.     

Role of the alpha(1) integrin cytoplasmic tail in the formation of focal complexes, actin organization, and in the control of cell migration

Integrins play a key role in cellular motility; an essential process for embryonic development and tissue morphogenesis, and also for pathological processes such as tumor cell invasion and metastasis. Recently, we showed that the cytoplasmic tail of integrin alpha(1) regulates the formation of focal complexes, F-actin cytoskeleton reorganization, and migration. We now report that the alpha(1) tail directly engages in collagen IV-mediated migration by regulation of the small GTPase Rac1. Deletion variants of the alpha(1) integrin differ in their ability to activate Rac1. Constitutively active Rac1 rescues motility in otherwise immotile cells expressing a truncated alpha(1) integrin without any cytoplasmic tail. In these cells, levels of GTP-Rac1 are constitutively elevated, but kept non-functional in the cytoplasm. The conserved GFFKR motif is sufficient to convey Rac1 activation, but downregulates the amount of GTP-Rac1 in the absence of the alpha(1)-specific sequence PLKKKMEK. This sequence is also required for the recruitment of PI3K to focal adhesions following Rac1 activation. Our results demonstrate that the short alpha(1) cytoplasmic tail is crucial for Rac1 activation and PI3K localization, which in turn results in cytoskeletal rearrangement and subsequent migration.     

Localization of Tie2 and phospholipase D in endothelial caveolae is involved in angiopoietin-1-induced MEK/ERK phosphorylation and migration in endothelial cells

Angiopoietin-1 (Ang1) and its receptor, Tie2, play critical roles in blood vessel formation. Ang1 triggers a variety of signaling events in endothelial cells leading to vasculogenic and angiogenic processes. However, the underlying mechanism for Ang1/Tie2 signaling is not fully understood. Here, we show that Tie2 and phospholipase D (PLD) are localized in the caveolae, specialized subdomains of the endothelial cell plasma membrane enriched with signaling molecules. Interestingly, Ang1 increased PLD activities in a dose- and time-dependent manner. Ang1-induced MEK/ERK activation was abrogated when PLD was inhibited, suggesting that PLD mediates Ang1-induced MEK/ERK activation. Moreover, PLD inhibitor, 1-butanol, inhibited Ang1-induced endothelial cell migration. Our results indicate that: (1) caveolae may be the platform for Tie2/PLD association in endothelial cells; (2) PLD is a new mediator of Ang1/Tie2-induced signaling pathway, and it participates in MAPK activation and endothelial cell migration.     

A palmitoylation switch mechanism regulates Rac1 function and membrane organization

The small GTPase Rac1 plays important roles in many processes, including cytoskeletal reorganization, cell migration, cell-cycle progression and gene expression. The initiation of Rac1 signalling requires at least two mechanisms: GTP loading via the guanosine triphosphate (GTP)/guanosine diphosphate (GDP) cycle, and targeting to cholesterol-rich liquid-ordered plasma membrane microdomains. Little is known about the molecular mechanisms governing this specific compartmentalization. We show that Rac1 can incorporate palmitate at cysteine 178 and that this post-translational modification targets Rac1 for stabilization at actin cytoskeleton-linked ordered membrane regions. Palmitoylation of Rac1 requires its prior prenylation and the intact C-terminal polybasic region and is regulated by the triproline-rich motif. Non-palmitoylated Rac1 shows decreased GTP loading and lower association with detergent-resistant (liquid-ordered) membranes (DRMs). Cells expressing no Rac1 or a palmitoylation-deficient mutant have an increased content of disordered membrane domains, and markers of ordered membranes isolated from Rac1-deficient cells do not correctly partition in DRMs. Importantly, cells lacking Rac1 palmitoylation show spreading and migration defects. These data identify palmitoylation as a mechanism for Rac1 function in actin cytoskeleton remodelling by controlling its membrane partitioning, which in turn regulates membrane organization.     

Regulation of Phospholipase D Activity and Phosphatidic Acid Production after Purinergic (P2Y6) Receptor Stimulation

Phosphatidic acid (PA) is a lipid second messenger located at the intersection of several lipid metabolism and cell signaling events including membrane trafficking, survival, and proliferation. Generation of signaling PA has long been primarily attributed to the activation of phospholipase D (PLD). PLD catalyzes the hydrolysis of phosphatidylcholine into PA. A variety of both receptor-tyrosine kinase and G-protein-coupled receptor stimulations have been shown to lead to PLD activation and PA generation. This study focuses on profiling the PA pool upon P2Y₆ receptor signaling manipulation to determine the major PA producing enzymes. Here we show that PLD, although highly active, is not responsible for the majority of stable PA being produced upon UDP stimulation of the P2Y₆ receptor and that PA levels are tightly regulated. By following PA flux in the cell we show that PLD is involved in an initial increase in PA upon receptor stimulation; however, when PLD is blocked, the cell compensates by increasing PA production from other sources. We further delineate the P2Y₆ signaling pathway showing that phospholipase Cβ3 (PLCβ3), PLCδ1, DGKζ and PLD are all downstream of receptor activation. We also show that DGKζ is a novel negative regulator of PLD activity in this system that occurs through an inhibitory mechanism with PKCα. These results further define the downstream events resulting in PA production in the P2Y₆ receptor signaling pathway.     

Tumor necrosis factor alpha stimulation of Rac1 activity. Role of isoprenylcysteine carboxylmethyltransferase

We have previously demonstrated that both isoprenylcysteine carboxylmethyltransferase (ICMT) and one of its substrates, the RhoGTPase Rac1, are critical for the tumor necrosis factor alpha (TNF alpha) stimulation of vascular cell adhesion molecule-1 expression in endothelial cells (EC). Here, we have shown that ICMT regulates TNF alpha stimulation of Rac1 activity. TNF alpha stimulation of EC increased the membrane association of Rac1, an event that is essential for Rac1 activity. ICMT inhibitor N-acetyl-S-farnesyl-L-cysteine (AFC) blocked the accumulation of Rac1 into the membrane both in resting and TNF alpha-stimulated conditions. Similarly, the membrane-associated Rac1 was lower in Icmt-deficient versus wild-type mouse embryonic fibroblasts (MEFs). TNF alpha also increased the level of GTP-Rac1, the active form of Rac1, in EC. AFC completely suppressed the TNF alpha stimulation of increase in GTP-Rac1 levels. Confocal microscopy revealed resting EC Rac1 was present in the plasma membrane and also in the perinuclear region. AFC mislocalized Rac1, both from the plasma membrane and the perinuclear region. Mislocalization of Rac1 was also observed in Icmt-deficient versus wild-type MEFs. To determine the consequences of ICMT inhibition, we investigated the effect of AFC on p38 mitogen-activated protein (MAP) kinase phosphorylation, which is downstream of Rac1. AFC inhibited the TNF alpha stimulation of p38 MAP kinase phosphorylation in EC. TNF alpha stimulation of p38 MAP kinase phosphorylation was also significantly attenuated in Icmt-deficient versus wild-type MEFs. To understand the mechanism of inhibition of Rac1 activity, we examined the effect of ICMT inhibition on the interaction of Rac1 with its inhibitor, Rho guanine nucleotide dissociation inhibitor (RhoGDI). The association of Rac1 with its inhibitor RhoGDI was dramatically increased in the Icmt-deficient versus wild-type MEFs both in resting as well as in TNF alpha-stimulated conditions, suggesting that RhoGDI was involved in inhibiting Rac1 activity under the conditions of ICMT inhibition. These results suggest that ICMT regulates Rac1 activity by controlling the interaction of Rac1 with RhoGDI. We hypothesize that ICMT regulates the release of Rac1 from RhoGDI.     

Cell migration is negatively modulated by ABCA1

Temporal and spatial changes of membrane lipid distribution in the plasma membrane are thought to be important for various cellular functions. ATP-Binding Cassette A1 (ABCA1) is a key lipid transporter for the generation of high density lipoprotein. Recently, we reported that ABCA1 maintains an asymmetric distribution of cholesterol in the plasma membrane. Here we report that ABCA1 suppresses cell migration by modulating signal pathways. ABCA1 knockdown in mouse embryonic fibroblasts accelerated cell migration and increased activation of Rac1 and its localization to detergent-resistant membranes. Phosphorylation of MEK and ERK also increased. Inhibition of Rac1 or MEK-ERK signals suppressed cell migration in ABCA1 knockdown cells. Because our experimental conditions for cell migration did not contain cholesterol or lipid acceptors for ABCA1, cellular cholesterol content was not changed. These data suggest that ABCA1 modulates cell migration via Rac1 and MEK-ERK signaling by altering lipid distribution in the plasma membrane.     

Yersinia pseudotuberculosis spatially controls activation and misregulation of host cell Rac1

Yersinia pseudotuberculosis binds host cells and modulates the mammalian Rac1 guanosine triphosphatase (GTPase) at two levels. Activation of Rac1 results from integrin receptor engagement, while misregulation is promoted by translocation of YopE and YopT proteins into target cells. Little is known regarding how these various factors interplay to control Rac1 dynamics. To investigate these competing processes, the localization of Rac1 activation was imaged microscopically using fluorescence resonance energy transfer. In the absence of translocated effectors, bacteria induced activation of the GTPase at the site of bacterial binding. In contrast, the entire cellular pool of Rac1 was inactivated shortly after translocation of YopE RhoGAP. Inactivation required membrane localization of Rac1. The translocated protease YopT had very different effects on Rac1. This protein, which removes the membrane localization site of Rac1, did not inactivate Rac1, but promoted entry of cleaved activated Rac1 molecules into the host cell nucleus, allowing Rac1 to localize with nuclear guanosine nucleotide exchange factors. As was true for YopE, membrane-associated Rac1 was the target for YopT, indicating that the two translocated effectors may compete for the same pool of target protein. Consistent with the observation that YopE inactivation requires membrane localization of Rac1, the presence of YopT in the cell interfered with the action of the YopE RhoGAP. As a result, interaction of target cells with a strain that produces both YopT and YopE resulted in two spatially distinct pools of Rac1: an inactive cytoplasmic pool and an activated nuclear pool. These studies demonstrate that competition between bacterial virulence factors for access to host substrates is controlled by the spatial arrangement of a target protein. In turn, the combined effects of translocated bacterial proteins are to generate pools of a single signaling molecule with distinct localization and activation states in a single cell.     

Phosphorylation of EBP50 negatively regulates β-PIX-dependent Rac1 activity in anoikis

We demonstrated a protein kinase C (PKC)-dependent phosphorylation of canine ezrin/radixin/moesin (ERM)-binding phosphoprotein 50 (EBP50) at serine 347/348 by site-directed mutagenesis and a phospho-specific antibody. Cell fractionation and confocal imaging revealed the relocation of EBP50 from the plasma membrane to cytosol that accompanied this phosphorylation event. Increased phosphorylation at these serine residues led to the dissociation of EBP50 from ezrin and β-PIX, which are two upstream regulators of Rac1 activation. Cells overexpressing an EBP50 mutant, mimicking serine 347/348 phosphorylation, became refractory to hepatocyte growth factor-induced cell spreading and scattering, which is normally mediated by Rac1 activation. Detachment of cells from the substratum also elicited an increase in EBP50 phosphorylation, apparently due to counteracting activities of PKC and protein phosphastase 2A, which resulted in decreased Rac1 activation and induction of anoikis. Cells overexpressing an EBP50 mutant defective in serine 347/348 phosphorylation did not undergo apoptosis in suspension culture. These studies reveal a signaling cascade in which different phosphorylation states and subcellular localization of EBP50 regulate Rac1 function.