Scaffold association factor B (SAFB) is required for expression of prenyltransferases and RAS membrane association.

Inhibiting membrane association of RAS has long been considered a rational approach to anticancer therapy, which led to the development of farnesyltransferase inhibitors (FTIs). However, FTIs proved ineffective against KRAS-driven tumors. To reveal alternative therapeutic strategies, we carried out a genome-wide CRISPR-Cas9 screen designed to identify genes required for KRAS4B membrane association. We identified five enzymes in the prenylation pathway and SAFB, a nuclear protein with both DNA and RNA binding domains. Silencing SAFB led to marked mislocalization of all RAS isoforms as well as RAP1A but not RAB7A, a pattern that phenocopied silencing FNTA, the prenyltransferase α subunit shared by farnesyltransferase and geranylgeranyltransferase type I. We found that SAFB promoted RAS membrane association by controlling FNTA expression. SAFB knockdown decreased GTP loading of RAS, abrogated alternative prenylation, and sensitized RAS-mutant cells to growth inhibition by FTI. Our work establishes the prenylation pathway as paramount in KRAS membrane association, reveals a regulator of prenyltransferase expression, and suggests that reduction in FNTA expression may enhance the efficacy of FTIs.

Molecular basis for autoinhibition of RIAM regulated by FAK in integrin activation.

RAP1-interacting adapter molecule (RIAM) mediates RAP1-induced integrin activation. The RAS-association (RA) segment of the RA-PH module of RIAM interacts with GTP-bound RAP1 and phosphoinositol 4,5 bisphosphate but this interaction is inhibited by the N-terminal segment of RIAM. Here we report the structural basis for the autoinhibition of RIAM by an intramolecular interaction between the IN region (aa 27-93) and the RA-PH module. We solved the crystal structure of IN-RA-PH to a resolution of 2.4-Å. The structure reveals that the IN segment associates with the RA segment and thereby suppresses RIAM:RAP1 association. This autoinhibitory configuration of RIAM can be released by phosphorylation at Tyr45 in the IN segment. Specific inhibitors of focal adhesion kinase (FAK) blocked phosphorylation of Tyr45, inhibited stimulated translocation of RIAM to the plasma membrane, and inhibited integrin-mediated cell adhesion in a Tyr45-dependent fashion. Our results reveal an unusual regulatory mechanism in small GTPase signaling by which the effector molecule is autoinhibited for GTPase interaction, and a modality of integrin activation at the level of RIAM through a FAK-mediated feedforward mechanism that involves reversal of autoinhibition by a tyrosine kinase associated with integrin signaling.

Rap1 and its effector RIAM are required for lymphocyte trafficking.

Regulation of integrins is critical for lymphocyte adhesion to endothelium and trafficking through secondary lymphoid organs. Inside-out signaling to integrins is mediated by the small GTPase Rap1. Two effectors of Rap1 regulate integrins, RapL and Rap1 interacting adaptor molecule (RIAM). Using mice conditionally deficient in both Rap1a and Rap1b and mice null for RIAM, we show that the Rap1/RIAM module is not required for T- or B-cell development but is essential for efficient adhesion to intercellular adhesion molecule (ICAM) 1 and vascular cell adhesion molecule (VCAM) 1 and for proper trafficking of lymphocytes to secondary lymphoid organs. Interestingly, in RIAM-deficient mice, whereas peripheral lymph nodes (pLNs) were depleted of both B and T cells and recirculating B cells were diminished in the bone barrow (BM), the spleen was hypercellular, albeit with a relative deficiency of marginal zone B cells. The abnormality in lymphocyte trafficking was accompanied by defective humoral immunity to T-cell-dependent antigens. Platelet function was intact in RIAM-deficient animals. These in vivo results confirm a role for RIAM in the regulation of some, but not all, leukocyte integrins and suggest that RIAM-regulated integrin activation is required for trafficking of lymphocytes from blood into pLNs and BM, where relatively high shear forces exist in high endothelial venules and sinusoids, respectively.

Dielectric relaxations of poly(N-isopropylacrylamide) microgels near the volume phase transition temperature: impact of cross-linking density distribution on the volume phase transition.

Dielectric relaxation behaviors of three types of thermally sensitive poly(N-isopropylacrylamide) (PNIPAM) microgels with different cross-linking density distributions were investigated in a frequency range from 40 Hz to 110 MHz at temperatures from 15 °C to 55 °C. After eliminating the electrode polarization at low frequency, two remarkable relaxations were observed, one in the kHz frequency range and the other in the MHz range. The low-frequency relaxation is attributed to the counterion polarization in the whole measuring temperature range, while the relaxation at high-frequency is probably dominated by different polarization mechanisms depending on below or above the volume phase transition temperature (VPTT): it is considered as micro-Brownian motion of side groups of PNIPAM when T < VPTT and interfacial polarization when T > VPTT. The temperature dependence of the dielectric parameters for both the relaxations presents an abrupt change around 32.5 °C, indicating the occurrence of phase transition. Based on the analysis and discussion about the micro-Brownian motion of the side groups, a possible microstructure for the microgels before and after the collapse of PNIPAM was suggested. A dielectric model to describe the collapsing microgel suspension was proposed, from which the electrical and structural parameters of the suspension were calculated. The information on the internal structure and hydration dynamic behavior of microgels was obtained by using the thermodynamic parameters which were calculated based on the Eyring equation. Our results reveal that the spatial distribution of the cross-linking density distribution has almost no effect on the volume phase transition temperature, but markedly affects the swelling capacity of PNIPAM microgels at low temperatures.

Genome-wide identification of the WRKY gene family in Camellia oleifera and expression analysis under phosphorus deficiency.

Camellia oleifera Abel. is an economically important woody edible-oil species that is mainly cultivated in hilly areas of South China. The phosphorus (P) deficiency in the acidic soils poses severe challenges for the growth and productivity of C. oleifera. WRKY transcription factors (TFs) have been proven to play important roles in biological processes and plant responses to various biotic/abiotic stresses, including P deficiency tolerance. In this study, 89 WRKY proteins with conserved domain were identified from the C. oleifera diploid genome and divided into three groups, with group II further classified into five subgroups based on the phylogenetic relationships. WRKY variants and mutations were detected in the gene structure and conserved motifs of CoWRKYs. Segmental duplication events were considered as the primary driver in the expanding process of WRKY gene family in C. oleifera. Based on transcriptomic analysis of two C. oleifera varieties characterized with different P deficiency tolerances, 32 CoWRKY genes exhibited divergent expression patterns in response to P deficiency stress. qRT-PCR analysis demonstrated that CoWRKY11, -14, -20, -29 and -56 had higher positive impact on P-efficient CL40 variety compared with P-inefficient CL3 variety. Similar expression trends of these CoWRKY genes were further observed under P deficiency with longer treatment period of 120d. The result indicated the expression sensitivity of CoWRKYs on the P-efficient variety and the C. oleifera cultivar specificity on the P deficiency tolerance. Tissue expression difference showed CoWRKYs may play a crucial role in the transportation and recycling P in leaves by affecting diverse metabolic pathways. The available evidences in the study conclusively shed light on the evolution of the CoWRKY genes in C. oleifera genome and provided a valuable resource for further investigation of functional characterization of WRKY genes involved to enhance the P deficiency tolerance in C. oleifera.

5-Fluoro-2-indolyl des-chlorohalopemide (FIPI), a phospholipase D pharmacological inhibitor that alters cell spreading and inhibits chemotaxis.

The signaling enzyme phospholipase D (PLD) and the lipid second messenger it generates, phosphatidic acid (PA), are implicated in many cell biological processes, including Ras activation, cell spreading, stress fiber formation, chemotaxis, and membrane vesicle trafficking. PLD production of PA is inhibited by the primary alcohol 1-butanol, which has thus been widely employed to identify PLD/PA-driven processes. However, 1-butanol does not always effectively reduce PA accumulation, and its use may result in PLD-independent deleterious effects. Consequently, identification of potent specific small-molecule PLD inhibitors would be an important advance for the field. We examine one such here, 5-fluoro-2-indolyl des-chlorohalopemide (FIPI), which was identified recently in an in vitro chemical screen for PLD2 inhibitors, and show that it rapidly blocks in vivo PA production with subnanomolar potency. We were surprised to find that several biological processes blocked by 1-butanol are not affected by FIPI, suggesting the need for re-evaluation of proposed roles for PLD. However, FIPI does inhibit PLD regulation of F-actin cytoskeleton reorganization, cell spreading, and chemotaxis, indicating potential utility for it as a therapeutic for autoimmunity and cancer metastasis.

Targeting phospholipase D with small-molecule inhibitors as a potential therapeutic approach for cancer metastasis.

Phospholipase D (PLD)1 and PLD2, the classic mammalian members of the PLD superfamily, have been linked over the past three decades to immune cell function and to cell biological processes required by cancer cells for metastasis. However, owing to the lack of effective small-molecule inhibitors, it has not been possible to validate these roles for the PLDs and to explore the possible utility of acute and chronic PLD inhibition in vivo. The first such inhibitors have recently been described and demonstrated to block neutrophil chemotaxis and invasion by breast cancer cells in culture, increasing the prospects for a new class of therapeutics for autoimmune disorders and several types of metastatic cancer.

[Pharmacokinetics of honokiol in rat after oral administration of Cortex of Magnolia officinalis and its compound preparation Houpu Sanwu Decoction].

OBJECTIVE: To study pharmacokinetics of honokiol in Cortex of Magnolia officinalis and its compound prescription Houpu Sanwu Decoction (HPSWD) in rat, and discuss the change on pharmacokinetic process affected by other components. METHODS: The rats were divided into two groups, one was supplied with HPSWD and the other was administrated with Cortex of Magnolia officinalis. Concentrations of honokiol in rat plasma were then determined using HPLC method and main pharmacokinetic parameters were estimated. RESULTS: The plasma concentrations of honokiol of both groups were conformed to the two-compartment model with first order absorption. There existed significant differences in AUC, C(max), T(max), CL/F among Cortex of Magnolia officinalis and HPSWD. CONCLUSION: The results indicate that Rhubarb and Immature Orange Fruit in HPSWD can influence the asorption, distribution and elimination of honokiol.

RhoA-mediated Phospholipase D1 signaling is not required for the formation of stress fibers and focal adhesions.

The small GTPase RhoA regulates a wide spectrum of cellular functions including transformation and cytoskeletal reorganization. A large number of proteins have been identified as targets of RhoA, but their specific roles in these processes are not clear. Phospholipase D (PLD) was shown to be one such target several years ago; more recent work from our laboratory and others has demonstrated that of the two mammalian PLD isozymes, PLD1 but not PLD2 is activated by RhoA and this activation proceeds through direct binding both in vitro and in vivo. In this study, using a series of RhoA mutants, we have defined a PLD1-specific interacting site on RhoA composed of the residues Asn41, Trp58 and Asp76, using the yeast two-hybrid system, co-immunoprecipitation, and a PLD in vivo assay. The results further substantiate our previous finding that RhoA activates PLD1 through direct interaction. These mutants were then used to investigate the role of PLD1 in the cytoskeletal reorganization stimulated by RhoA signaling. Our results show that PLD1 is not required for the RhoA-mediated stress fiber and focal adhesion formation. The lack of importance of PLD1 signaling in RhoA-mediated cytoskeletal reorganization is further supported by the observation that PLD1 depletion using an shRNA approach and tetracycline-induced overexpression of the wild-type and the catalytically inactive mutant of PLD1 in stable cell lines do not alter stress fiber and focal adhesion formation.

Interaction between Brk kinase and insulin receptor substrate-4.

Breast tumor kinase (Brk) is a member of the Frk family of nonreceptor tyrosine kinases that is overexpressed in a high percentage of human breast tumors. The downstream substrates and effectors of Brk remain largely unidentified. In this study, we carried out immunoprecipitation and mass spectrometry experiments to identify new Brk binding partners. One interacting protein was insulin receptor substrate 4 (IRS-4), a member of the IRS family. We confirmed that Brk associates with IRS-4 in resting and insulin-like growth factor 1 (IGF-1)-stimulated HEK 293 cells. The SH3 and SH2 domains of Brk are both involved in the association. The tyrosine phosphorylation of Brk increases after stimulation with IGF-1, and in MCF-7 breast cancer cells we show that the presence of IRS-4 enhances this effect. Finally, we demonstrate that endogenous Brk and IRS-4 interact in A431 human epidermoid carcinoma cells.

Microwave ablation versus laser ablation in occluding lateral veins in goats.

Increasing number of endovenous techniques are available for the treatment of saphenous vein reflux and endovenous laser ablation (EVLA) is a frequently used method. A newly developed alternative, based on thermal therapy, is endovenous microwave ablation (EMA). This study evaluated the effect of the two procedures, in terms of coagulation and histological changes, in occluding lateral veins in goats. Twelve animals were randomized into two group, with 6 treated with EMA (EMA group), and the rest 6 with EVLA (EVLA group). Results of coagulation, including coagulation, fibrinolysis and platelet activation, were assessed at three or four different time points: before, immediately after, 24 h (and 48 h) after ablation. The diameter change, a measure of efficacy, was ultrasonographically measured before and 1 month after the ablation. Histological changes were grossly and microscopically evaluated immediately, 1 and 3 month(s) after the ablation. The length of the ablated vein and preoperative average diameter were comparable between the two groups. In both EMA and EVLA groups, several coagulation parameters, fibrinolysis and platelet activation parameters only underwent slight changes. Ultrasound imaging displayed that the diameter reduction of the veins treated by EMA was significantly larger than by EVLA, in consistent with the results of macroscopic examination. Microscopic examination revealed necrosis and thickening of the vein wall, and occlusion of the lumen within 3 months after ablation in both EMA and EVLA groups. It is concluded that EMA is a minimally invasive therapy, which appears to be safe and effective for treatment of lateral veins in goats.

VPS35 binds farnesylated N-Ras in the cytosol to regulate N-Ras trafficking.

Ras guanosine triphosphatases (GTPases) regulate signaling pathways only when associated with cellular membranes through their C-terminal prenylated regions. Ras proteins move between membrane compartments in part via diffusion-limited, fluid phase transfer through the cytosol, suggesting that chaperones sequester the polyisoprene lipid from the aqueous environment. In this study, we analyze the nature of the pool of endogenous Ras proteins found in the cytosol. The majority of the pool consists of farnesylated, but not palmitoylated, N-Ras that is associated with a high molecular weight (HMW) complex. Affinity purification and mass spectrographic identification revealed that among the proteins found in the HMW fraction is VPS35, a latent cytosolic component of the retromer coat. VPS35 bound to N-Ras in a farnesyl-dependent, but neither palmitoyl- nor guanosine triphosphate (GTP)-dependent, fashion. Silencing VPS35 increased N-Ras's association with cytoplasmic vesicles, diminished GTP loading of Ras, and inhibited mitogen-activated protein kinase signaling and growth of N-Ras-dependent melanoma cells.

Role of withdrawal in reinstatement of morphine-conditioned place preference.

RATIONALE: Relapse is a major characteristic of drug addiction and the primary problem in treating drug abuse. Based on the negative reinforcement view of addiction, in which the motivation to take drugs is thought to result from the desire to avoid the aversive effect of drug withdrawal, it has been theorized that withdrawal symptoms play a major role in the maintenance of and relapse to drug taking. However, the role of withdrawal in relapse has not yet been systemically investigated in the reinstatement model. OBJECTIVES: Using a conditioned place preference (CPP) paradigm, we examined the role of different morphine withdrawal states (spontaneous withdrawal, naloxone-precipitated withdrawal, and conditioned withdrawal) in relapse to drug seeking. METHODS: Rats alternately received morphine (10 mg/kg, s.c.) and saline for 8 days to acquire the CPP. The morphine CPP disappeared after a 2-week extinction phase of saline-paired training. Rats were then chronically administered morphine to induce physical dependence. The different withdrawal states were induced and their roles in the reinstatement of extinguished CPP were assessed. During conditioned withdrawal, trunk blood samples were taken and the corticosterone level was measured by radioimmunoassay. To examine the role of corticotropin-releasing factor (CRF) receptor antagonist on conditioned-withdrawal-induced reinstatement of CPP, different doses of alpha-helical CRF (0.1 and 1 mug, i.c.v.) were administered 30 min prior to the CPP testing. RESULTS: The results show that morphine spontaneous withdrawal and naloxone-precipitated morphine withdrawal were ineffective in reinstatement morphine CPP. However, the withdrawal cues significantly elicited the reinstatement of CPP and increased corticosterone level. Moreover, pretreatment with the CRF receptor antagonist alpha-helical CRF (1 mug, i.c.v.) significantly attenuated the effects of withdrawal cues on reinstatement of CPP and corticosterone levels. CONCLUSION: These findings demonstrate that the cues associated with previous drug withdrawal play a major role in drug relapse and that activation of the CRF receptor is involved in conditioned-withdrawal-induced reinstatement. The present study suggests that CRF receptor antagonists might be of value in the treatment and prevention of relapse to drug seeking after long-term abstinence.

Reactivation of morphine conditioned place preference by drug priming: role of environmental cues and sensitization.

RATIONALE: Relapse is a major characteristic of drug addiction and remains the primary problem in treating drug abuse. Despite a great deal of research, the exact factors that determine renewed drug-seeking and persistent craving for them remain unclear. OBJECTIVE: The present study was designed to evaluate the role of environmental cues and behavioral sensitization in reactivation of place preference following long-term extinction of morphine conditioned place preference (CPP) in rats. METHODS: After being injected with morphine and saline alternately for 6 days to induce morphine CPP, the rats were subjected to extinction of conditioning for 21 days. The rats were then administered various doses of morphine, heroin, or cocaine and confined in the previous drug- or saline-paired compartment. CPP was determined. Some rats were treated with scopolamine or naloxone prior to administration of these three drugs. RESULTS: Morphine CPP disappeared following a 21-day extinction. A single injection of morphine, heroin, or cocaine evoked place preference for the previous drug-paired side. However, place preference for the previous vehicle-paired side was induced after the animals received a single injection of morphine, heroin or cocaine and confined to the previous vehicle-paired compartment. Administration of naloxone prior to drug treatment significantly attenuated the place preference induced by morphine or heroin, but had no significant effect on the place preference elicited by cocaine. Administration of the cholinergic antagonist scopolamine before morphine, heroin and cocaine inhibited the expression of place preference. CONCLUSIONS: Environment-related cues and behavioral sensitization play critical roles in the incentive motivation underlying drug-seeking 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.

Rap1-interacting adapter molecule (RIAM) associates with the plasma membrane via a proximity detector.

Adaptive immunity depends on lymphocyte adhesion that is mediated by the integrin lymphocyte functional antigen 1 (LFA-1). The small guanosine triphosphatase Rap1 regulates LFA-1 adhesiveness through one of its effectors, Rap1-interacting adapter molecule (RIAM). We show that RIAM was recruited to the lymphocyte plasma membrane (PM) through its Ras association (RA) and pleckstrin homology (PH) domains, both of which were required for lymphocyte adhesion. The N terminus of RIAM inhibited membrane translocation. In vitro, the RA domain bound both Rap1 and H-Ras with equal but relatively low affinity, whereas in vivo only Rap1 was required for PM association. The PH domain bound phosphoinositol 4,5-bisphosphate (PI(4,5)P(2)) and was responsible for the spatial distribution of RIAM only at the PM of activated T cells. We determined the crystal structure of the RA and PH domains and found that, despite an intervening linker of 50 aa, the two domains were integrated into a single structural unit, which was critical for proper localization to the PM. Thus, the RA-PH domains of RIAM function as a proximity detector for activated Rap1 and PI(4,5)P(2).

Phosphatidylinositol-4-phosphate-5-kinase alpha deficiency alters dynamics of glucose-stimulated insulin release to improve glucohomeostasis and decrease obesity in mice.

OBJECTIVE: Phosphatidylinositol-4-phosphate-5-kinase (PI4P5K) has been proposed to facilitate regulated exocytosis and specifically insulin secretion by generating phosphatidylinositol-4,5-bisphosphate (PIP(2)). We sought to examine the role of the α isoform of PI4P5K in glucohomeostasis and insulin secretion. RESEARCH DESIGN AND METHODS: The response of PI4P5Kα(-/-) mice to glucose challenge and a type 2-like diabetes-inducing high-fat diet was examined in vivo. Glucose-stimulated responses and PI4P5Kα(-/-) pancreatic islets and ß-cells were characterized in culture. RESULTS: We show that PI4P5Kα(-/-) mice exhibit increased first-phase insulin release and improved glucose clearance, and resist high-fat diet-induced development of type 2-like diabetes and obesity. PI4P5Kα(-/-) pancreatic islets cultured in vitro exhibited decreased numbers of insulin granules docked at the plasma membrane and released less insulin under quiescent conditions, but then secreted similar amounts of insulin on glucose stimulation. Stimulation-dependent PIP(2) depletion occurred on the plasma membrane of the PI4P5Kα(-/-) pancreatic ß-cells, accompanied by a near-total loss of cortical F-actin, which was already decreased in the PI4P5Kα(-/-) ß-cells under resting conditions. CONCLUSIONS: Our findings suggest that PI4P5Kα plays a complex role in restricting insulin release from pancreatic ß-cells through helping to maintain plasma membrane PIP(2) levels and integrity of the actin cytoskeleton under both basal and stimulatory conditions. The increased first-phase glucose-stimulated release of insulin observed on the normal diet may underlie the partial protection against the elevated serum glucose and obesity seen in type 2 diabetes-like model systems.

Oral administration and external application of Chinese drugs combined with micro-invasive operation for the treatment of varicose ulcers in the lower extremities.

OBJECTIVE: To evaluate the clinical therapeutic effects of oral administration and external application of Chinese drugs combined with micro-invasive surgery for the treatment of varicose ulcers in the lower extremities (ecthyma). METHODS: A total of 152 patients (163 limbs) suffering from varicose ulcers on the lower limbs were assigned to two groups according to the patients' willingness. The 102 cases (109 limbs) in the treatment group underwent the method of endovenous microwave closure of communicating veins combined with oral administration and external application of Chinese drugs before and after the operation. The 50 cases (54 limbs) in the control group, were treated with oral administration and external application of Chinese drugs only. Clinical manifestations, including the condition of ulcer healing, the improvement conditions of alogotrophy, edema and other symptoms, were observed before and after 3 months of treatment. The clinical healing rate, the ulcer healing time, and the ulcer recurrence rate, were compared between the two groups. All the patients were followed-up 3 months after treatment. RESULTS: The follow-up was carried out for 3 to 42 months (mean 30.5 months). In the treatment group, 99 patients (106 limbs) were clinically cured, and the clinical healing rate was 97.06%; the ulcer healing time was 9-101 days (average 31.25+/-8.28 days) and 3 cases (5 limbs) had recurrence; the ulcer recurrence rate was 5.81%. In the control group, 40 patients (43 limbs) were clinically cured, with a clinical healing rate of 80.00%; the ulcer healing time was 10-152 days (average 50.60+/-12.31 day) and 5 cases (7 limbs) recurred, with the ulcer recurrence rate being 20.59%. The effects in the treatment group were obviously better than those in the control group (P<0.01 or P<0.05). CONCLUSION: The oral administration and external application of Chinese drugs combined with micro-invasive surgery for the treatment of varicose ulcers in the lower extremities has a significant curative effect, with a higher clinical healing rate, shorter ulcer healing time and lower ulcer recurrence rate than either treatment alone.

Sequential regulation of DOCK2 dynamics by two phospholipids during neutrophil chemotaxis.

During chemotaxis, activation of the small guanosine triphosphatase Rac is spatially regulated to organize the extension of membrane protrusions in the direction of migration. In neutrophils, Rac activation is primarily mediated by DOCK2, an atypical guanine nucleotide exchange factor. Upon stimulation, we found that DOCK2 rapidly translocated to the plasma membrane in a phosphatidylinositol 3,4,5-trisphosphate-dependent manner. However, subsequent accumulation of DOCK2 at the leading edge required phospholipase D-mediated synthesis of phosphatidic acid, which stabilized DOCK2 there by means of interaction with a polybasic amino acid cluster, resulting in increased local actin polymerization. When this interaction was blocked, neutrophils failed to form leading edges properly and exhibited defects in chemotaxis. Thus, intracellular DOCK2 dynamics are sequentially regulated by distinct phospholipids to localize Rac activation during neutrophil chemotaxis.

A nuclear function of beta-arrestin1 in GPCR signaling: regulation of histone acetylation and gene transcription.

Chromatin modification is considered to be a fundamental mechanism of regulating gene expression to generate coordinated responses to environmental changes, however, whether it could be directly regulated by signals mediated by G protein-coupled receptors (GPCRs), the largest surface receptor family, is not known. Here, we show that stimulation of delta-opioid receptor, a member of the GPCR family, induces nuclear translocation of beta-arrestin 1 (betaarr1), which was previously known as a cytosolic regulator and scaffold of GPCR signaling. In response to receptor activation, betaarr1 translocates to the nucleus and is selectively enriched at specific promoters such as that of p27 and c-fos, where it facilitates the recruitment of histone acetyltransferase p300, resulting in enhanced local histone H4 acetylation and transcription of these genes. Our results reveal a novel function of betaarr1 as a cytoplasm-nucleus messenger in GPCR signaling and elucidate an epigenetic mechanism for direct GPCR signaling from cell membrane to the nucleus through signal-dependent histone modification.