Disheveled, Egl-10, Pleckstrin Research Articles

DEPDC1B is a key regulator of myoblast proliferation in mouse and man.

ObjectivesDISHEVELLED, EGL‐10, PLECKSTRIN (DEP) domain‐containing 1B (DEPDC1B) promotes dismantling of focal adhesions and coordinates detachment events during cell cycle progression. DEPDC1B is overexpressed in several cancers with expression inversely correlated with patient survival. Here, we analysed the role of DEPDC1B in the regulation of murine and human skeletal myogenesis.Materials and methodsExpression dynamics of DEPDC1B were examined in murine and human myoblasts and rhabdomyosarcoma cells in vitro by RT‐qPCR and/or immunolabelling. DEPDC1B function was mainly tested via siRNA‐mediated gene knockdown.Results DEPDC1B was expressed in proliferating murine and human myoblasts, with expression then decreasing markedly during myogenic differentiation. SiRNA‐mediated knockdown of DEPDC1B reduced myoblast proliferation and induced entry into myogenic differentiation, with deregulation of key cell cycle regulators (cyclins, CDK, CDKi). DEPDC1B and β‐catenin co‐knockdown was unable to rescue proliferation in myoblasts, suggesting that DEPDC1B functions independently of canonical WNT signalling during myogenesis. DEPDC1B can also suppress RHOA activity in some cell types, but DEPDC1B and RHOA co‐knockdown actually had an additive effect by both further reducing proliferation and enhancing myogenic differentiation. DEPDC1B was expressed in human Rh30 rhabdomyosarcoma cells, where DEPDC1B or RHOA knockdown promoted myogenic differentiation, but without influencing proliferation.ConclusionDEPDC1B plays a central role in myoblasts by driving proliferation and preventing precocious myogenic differentiation during skeletal myogenesis in both mouse and human.

Identification of DEP domain-containing proteins by a machine learning method and experimental analysis of their expression in human HCC tissues.

The Dishevelled/EGL-10/Pleckstrin (DEP) domain-containing (DEPDC) proteins have seven members. However, whether this superfamily can be distinguished from other proteins based only on the amino acid sequences, remains unknown. Here, we describe a computational method to segregate DEPDCs and non-DEPDCs. First, we examined the Pfam numbers of the known DEPDCs and used the longest sequences for each Pfam to construct a phylogenetic tree. Subsequently, we extracted 188-dimensional (188D) and 20D features of DEPDCs and non-DEPDCs and classified them with random forest classifier. We also mined the motifs of human DEPDCs to find the related domains. Finally, we designed experimental verification methods of human DEPDC expression at the mRNA level in hepatocellular carcinoma (HCC) and adjacent normal tissues. The phylogenetic analysis showed that the DEPDCs superfamily can be divided into three clusters. Moreover, the 188D and 20D features can both be used to effectively distinguish the two protein types. Motif analysis revealed that the DEP and RhoGAP domain was common in human DEPDCs, human HCC and the adjacent tissues that widely expressed DEPDCs. However, their regulation was not identical. In conclusion, we successfully constructed a binary classifier for DEPDCs and experimentally verified their expression in human HCC tissues.

The cellular story of dishevelleds.

Dishevelled (DVL) proteins, three of which have been identified in humans, are highly conserved components of canonical and noncanonical Wnt signaling pathways. These multifunctional proteins, originally discovered in the fruit fly, through their different domains mediate complex signal transduction: DIX (dishevelled, axin) and PDZ (postsynaptic density 95, discs large, zonula occludens-1) domains serve for canonical beta-catenin signaling, while PDZ and DEP (dishevelled, Egl-10, pleckstrin) domains serve for non-canonical signaling. In canonical or beta-catenin signaling, DVL forms large molecular supercomplexes at the plasma membrane consisting of Wnt-Fz-LRP5/6-DVL-AXIN. This promotes the disassembly of the beta-catenin destruction machinery, beta-catenin accumulation, and consequent activation of Wnt signaling. Therefore, DVLs are considered to be key regulators that rescue cytoplasmic beta-catenin from degradation. The potential medical importance of DVLs is in both human degenerative disease and cancer. The overexpression of DVL has been shown to potentiate the activation of Wnt signaling and it is now apparent that up-regulation of DVLs is involved in several types of cancer.

Inhibition of DEPDC1A, a Bad Prognostic Marker in Multiple Myeloma, Delays Growth and Induces Mature Plasma Cell Markers in Malignant Plasma Cells

High throughput DNA microarray has made it possible to outline genes whose expression in malignant plasma cells is associated with short overall survival of patients with Multiple Myeloma (MM). A further step is to elucidate the mechanisms encoded by these genes yielding to drug resistance and/or patients’ short survival. We focus here on the biological role of the DEP (for Disheveled, EGL-10, Pleckstrin) domain contained protein 1A (DEPDC1A), a poorly known protein encoded by DEPDC1A gene, whose high expression in malignant plasma cells is associated with short survival of patients. Using conditional lentiviral vector delivery of DEPDC1A shRNA, we report that DEPDC1A knockdown delayed the growth of human myeloma cell lines (HMCLs), with a block in G2 phase of the cell cycle, p53 phosphorylation and stabilization, and p21Cip1 accumulation. DEPDC1A knockdown also resulted in increased expression of mature plasma cell markers, including CXCR4, IL6-R and CD38. Thus DEPDC1A could contribute to the plasmablast features of MMCs found in some patients with adverse prognosis, blocking the differentiation of malignant plasma cells and promoting cell cycle.

Subcellular localization of regulator of G protein signaling RGS7 complex in neurons and transfected cells

The R7 family of regulators of G protein signaling (RGS) is involved in many functions of the nervous system. This family includes RGS6, RGS7, RGS9, and RGS11 gene products and is defined by the presence of the characteristic first found in Disheveled, Egl-10, Pleckstrin (DEP), DEP helical extension (DHEX), Gγ-like, and RGS domains. Herein, we examined the subcellular localization of RGS7, the most broadly expressed R7 member. Our immunofluorescence studies of retinal and dorsal root ganglion neurons showed that RGS7 concentrated at the plasma membrane of cell bodies, in structures resembling lamellipodia or filopodia along the processes, and at the dendritic tips. At the plasma membrane of dorsal root ganglia neurons, RGS7 co-localized with its known binding partners R7 RGS binding protein (R7BP), Gαo, and Gαq. More than 50% of total RGS7-specific immunofluorescence was present in the cytoplasm, primarily within numerous small puncta that did not co-localize with R7BP. No specific RGS7 or R7BP immunoreactivity was detected in the nuclei. In transfected cell lines, ectopic RGS7 had both diffuse cytosolic and punctate localization patterns. RGS7 also localized in centrosomes. Structure-function analysis showed that the punctate localization was mediated by the DEP/DHEX domains, and centrosomal localization was dependent on the DHEX domain.

CAMP regulates DEP domain-mediated binding of the guanine nucleotide exchange factor Epac1 to phosphatidic acid at the plasma membrane

Epac1 is a cAMP-regulated guanine nucleotide exchange factor for the small G protein Rap. Upon cAMP binding, Epac1 undergoes a conformational change that results in its release from autoinhibition. In addition, cAMP induces the translocation of Epac1 from the cytosol to the plasma membrane. This relocalization of Epac1 is required for efficient activation of plasma membrane-located Rap and for cAMP-induced cell adhesion. This translocation requires the Dishevelled, Egl-10, Pleckstrin (DEP) domain, but the molecular entity that serves as the plasma membrane anchor and the possible mechanism of regulated binding remains elusive. Here we show that Epac1 binds directly to phosphatidic acid. Similar to the cAMP-induced Epac1 translocation, this binding is regulated by cAMP and requires the DEP domain. Furthermore, depletion of phosphatidic acid by inhibition of phospholipase D1 prevents cAMP-induced translocation of Epac1 as well as the subsequent activation of Rap at the plasma membrane. Finally, mutation of a single basic residue within a polybasic stretch of the DEP domain, which abolishes translocation, also prevents binding to phosphatidic acid. From these results we conclude that cAMP induces a conformational change in Epac1 that enables DEP domain-mediated binding to phosphatidic acid, resulting in the tethering of Epac1 at the plasma membrane and subsequent activation of Rap.

Role of Dynamics in the Autoinhibition and Activation of the Exchange Protein Directly Activated by Cyclic AMP (EPAC)

The exchange protein directly activated by cAMP (EPAC) is a key receptor of cAMP in eukaryotes and controls critical signaling pathways. Currently, no residue resolution information is available on the full-length EPAC dynamics, which are known to be pivotal determinants of allostery. In addition, no information is presently available on the intermediates for the classical induced fit and conformational selection activation pathways. Here these questions are addressed through molecular dynamics simulations on five key states along the thermodynamic cycle for the cAMP-dependent activation of a fully functional construct of EPAC2, which includes the cAMP-binding domain and the integral catalytic region. The simulations are not only validated by the agreement with the experimental trends in cAMP-binding domain dynamics determined by NMR, but they also reveal unanticipated dynamic attributes, rationalizing previously unexplained aspects of EPAC activation and autoinhibition. Specifically, the simulations show that cAMP binding causes an extensive perturbation of dynamics in the distal catalytic region, assisting the recognition of the Rap1b substrate. In addition, analysis of the activation intermediates points to a possible hybrid mechanism of EPAC allostery incorporating elements of both the induced fit and conformational selection models. In this mechanism an entropy compensation strategy results in a low free-energy pathway of activation. Furthermore, the simulations indicate that the autoinhibitory interactions of EPAC are more dynamic than previously anticipated, leading to a revised model of autoinhibition in which dynamics fine tune the stability of the autoinhibited state, optimally sensitizing it to cAMP while avoiding constitutive activation.

β-Arrestin2 Plays Permissive Roles in the Inhibitory Activities of RGS9-2 on G Protein-Coupled Receptors by Maintaining RGS9-2 in the Open Conformation

Together with G protein-coupled receptor (GPCR) kinases (GRKs) and β-arrestins, RGS proteins are the major family of molecules that control the signaling of GPCRs. The expression pattern of one of these RGS family members, RGS9-2, coincides with that of the dopamine D(3) receptor (D(3)R) in the brain, and in vivo studies have shown that RGS9-2 regulates the signaling of D2-like receptors. In this study, β-arrestin2 was found to be required for scaffolding of the intricate interactions among the dishevelled-EGL10-pleckstrin (DEP) domain of RGS9-2, Gβ5, R7-binding protein (R7BP), and D(3)R. The DEP domain of RGS9-2, under the permission of β-arrestin2, inhibited the signaling of D(3)R in collaboration with Gβ5. β-Arrestin2 competed with R7BP and Gβ5 so that RGS9-2 is placed in the cytosolic region in an open conformation which is able to inhibit the signaling of GPCRs. The affinity of the receptor protein for β-arrestin2 was a critical factor that determined the selectivity of RGS9-2 for the receptor it regulates. These results show that β-arrestins function not only as mediators of receptor-G protein uncoupling and initiators of receptor endocytosis but also as scaffolding proteins that control and coordinate the inhibitory effects of RGS proteins on the signaling of certain GPCRs.

MrflbA, encoding a putative FlbA, is involved in aerial hyphal development and secondary metabolite production in Monascus ruber M-7

FlbA (fluffy low brlA expression), a regulator of the G protein signalling (RGS) pathway, has been implicated in the control of hyphal development, sporulation, mycotoxin/pigment production in many kinds of filamentous fungi and yeasts. In the current study, a FlbA-like protein gene mrflbA (Monascus ruber flbA) was isolated, sequenced, and disrupted in order to investigate the RGS function in M. ruber. The results revealed that the derived protein of the mrflbA gene consisted of 734 amino acids and had the conserved RGS domain at the C-terminus and two DEP (dishevelled, Egl-10, pleckstrin) domains at the N-terminus similar to the structure of RGS proteins in other filamentous fungi. Deletion of the mrflbA gene resulted in the formation of an abnormal colony phenotype with fluffy aerial hyphae that autolyzed as the colony grew on potato dextrose agar (PDA) at 28°C. Additionally, mrflbA deletion could repress conidial germination and pigment/citrinin production in M. ruber M-7. Real-time RT-PCR analysis demonstrated that the transcription level of the G protein α subunit (Gα) was remarkably increased in the mrflbA deletion strain. These results suggest that mrflbA is involved in the modulation of aerial hyphal development and secondary metabolism, as well as, negative regulation of Gα subunit expression in M. ruber M-7.

RGS9‐2 mediates specific inhibition of agonist‐induced internalization of D2‐dopamine receptors

Regulator of G protein signaling 9-2 (RGS9-2), a member of the RGS family of GTPase accelerating proteins, is expressed specifically in the striatum, a brain region involved in controlling movement, motivation, mood and addiction. RGS9-2 can be found co-localized with D(2)-class dopamine receptors in medium spiny striatal neurons and altered functioning of both RGS9-2 and D(2)-like dopamine receptors have been implicated in schizophrenia, movement disorders and reward responses. Previously we showed that RGS9-2 can specifically co-localize with D(2)-dopamine receptors (D2R). Here we provide further evidence of the specificity of RGS9-2 for regulating D2R cellular functions: the expression of RGS9-2 inhibits dopamine-mediated cellular internalization of D2R, while the expression of another RGS protein, RGS4, had no effect. In addition, the agonist-mediated internalization of the G protein coupled delta opioid receptor was unaffected by RGS9-2 expression. We utilized mutant constructs of RGS9-2 to show that the RGS9-2 DEP (for Disheveled, EGL-10, Pleckstrin homology) domain and the GTPase accelerating activity of RGS9-2 were necessary for mediating specific inhibition of D2R internalization.

Domain–domain interaction of P-Rex1 is essential for the activation and inhibition by G protein βγ subunits and PKA

PtdIns(3, 4, 5)P3-dependent Rac exchanger (P-Rex) 1 is a guanine nucleotide exchange factor (GEF) for the small GTPase Rac. P-Rex1 is activated by G protein βγ subunits (Gβγ), and the Gβγ-induced activation is inhibited by cAMP-dependent protein kinase A (PKA). However, the details of regulatory mechanism of P-Rex1 remain to be clarified. In the present study, we investigated the mechanism of activation and inhibition of P-Rex1 using various truncated and alanine-substituted mutants and found that the domain–domain interaction of P-Rex1 is important for Gβγ-induced activation and PKA-induced inhibition. Immunoprecipitation analysis showed that the second Disheveled/EGL-10/Pleckstrin (DEP) and first PSD-95/Dlg/ZO-1 (PDZ) domains of P-Rex1 associate with the inositol polyphosphate-4-phosphatase (IP4P) domain. Carboxyl-terminal truncation on the IP4P domain or mutations in the protein-binding pocket of the first PDZ domain abolished the association. Analysis of in vitro guanine nucleotide exchange assay, PAK1/2 phosphorylation, and Rac-specific actin reorganization revealed that Gβγ could activate a complex of the P-Rex1 mutant lacking the IP4P domain and the isolated IP4P domain as well as full-length P-Rex1. Moreover, PKA phosphorylation prevented the domain–domain interaction and Gβγ-binding. These results provide a new insight into the regulation of other Rho-family GEFs and cell responses induced by the heterotrimeric G protein.

PI3K regulates pleckstrin-2 in T-cell cytoskeletal reorganization

AbstractPleckstrin-2 is composed of 2 pleckstrin homology (PH) domains and a disheveled–Egl-10–pleckstrin (DEP) domain. A lipid-binding assay revealed that pleckstrin-2 binds with greatest affinity to D3 and D5 phosphoinositides. Pleckstrin-2 expressed in Jurkat T cells bound to the cellular membrane and enhanced actin-dependent spreading only after stimulation of the T-cell antigen receptor or the integrin α4β1. A pleckstrin-2 variant containing point mutations in both PH domains failed to associate with the Jurkat membrane and had no effect on spreading under the same conditions. Although still membrane bound, a pleckstrin-2 variant containing point mutations in the DEP domain demonstrated a decreased ability to induce membrane ruffles and spread. Pleckstrin-2 also colocalized with actin at the immune synapse and integrin clusters via its PH domains. Although pleckstrin-2 can bind to purified D3 and D5 phosphoinositides, the intracellular membrane association of pleckstrin-2 and cell spreading are dependent on D3 phosphoinositides, because these effects were disrupted by pharmacologic inhibition of phosphatidylinositol 3-kinase (PI3K). Our results indicate that pleckstrin-2 uses its modular domains to bind to membrane-associated phosphatidylinositols generated by PI3K, whereby it coordinates with the actin cytoskeleton in lymphocyte spreading and immune synapse formation.

The Membrane Anchor R7BP Controls the Proteolytic Stability of the Striatal Specific RGS Protein, RGS9-2

A member of the RGS (regulators of G protein signaling) family, RGS9-2 is a critical regulator of G protein signaling pathways that control locomotion and reward signaling in the brain. RGS9-2 is specifically expressed in striatal neurons where it forms complexes with its newly discovered partner, R7BP (R7 family binding protein). Interaction with R7BP is important for the subcellular targeting of RGS9-2, which in native neurons is found in plasma membrane and its specializations, postsynaptic densities. Here we report that R7BP plays an additional important role in determining proteolytic stability of RGS9-2. We have found that co-expression with R7BP dramatically elevates the levels of RGS9-2 and its constitutive subunit, Gbeta5. Measurement of the RGS9-2 degradation kinetics in cells indicates that R7BP markedly reduces the rate of RGS9-2.Gbeta5 proteolysis. Lentivirus-mediated RNA interference knockdown of the R7BP expression in native striatal neurons results in the corresponding decrease in RGS9-2 protein levels. Analysis of the molecular determinants that mediate R7BP/RGS9-2 binding to result in proteolytic protection have identified that the binding site for R7BP in RGS proteins is formed by pairing of the DEP (Disheveled, EGL-10, Pleckstrin) domain with the R7H (R7 homology), a domain of previously unknown function that interacts with four putative alpha-helices of the R7BP core. These findings provide a mechanism for the regulation of the RGS9 protein stability in the striatal neurons.

The effect of the RGS7‐Gβ5‐R7BP complex on GPCR mediated calcium mobilization

G protein coupled receptors (GPCRs) are major targets for therapeutic drugs, hence understanding the mechanism of signal transduction through these receptors is crucial for the development of novel therapies. Regulators of G protein signaling (RGS) proteins accelerate the deactivation of G proteins by acting as GTPase activating proteins (GAPs) towards Gα subunits. The R7 subfamily of RGS proteins contains a Dishevelled, Egl-10, Pleckstrin (DEP) and Gγ like (GGL) domain along with the characteristic RGS domain. It is via the GGL domain that R7 RGS proteins form a stable complex with the Gβ subunit, Gβ5 in vivo. Recently, an additional member of this complex R7 binding protein (R7BP) has been identified. R7BP binds to all R7 RGS proteins via the DEP domain. This interaction facilitates the recruitment of the RGS7-Gβ5 complex to the plasma membrane. Previously we demonstrated that RGS7-Gβ5 attenuates Ca2+ mobilization from M3 receptor in transfected cells. In this study, using Fura 2, we show that RGS7-Gβ5 acts in a receptor-specific manner, as it does not have an effect on M1, another Gq-coupled GPCR. We also investigated the effect of R7BP on the activity of RGS7-Gβ5 and found that it diminishes the negative regulation of M3 receptor signaling. Additional insights into this molecular mechanism were obtained via microscopy and GST pulldown assays. This research is supported by NIH grant GM 060019 (V.Z.S.).

Dishevelled mediates ephrinB1 signalling in the eye field through the planar cell polarity pathway

An important step in retinal development is the positioning of progenitors within the eye field where they receive the local environmental signals that will direct their ultimate fate. Recent evidence indicates that ephrinB1 functions in retinal progenitor movement, but the signalling pathway is unclear. We present evidence that ephrinB1 signals through its intracellular domain to control retinal progenitor movement into the eye field by interacting with Xenopus Dishevelled (Xdsh), and by using the planar cell polarity (PCP) pathway. Blocking Xdsh translation prevents retinal progeny from entering the eye field, similarly to the morpholino-mediated loss of ephrinB1 (ref. 2). Overexpression of Xdsh can rescue the phenotype induced by loss of ephrinB1, and this rescue (as well as a physical association between Xdsh and ephrinB1) is completely dependent on the DEP (Dishevelled, Egl-10, Pleckstrin) domain of Xdsh. Similar gain- and loss-of-function experiments suggest that Xdsh associates with ephrinB1 and mediates ephrinB1 signalling through downstream members of the PCP pathway during eye field formation.

PI3K Regulates Pleckstrin-2 in T-Cell Cytoskeletal Re-Organization.

Pleckstrin-2, a paralog of pleckstrin-1, is composed of two Pleckstrin Homology (PH) domains and a Disheveled-Egl 10-Pleckstrin (DEP) domain. Several studies have shown that PH domains mediate binding of their host proteins to inositol phosphates and phospholipids, and thus regulate protein function. PH domains are found in many molecules involved in cellular signaling, cytoskeletal organization, membrane trafficking, and phospholipid modification. Proteins containing the DEP domain also regulate a broad range of cellular functions and evidence is emerging that several signaling proteins may rely on their DEP domains for membrane association. We speculated that the function of pleckstrin-2 is dependent upon its ability to bind to specific polyphosphatidylinositols. A lipid-binding assay revealed that pleckstrin-2 binds with greatest affinity to the products of phosphatidylinositol 3-kinase (PI3K) and phosphatidylinositol 5-kinase. The individual PH domains of pleckstrin-2 bind to the same products but with lower affinity, implying that both PH domains cooperate for maximal lipid affinity of the full-length protein. To examine the effect of pleckstrin-2 in human T-cells, Jurkat T-cells were transfected with GFP-tagged plasmids that direct the expression of pleckstrin-2 variants. Using confocal video microscopy we demonstrated that upon activation of the T-cell antigen receptor or the integrin α4β1, pleckstrin-2 rapidly moves from the cytoplasm to the cellular membrane and enhances membrane ruffling. Quantitation of cell footprint size revealed a two-fold increase in cell spreading. Furthermore, the membrane association of pleckstrin-2 and its resultant cell spreading were dependent on D3-phosphoinositides since these effects were disrupted by pharmacologic inhibition of PI3K with either wortmannin or LY294002. Consistent with this observation, a pleckstrin-2 variant containing point mutations in both of its PH domains failed to associate with the cell membrane and had no effect on spreading under the same conditions, suggesting that pleckstrin-2 membrane association occurs through a pathway dependent on the phospholipid-binding pocket of its PH domains. Although still membrane-bound, a pleckstin-2 variant containing point mutations in the second β-turn and second α-helical coil of the DEP domain demonstrated a decreased ability of pleckstrin-2 to induce membrane ruffles and lamellipodia, without decreasing filopodia formation. The cell footprint size of the DEP domain mutants was also decreased compared to that of wild type pleckstrin-2. These results suggest that the pleckstrin-2 DEP domain may function to promote actin-rich membrane extensions and ruffling. The localization of receptors, signaling intermediates, and cytoskeletal components at the T-cell/APC interface is thought to be a major determinant of efficient T-cell activation. Our data indicate that in T-lymphocytes, pleckstrin-2 uses modular motifs to bind to membrane-associated phosphatidylinositols, such as those generated by PI3K, to organize the actin cytoskeleton and to promote lymphocyte spreading.

Structure and dynamics of the human pleckstrin DEP domain: Distinct molecular features of a novel DEP domain subfamily

Pleckstrin1 is a major substrate for protein kinase C in platelets and leukocytes, and comprises a central DEP (disheveled, Egl-10, pleckstrin) domain, which is flanked by two PH (pleckstrin homology) domains. DEP domains display a unique alpha/beta fold and have been implicated in membrane binding utilizing different mechanisms. Using multiple sequence alignments and phylogenetic tree reconstructions, we find that 6 subfamilies of the DEP domain exist, of which pleckstrin represents a novel and distinct subfamily. To clarify structural determinants of the DEP fold and to gain further insight into the role of the DEP domain, we determined the three-dimensional structure of the pleckstrin DEP domain using heteronuclear NMR spectroscopy. Pleckstrin DEP shares main structural features with the DEP domains of disheveled and Epac, which belong to different DEP subfamilies. However, the pleckstrin DEP fold is distinct from these structures and contains an additional, short helix alpha4 inserted in the beta4-beta5 loop that exhibits increased backbone mobility as judged by NMR relaxation measurements. Based on sequence conservation, the helix alpha4 may also be present in the DEP domains of regulator of G-protein signaling (RGS) proteins, which are members of the same DEP subfamily. In pleckstrin, the DEP domain is surrounded by two PH domains. Structural analysis and charge complementarity suggest that the DEP domain may interact with the N-terminal PH domain in pleckstrin. Phosphorylation of the PH-DEP linker, which is required for pleckstrin function, could regulate such an intramolecular interaction. This suggests a role of the pleckstrin DEP domain in intramolecular domain interactions, which is distinct from the functions of other DEP domain subfamilies found so far.

Null Mutation of Pleckstrin2, a PI3K Effector, Impairs Platelet Secretion.

Pleckstrin-2, a widely expressed paralog of pleckstrin, is composed of two Pleckstrin Homology (PH) domains and Disheveled-Egl 10-Pleckstrin (DEP) domain. Although the activity of pleckstrin is regulated by its' phosphorylation state, pleckstrin-2 is not a phospho-protein suggesting that it possesses a different mechanism of regulation. Previous reports have shown that many PH domains mediate protein binding to inositol phosphates and phospholipids, thus regulating protein function. Therefore, we speculated that localized production of specific polyphosphatidylinositols might bind, and activate, pleckstrin-2. Using a lipid-binding assay, we found that pleckstrin-2 bound with greatest affinity to the products of phosphatidylinositol 3-kinase and phosphatidylinositol 5-kinase. The individual PH domains of pleckstrin-2 bound the same products but with lower affinity, implying that both PH domains cooperate for maximal lipid affinity of the full-length protein. GFP-tagged pleckstrin-2 had a cytoplasmic distribution in non-adherent Jurkat cells, but through a pathway dependent on the phospholipid-binding pocket of its PH domains, rapidly moved to the cell membrane following adhesion to immobilized fibronectin. Once bound to the cell membrane, pleckstrin-2 enhanced Jurkat cell spreading 2-fold and increased membrane ruffling. The membrane association of pleckstrin-2, and its resultant cell spreading, were dependent on D3-phosphoinositides since these effects were disrupted by pharmacologic inhibition of PI3K with either wortmannin or LY294002. To investigate the role of this protein within platelets, we generated mice containing a null mutation within the pleckstrin-2 gene. Pleckstrin-2 null mice were born at the expected frequency, and had no overt spontaneous hemorrhagic events. Mice lacking pleckstrin-2 had normal platelet counts and morphologic appearance of their megakaryocytes. Following stimulation of the PAR-4 (thrombin) receptor, pleckstrin-2 knockout platelets displayed normal assembly of filamentous actin. However, pleckstrin-2 null platelets had impaired aggregation following stimulation by collagen or submaximal doses of the PAR-4 activating peptide. Since pleckstrin-2 deficient platelets aggregated normally in response to ADP, these results suggested that these platelets might have an impaired ability to secrete dense granules. Accordingly, we found that pleckstrin-2 null platelets had a defect in their ability to secrete ATP in response to stimulation by 5μM collagen or 200μM of the PAR-4 activating peptide. However, pleckstrin-2 knockout platelets did incorporate 14C-serotonin as efficiently as wild type platelets. This latter observation suggested that the secretion defect in pleckstrin-2 null platelets was not attributable to a deficiency of dense granules, but instead is due to a defect in exocytosis of granules. Together, these data suggest that the PH domains of pleckstrin-2 cooperatively bind PI3K generated phospholipids on the cell membrane, and help mediate platelet secretion.

Palmitoylation and Plasma Membrane Targeting of RGS7 Are Promoted by αo

Regulator of G protein signaling (RGS) proteins modulate G protein signaling by acting as GTPase-activating proteins for G protein alpha-subunits. RGS7 belongs to a subfamily of RGS proteins that exist as dimers with the G protein beta(5)-subunit. In this report, we addressed the mechanisms of plasma membrane localization of beta(5)RGS7. When expressed in human embryonic kidney 293 cells, beta(5)RGS7 was found to be cytoplasmic and soluble. Expression of alpha(o) promoted a strong redistribution of beta(5)RGS7 to the plasma membrane. Expression of alpha(q), however, failed to affect the subcellular localization of beta(5)RGS7. The constitutively active mutant alpha(o)R179C, like wild-type alpha(o), strongly recruited beta(5)RGS7 to plasma membranes; however, inactive alpha(o)G204A, RGS-insensitive alpha(o)G184S, and lipidation-deficient alpha(o)G2A were all defective in the ability to promote plasma membrane localization of beta(5)RGS7. In addition, palmitoylation of RGS7 was demonstrated, and palmitoylation required expression of alpha(o) or alpha(o)R179C. To examine potential palmitoylation sites of RGS7, several cysteines were substituted with serines. beta(5)RGS7C133S failed to localize to plasma membranes when coexpressed with alpha(o), suggesting cysteine 133 of RGS7 as a putative palmitoylation site. Finally, deletion of amino acids 76 to 128 of RGS7, which includes part of the disheveled, EGL-10, pleckstrin (DEP) domain, prevented alpha(o)-mediated plasma membrane recruitment of beta(5)RGS7. These findings are the first to demonstrate Galpha-regulated plasma membrane localization and palmitoylation of beta(5)RGS7 and suggest that membrane targeting of beta(5)RGS7 is a complex process requiring at least RGS domain-mediated interaction with alpha(o) and RGS7 palmitoylation.

RGS6 Interacts with DMAP1 and DNMT1 and Inhibits DMAP1 Transcriptional Repressor Activity

RGS6 is a member of a subfamily of mammalian RGS proteins that possess DEP (disheveled, Egl-10, pleckstrin) and GGL (G protein gamma subunit-like) domains in addition to the hallmark RGS domain. RGS proteins negatively regulate heterotrimeric G protein signaling by virtue of the GTPase-activating protein activity of their RGS domains. RGS6 exists in multiple splice forms with a long (6L) or short (6S) N terminus, a complete or incomplete GGL domain, in combination with various C-terminal domains. Green fluorescent protein-tagged RGS6L and RGS6S forms exhibit predominantly cytoplasmic and nuclear patterns of distribution in COS-7 cells, respectively, and traffic from these sites to nucleoli in response to stress signaling. We undertook a yeast two-hybrid screen for nuclear RGS6-binding proteins and here identify DMAP1 as an RGS6-interacting protein. DMAP1 is a component of the Dnmt1 complex involved in repression of newly replicated genes. The domains of interaction were mapped to the N-terminal region of the GGL domain of RGS6, a region distinct from its Gbeta5 binding region, and the C-terminal domain of DMAP1. Gbeta5 and DMAP1 did not compete for each other's interaction with RGS6. Co-immunoprecipitation studies in COS-7 cells showed that RGS6L and RGS6S, but not RGS6LDelta258-293 deletion mutant lacking a DMAP1-binding module, co-immunoprecipitate DMAP1 as well as Dnmt1 in a DMAP1-dependent manner. A recombinant GGL domain of RGS6 precipitated endogenous DMAP1 and Dnmt1 in neuroblastoma cell lysates and endogenous DMAP1 co-immunoprecipitated with RGS6L from mouse brain. Co-expression of DMAP1 with RGS6L promoted nuclear migration of RGS6L and its co-localization with DMAP1, a response not observed with RGS6LDelta258-293. RGS6 inhibited the transcriptional repressor activity of DMAP1. RGS6 is the first member of the RGS protein family shown to interact with proteins involved in transcriptional regulation.