Search

SignificanceWhy women carrying a pathogenic germlineBRCA1mutation are predisposed to ovarian and breast cancer remains elusive. This study points to ovarian progesterone as a culprit. Generally,BRCA1-mutation carriers exhibit high yet individually varying levels of progesterone during the menstrual cycle. Although not allBRCA1-mutation carriers develop these cancers, all of them are advised to undergo prophylactic surgeries at a young age (under 40 y to 45 y) to prevent ovarian and breast cancer. Insights from robust in vivo findings in this study offer a novel concept: Targeting progesterone signaling with antiprogestins could be an effective nonsurgical prophylactic option for ovarian and breast cancer prevention for these high-risk women.

Specific cell shapes are fundamental to the organization and function of multicellular organisms. Fibroblast Growth Factor (FGF) signaling induces the elongation of lens fiber cells during vertebrate lens development. Nonetheless, exactly how this extracellular FGF signal is transmitted to the cytoskeletal network has previously not been determined. Here, we show that the Crk family of adaptor proteins, Crk and Crkl, are required for mouse lens morphogenesis but not differentiation. Genetic ablation and epistasis experiments demonstrated that Crk and Crkl play overlapping roles downstream of FGF signaling in order to regulate lens fiber cell elongation. Upon FGF stimulation, Crk proteins were found to interact with Frs2, Shp2 and Grb2. The loss of Crk proteins was partially compensated for by the activation of Ras and Rac signaling. These results reveal that Crk proteins are important partners of the Frs2/Shp2/Grb2 complex in mediating FGF signaling, specifically promoting cell shape changes., eLife digest As an embryo develops, its cells divide multiple times to transform into the specialized cell types that form our tissues and organs. To carry out specific roles, cells need to be of a certain shape. For example, in mammals, the cells that make up the main portion of the eye lens, develop into a fiber-like shape to be perfectly aligned with each other. This enables them to transmit light to the retina at the rear end of the eye. To do so, the lens cells increase over 1000 times in length with the help of a group of proteins called the Fibroblast Growth Factor, or FGF for short. The FGF pathway includes a network of interacting proteins that transmit signals to molecules inside the lens cells to control how they specialize and grow. However, until now it was not clear how it does this. Here, Zhang et al. used mouse lens-cells grown in the laboratory to investigate how FGF signaling causes cells to change their structure. The experiments revealed two related proteins called Crk and Crkl that linked the FGF pathway with another signaling system. When these two proteins were removed from the lens cells, the lens cells were still able to specialize, but could no longer grow in length. This suggests that these two processes are independent of each other. Moreover, Crk and Crkl helped the cells to change shape by increasing the amount of another group of proteins called Ras, which are known to both help cells to specialize and to regulate their shape. Zhang et al. discovered that the amount of Ras proteins determined whether cells specialized or modified their shape by changing the organization of proteins in the cell. Millions of children are born with cataracts, a disease caused when lens cells fail to shape properly. A better knowledge of FGF signaling may help to understand how cataracts develop and inspire future treatments. Moreover, the pathways identified in this study could also apply to other organs and diseases in which FGF signaling is active.

Protective effects of prostacyclin (PC) or its stable analog beraprost against agonist-induced lung vascular inflammation have been associated with elevation of intracellular cAMP and Rac GTPase signaling which inhibited the RhoA GTPase-dependent pathway of endothelial barrier dysfunction. This study investigated a distinct mechanism of PC-stimulated lung vascular endothelial (EC) barrier recovery and resolution of LPS-induced inflammation mediated by small GTPase Rap1. Efficient barrier recovery was observed in LPS-challenged pulmonary EC after prostacyclin administration even after 15h of initial inflammatory insult and was accompanied by the significant attenuation of p38 MAP kinase and NFκB signaling and decreased production of IL-8 and soluble ICAM1. These effects were reproduced in cells post-treated with 8CPT, a small molecule activator of Rap1-specific nucleotide exchange factor Epac. By contrast, pharmacologic Epac inhibitor, Rap1 knockdown, or knockdown of cell junction-associated Rap1 effector afadin attenuated EC recovery caused by PC or 8CPT post-treatment. The key role of Rap1 in lung barrier restoration was further confirmed in the murine model of LPS-induced acute lung injury. Lung injury was monitored by measurements of bronchoalveolar lavage protein content, cell count, and Evans blue extravasation and live imaging of vascular leak over 6days using a fluorescent tracer. The data showed significant acceleration of lung recovery by PC and 8CPT post-treatment, which was abrogated in Rap1a−/− mice. These results suggest that post-treatment with PC triggers the Epac/Rap1/afadin-dependent mechanism of endothelial barrier restoration and downregulation of p38MAPK and NFκB inflammatory cascades, altogether leading to accelerated lung recovery.

Activation of Rap1 GTPase can improve the integrity of the barrier of the retina pigment epithelium (RPE) and reduce choroidal neovascularization (CNV). Inhibition of NADPH oxidase activation also reduces CNV. We hypothesize that Rap1 inhibits NADPH oxidase-generated ROS and thereby reduces CNV formation. Using a murine model of laser-induced CNV, we determined that reduced Rap1 activity in RPE/choroid occurred with CNV formation and that activation of Rap1 by 2′-O-Me-cAMP (8CPT)-reduced laser-induced CNV via inhibiting NADPH oxidase-generated ROS. In RPE, inhibition of Rap1 by Rap1 GTPase-activating protein (Rap1GAP) increased ROS generation, whereas activation of Rap1 by 8CPT reduced ROS by interfering with the assembly of NADPH oxidase membrane subunit p22phox with NOX4 or cytoplasmic subunit p47phox. Activation of NADPH oxidase with Rap1GAP reduced RPE barrier integrity via cadherin phosphorylation and facilitated choroidal EC migration across the RPE monolayer. Rap1GAP-induced ROS generation was inhibited by active Rap1a, but not Rap1b, and activation of Rap1a by 8CPT in Rap1b−/− mice reduced laser-induced CNV, in correlation with decreased ROS generation in RPE/choroid. These findings provide evidence that active Rap1 reduces CNV by interfering with the assembly of NADPH oxidase subunits and increasing the integrity of the RPE barrier.—Wang, H., Jiang, Y., Shi, D., Quilliam, L. A., Chrzanowska-Wodnicka, M., Wittchen, E. S., Li, D. Y., Hartnett, M. E. Activation of Rap1 inhibits NADPH oxidase-dependent ROS generation in retinal pigment epithelium and reduces choroidal neovascularization.

Phagocyte NADPH oxidase plays a key role in pathogen clearance via reactive oxygen species (ROS) production. Defects in oxidase function result in chronic granulomatous disease with hallmark recurrent microbial infections and inflammation. The oxidase’s role in the adaptive immune response is not well understood. Class II presentation of cytoplasmic and exogenous Ag to CD4+ T cells was impaired in human B cells with reduced oxidase p40phox subunit expression. Naturally arising mutations, which compromise p40phox function in a chronic granulomatous disease patient, also perturbed class II Ag presentation and intracellular ROS production. Reconstitution of patient B cells with a wild-type, but not a mutant, p40phox allele restored exogenous Ag presentation and intracellular ROS generation. Remarkably, class II presentation of epitopes from membrane Ag was robust in p40phox-deficient B cells. These studies reveal a role for NADPH oxidase and p40phox in skewing epitope selection and T cell recognition of self Ag.

Intracellular signaling by the second messenger cyclic AMP (cAMP) activates the Ras-related small GTPase Rap1 through the guanine exchange factor Epac. This activation leads to effector protein interactions, activation, and biological responses in the vasculature, including vasorelaxation. In vascular smooth muscle cells derived from human dermal arterioles (microVSM), Rap1 selectively regulates expression of G protein-coupled α2C-adrenoceptors (α2C-ARs) through JNK-c-jun nuclear signaling. The α2C-ARs are generally retained in the trans-Golgi compartment and mobilize to the cell surface and elicit vasoconstriction in response to cellular stress. The present study used human microVSM to examine the role of Rap1 in receptor localization. Complementary approaches included murine microVSM derived from tail arteries of C57BL6 mice that express functional α2C-ARs and mice deficient in Rap1A (Rap1A-null). In human microVSM, increasing intracellular cAMP by direct activation of adenylyl cyclase by forskolin (10 μM) or selectively activating Epac-Rap signaling by the cAMP analog 8-pCPT-2′- O-Me-cAMP (100 μM) activated RhoA, increased α2C-AR expression, and reorganized the actin cytoskeleton, increasing F-actin. The α2C-ARs mobilized from the perinuclear region to intracellular filamentous structures and to the plasma membrane. Similar results were obtained in murine wild-type microVSM, coupling Rap1-Rho-actin dynamics to receptor relocalization. This signaling was impaired in Rap1A-null murine microVSM and was rescued by delivery of constitutively active (CA) mutant of Rap1A. When tested in heterologous HEK293 cells, Rap1A-CA or Rho-kinase (ROCK-CA) caused translocation of functional α2C-ARs to the cell surface (∼4- to 6-fold increase, respectively). Together, these studies support vascular bed-specific physiological role of Rap1 and suggest a role in vasoconstriction in microVSM.

Constitutive activation of M-Ras has previously been reported to cause morphologic and growth transformation of murine cells, suggesting that M-Ras plays a role in tumorigenesis. Cell transformation by M-Ras correlated with weak activation of the Raf/MEK/ERK pathway, although contributions from other downstream effectors were suggested. Recent studies indicate that signaling events distinct from the Raf/MEK/ERK cascade are critical for human tumorigenesis. However, it is unknown what signaling events M-Ras triggers in human cells. Using constitutively active M-Ras (Q71L) containing additional mutations within its effector-binding loop, we found that M-Ras induces MEK/ERK-dependent and -independent Elk1 activation as well as phosphatidylinositol 3 kinase (PI3K)/Akt and JNK/cJun activation in human MCF-7 breast cancer cells. Among several human cell lines examined, M-Ras-induced MEK/ERK-independent Elk1 activation was only detected in MCF-7 cells, and correlated with Rlf/M-Ras interaction and Ral/JNK activation. Supporting a role for M-Ras signaling in breast cancer, EGF activated M-Ras and promoted its interaction with endogenous Rlf. In addition, constitutive activation of M-Ras induced estrogen-independent growth of MCF-7 cells that was dependent on PI3K/Akt, MEK/ERK, and JNK activation. Thus, our studies demonstrate that M-Ras signaling activity differs between human cells, highlighting the importance of defining Ras protein signaling within each cell type, especially when designing treatments for Ras-induced cancer. These findings also demonstrate that M-Ras activity may be important for progression of EGFR-dependent tumors.

Second-phase insulin release requires the sustained mobilization of insulin granules from internal storage pools to the cell surface for fusion with the plasma membrane. However, the detailed mechanisms underlying this process remain largely unknown. GTP-loading of the small GTPase Cdc42 is the first glucose-specific activation step in the process, although how glucose triggers Cdc42 activation is entirely unknown. In a directed candidate screen for guanine nucleotide exchange factors (GEFs), which directly activate small GTPases, Cool-1/βPix was identified in pancreatic islet beta cells. In support of its role as the beta cell Cdc42 GEF, βPix coimmunoprecipitated with Cdc42 in human islets and MIN6 beta cells in a glucose-dependent manner, peaking just prior to Cdc42 activation. Furthermore, RNAi-mediated βPix reduction by 50% corresponded to full ablation of glucose-induced Cdc42 activation and significant attenuation of basal and glucose-stimulated insulin secretion. Of the two Cdc42 guanine nucleotide dissociation inhibitor (GDI) proteins identified in beta cells, βPix competed selectively with caveolin-1 (Cav-1) but not RhoGDI in coimmunoprecipitation and GST-Cdc42-GDP interaction assays. However, a phospho-deficient Cav-1-Y14F mutant failed to compete with βPix; Cav-1Tyr14 is an established phosphorylation site for Src kinase. Taken together, these data support a new model, wherein glucose stimulates Cav-1 and induces its dissociation from Cdc42, possibly via Src kinase activation to phosphorylate Cav-1Tyr14, to promote Cdc42-βPix binding and Cdc42 activation, and to trigger downstream signaling and ultimately sustain insulin release.

Phosphatases of the regenerating liver (PRL) play oncogenic roles in cancer development and metastasis. Although previous studies indicate that PRL-1 promotes cell growth and migration by activating both the ERK1/2 and RhoA pathways, the mechanism by which it activates these signaling events remains unclear. We have identified a PRL-1-binding peptide (Peptide 1) that shares high sequence identity with a conserved motif in the Src homology 3 (SH3) domain of p115 Rho GTPase-activating protein (GAP). p115 RhoGAP directly binds PRL-1 in vitro and in cells via its SH3 domain. Structural analyses of the PRL-1·Peptide 1 complex revealed a novel protein-protein interaction whereby a sequence motif within the PxxP ligand-binding site of the p115 RhoGAP SH3 domain occupies a folded groove within PRL-1. This prevents the canonical interaction between the SH3 domain of p115 RhoGAP and MEKK1 and results in activation of ERK1/2. Furthermore, PRL-1 binding activates RhoA signaling by inhibiting the catalytic activity of p115 RhoGAP. The results demonstrate that PRL-1 binding to p115 RhoGAP provides a coordinated mechanism underlying ERK1/2 and RhoA activation.

Rap1 GTPases control immune synapse formation and signaling in lymphocytes. However, the precise molecular mechanism by which Rap1 regulates natural killer (NK) cell activation is not known. Using Rap1a or Rap1b knockout mice, we identify Rap1b as the major isoform in NK cells. Its absence significantly impaired LFA1 polarization, spreading, and microtubule organizing center (MTOC) formation in NK cells. Neither Rap1 isoform was essential for NK cytotoxicity. However, absence of Rap1b impaired NKG2D, Ly49D, and NCR1-mediated cytokine and chemokine production. Upon activation, Rap1b colocalized with the scaffolding protein IQGAP1. This interaction facilitated sequential phosphorylation of B-Raf, C-Raf, and ERK1/2 and helped IQGAP1 to form a large signalosome in the perinuclear region. These results reveal a previously unrecognized role for Rap1b in NK cell signaling and effector functions.

This study shows that Epac2, a cAMP-activated Rap-GEF, acts downstream of D1/D5 dopamine receptor signaling to regulate dendritic spines and synaptic transmission. The authors also show that rare mutations of the EPAC2 gene that are associated with autism cause defects in Epac2-mediated spine remodeling. Dynamic remodeling of spiny synapses is crucial for cortical circuit development, refinement and plasticity, whereas abnormal morphogenesis is associated with neuropsychiatric disorders. We found that activation of Epac2, a PKA-independent cAMP target and Rap guanine-nucleotide exchange factor (GEF), in cultured rat cortical neurons induced spine shrinkage, increased spine motility, removed synaptic GluR2/3-containing AMPA receptors and depressed excitatory transmission, whereas its inhibition promoted spine enlargement and stabilization. Epac2 was required for dopamine D1-like receptor–dependent spine shrinkage and GluR2 removal from spines. Epac2 interaction with neuroligin promoted its membrane recruitment and enhanced its GEF activity. Rare missense mutations in the EPAC2 (also known as RAPGEF4) gene, previously found in individuals with autism, affected basal and neuroligin-stimulated GEF activity, dendritic Rap signaling, synaptic protein distribution and spine morphology. Thus, we identify a previously unknown mechanism that promotes dynamic remodeling and depression of spiny synapses, disruption of which may contribute to some aspects of disease.

The Ras-related GTPases Rap1a and 1b have been implicated in multiple biological events including cell adhesion, free radical production, and cancer. To gain a better understanding of Rap1 function in mammalian physiology, we deleted the Rap1a gene. Although loss of Rap1a expression did not initially affect mouse size or viability, upon backcross into C57BL/6J mice some Rap1a−/− embryos died in utero. T cell, B cell, or myeloid cell development was not disrupted in Rap1a −/− mice. However, macrophages from Rap1a null mice exhibited increased haptotaxis on fibronectin and vitronectin matrices that correlated with decreased adhesion. Chemotaxis of lymphoid and myeloid cells in response to CXCL12 or CCL21 was significantly reduced. In contrast, an increase in FcR-mediated phagocytosis was observed. Because Rap1a was previously copurified with the human neutrophil NADPH oxidase, we addressed whether GTPase loss affected superoxide production. Neutrophils from Rap1a−/− mice had reduced fMLP-stimulated superoxide production as well as a weaker initial response to phorbol ester. These results suggest that, despite 95% amino acid sequence identity, similar intracellular distribution, and broad tissue distribution, Rap1a and 1b are not functionally redundant but rather differentially regulate certain cellular events.

Epac-1 and -2 (exchange proteins directly activated by cyclic AMP) are guanine-nucleotide exchange factors for the GTPases Rap1 and -2. Epac2 but not Epac1 was found to possess a RA (Ras association) domain similar to that found in the Ras effector Ral-GDS. This domain specifically bound Ras-GTP, enabling oncogenic Ras to translocate Epac2 from the cytosol to the plasma membrane. Consequently, a small pool of plasma membrane-bound Rap1 was activated at the expense of bulk Rap1 located on intracellular organelles. Whereas translocation of Epac2 was not mimicked by challenge with epidermal growth factor alone, costimulation with forskolin, prostaglandin E2, or an Epac-selective cyclic AMP analog-induced rapid relocation of GFP-Epac2 but not -Epac1 to the plasma membrane in a Ras-dependent manner. Deletion of the cyclic AMP-binding domain overcame the need for nucleotide, suggesting that this domain normally masked the RA domain in the resting GEF. Thus, Epac2 can respond to costimulation by agonists that jointly elevate Ras-GTP and cyclic AMP levels, activating a specific pool of Rap1 at the plasma membrane. Therefore, despite its previous description as a Ras antagonist or independently functioning GTPase, Rap1/Krev-1 may additionally act downstream of Ras in cells that express the cyclic AMP-regulated GEF, Epac2.

The detection of Ras-family GTPase activity is important in the determination of cell signaling events elicited by numerous ligands and cellular processes. This has been made much easier in recent years by the use of glutathione S-transferase (GST)-fused Ras binding domains. These domains from downstream effectors such as Raf and RalGDS preferentially bind the GTP-bound Ras proteins enabling their extraction and subsequent quantification by immunoblotting. Despite this advance, effectors that efficiently discriminate between GTP- and GDP-bound states are not available for many Ras-family members. While this hampers the ability to detect activity in tissue specimens, it is still possible to metabolically label cells with (32)Pi to load the GTP/GDP pool with labeled nucleotides, immunoprecipitate the Ras protein and detect the bound label following thin layer chromatographic separation and exposure to film or a phosphorimager. Using a transfection system and antibodies that recognize epitope tags one can test the ability of a protein to work as a GEF or GAP for a certain GTPase. Alternatively, if an immunoprecipitating antibody is available to the target GTPase, then analysis of endogenous GTP/GDP ratio is possible. Here we describe the detection of M-Ras and Rap1 activity by GST-RBD pull-down as well as that of Rheb and epitope-tagged R-Ras by classical metabolic labeling and immunoprecipitation.

AND-34, a 95-kDa protein with modest homology to Ras GDP exchange factors, associates with the focal adhesion protein p130Cas. Overexpression of AND-34 confers anti-estrogen resistance in breast cancer cell lines, a property linked to its ability to activate Rac. Here, we show that both the GDP exchange factor–like domain and the SH2 domain of AND-34 are required for Rac activation and for resistance to the estrogen receptor (ER) antagonist ICI 182,780. As phosphatidylinositol 3-kinase (PI3K) signaling can regulate Rac activation, we examined the effects of AND-34 on PI3K. Overexpression of AND-34 in MCF-7 cells increased PI3K activity and augmented Akt Ser473 phosphorylation and kinase activity. Inhibition of PI3K with LY294002 or a dominant-negative p85 construct blocked AND-34-mediated Rac and Akt activation. Although R-Ras can activate PI3K, transfection with constitutively active R-Ras failed to induce Rac activation and AND-34 overexpression failed to induce R-Ras activation. Treatment of either vector-only or AND-34-transfected ZR-75-1 cells with ICI 182,780 markedly diminished ERα levels, suggesting that AND-34-induced anti-estrogen resistance is likely to occur by an ERα-independent mechanism. Treatment of a ZR-75-1 breast cancer cell line stably transfected with AND-34 plus 2 μmol/L LY294002 or 10 μmol/L NSC23766, a Rac-specific inhibitor, abrogated AND-34-induced resistance to ICI 182,780. Our studies suggest that AND-34-mediated PI3K activation induces Rac activation and anti-estrogen resistance in human breast cancer cell lines.

The Ras-related GTPase Rap1 stimulates integrin-mediated adhesion and spreading in various mammalian cell types. Here, we demonstrate that Rap1 regulates cell spreading by localizing guanine nucleotide exchange factors (GEFs) that act via the Rho family GTPase Rac1. Rap1a activates Rac1 and requires Rac1 to enhance spreading, whereas Rac1 induces spreading independently of Rap1. Active Rap1a binds to a subset of Rac GEFs, including VAV2 and Tiam1 but not others such as SWAP-70 or COOL-1. Overexpressed VAV2 and Tiam1 specifically require Rap1 to promote spreading, even though Rac1 is activated independently of Rap1. Rap1 is necessary for the accumulation of VAV2 in membrane protrusions at the cell periphery. In addition, if VAV2 is artificially localized to the cell edge with the subcellular targeting domain of Rap1a, it increases cell spreading independently of Rap1. These results lead us to propose that Rap1 promotes cell spreading by localizing a subset of Rac GEFs to sites of active lamellipodia extension.

The workshop on ‘Exchange Factors’ was held at Instituto Juan March de Estudios e Investigaciones, Madrid, Spain, during November 4–6, 2002. It was organized by Xose R. Bustelo, J. Silvio Gutkind and Piero Crespo. ![][1] The Center for International Meetings on Biology workshop on ‘Exchange Factors’ provided an up‐to‐date view of how guanine‐nucleotide‐exchange factors (GEFs) affect the biochemical pathways that regulate the Ras superfamily of small GTPases, and how these molecules function as part of an interrelated network of regulatory proteins that mediate the biological responses of Ras‐like GTPases (Fig. 1). Reflecting the complex nature of GEF functions, the meeting focused on their structure–function relationships, regulation and biological functions. Particular emphasis was placed on a subset of regulators of the Ras and Rho subfamilies that contain catalytic CDC25 homology domains or Dbl homology (DH)/pleckstrin homology (PH) cassettes, respectively (Fig. 2; Quilliam et al ., 2002; Zheng, 2001). Figure 1. A biochemical model for guanine‐nucleotide‐exchange factor activation of Ras‐like GTPases. The cycle between active (GTP‐bound) and inactive (GDP‐bound) Ras proteins is regulated by guanine‐nucleotide‐exchange factors (GEFs) and GTPase‐activating proteins (GAPs). Extracellular signals, conveyed through specific cell‐surface receptors, modulate GEF activity, which in turn regulates the activity of individual Ras GTPases. The active Ras proteins are able to interact with many effectors, leading to diverse biological responses. Figure 2. Many guanine‐nucleotide‐exchange factors are involved in signalling to small GTPases. The multifunctional domain features of the representative guanine‐nucleotide‐exchange factors (GEFs) discussed at the meeting reflect their diverse biochemical modes of regulation (signal convergence and divergence) and biological function. Due to space limitations, only a subset of these is presented here. GEFs that regulate Ras and Rap proteins are aligned according to their CDC25 domains, whereas GEFs that regulate Rho and Rac proteins are aligned on the basis of their Dbl homology (DH) and pleckstrin homology (PH) … [1]: /embed/graphic-1.gif

Rho family GTPases activate intracellular kinase cascades to modulate transcription of multiple genes. Previous studies have examined the roles of the ubiquitously expressed Rho GTPase, Rac1, in regulation of gene expression in cell lines and implicated NF-kappaB, serum response factor, and kinase signaling pathways in this regulation. To understand the role of the closely related but hematopoiesis-specific Rho GTPase, Rac2, in regulation of gene transcription, we compared the gene expression profiles between wild-type and Rac2(-/-) bone marrow-derived mast cells. Our data demonstrate remarkable specificity in the regulation of gene expression by Rac2 versus Rac1. Microarray analysis demonstrated that expression of 38 known genes was significantly altered in Rac2(-/-) mast cells after cytokine stimulation compared with those in wild-type cells. Of these, the expression of the mouse mast cell protease 7 (MMCP-7) gene in wild-type cells was highly induced at the transcriptional level after stimulation with stem cell factor (SCF). In spite of compensatorily increased expression of Rac1 in Rac2-deficient cells, SCF-induced MMCP-7 transcription did not occur. Surprisingly, the loss of MMCP-7 induction was not due to decreased activation of NF-kappaB, a transcription factor postulated to lie downstream of Rac1 and known to play a critical role in hematopoietic cell differentiation and proliferation. However, the activities of c-Jun N-terminal kinases (JNKs) were markedly decreased in Rac2(-/-) mast cells. Our results suggest that cytokine-stimulated activation of MMCP-7 gene transcription is selectively regulated by a Rac2-dependent JNK signaling pathway in primary mast cells and imply a remarkable specificity in the regulation of transcriptional activity by these two highly related Rho GTPases.

The recent discovery of Epac, a novel cAMP receptor protein, opens up a new dimension in studying cAMP-mediated cell signaling. It is conceivable that many of the cAMP functions previously attributed to cAMP-dependent protein kinase (PKA) are in fact also Epac-dependent. The finding of an additional intracellular cAMP receptor provides an opportunity to further dissect the divergent roles that cAMP exerts in different cell types. In this study, we probed cross-talk between cAMP signaling and the phosphatidylinositol 3-kinase/PKB pathways. Specifically, we examined the modulatory effects of cAMP on PKB activity by monitoring the specific roles that Epac and PKA play individually in regulating PKB activity. Our study suggests a complex regulatory scheme in which Epac and PKA mediate the opposing effects of cAMP on PKB regulation. Activation of Epac leads to a phosphatidylinositol 3-kinase-dependent PKB activation, while stimulation of PKA inhibits PKB activity. Furthermore, activation of PKB by Epac requires the proper subcellular targeting of Epac. The opposing effects of Epac and PKA on PKB activation provide a potential mechanism for the cell type-specific differential effects of cAMP. It is proposed that the net outcome of cAMP signaling is dependent upon the dynamic abundance and distribution of intracellular Epac and PKA.

Rap1 is a Ras family GTPase with a well documented role in ERK/MAP kinase signaling and integrin activation. Stimulation of the G-protein-coupled receptor PAR-1 with thrombin in human 1321N1 glioblastoma cells led to a robust increase in Rap1 activation. This response was sustained for up to 6 h and mediated through RhoA and phospholipase D (PLD). Thrombin treatment also induced a 5-fold increase in cell adhesion to fibronectin, which was blocked by down-regulating PLD or Rap1A or by treatment with a β1integrin neutralizing antibody. In addition, thrombin treatment led to increases in phospho-focal adhesion kinase (tyrosine 397), ERK1/2 phosphorylation and cell proliferation, which were significantly inhibited in cells treated with β1integrin antibody or Rap1A siRNA. To assess the role of Rap1A in tumor formation in vivo, we compared growth of 1321N1 cells stably expressing control, Rap1A or Rap1B shRNA in a mouse xenograft model. Deletion of Rap1A, but not of Rap1B, reduced tumor mass by >70% relative to control. Similar observations were made with U373MG glioblastoma cells in which Rap1A was down-regulated. Collectively, these findings implicate a Rap1A/β1integrin pathway, activated downstream of G-protein-coupled receptor stimulation and RhoA, in glioblastoma cell proliferation. Moreover, our data demonstrate a critical role for Rap1A in glioblastoma tumor growth in vivo. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

RhoA is involved in multiple cellular processes, including cytoskeletal organization, gene expression, and transformation. These processes are mediated by a variety of downstream effector proteins. However, which effectors are involved in cellular transformation and how these proteins are activated following interaction with Rho remains to be established. A unique feature that distinguishes the Rho family from other Ras-related GTPases is the insert region, which may confer Rho-specific signaling events. Here we report that deletion of the insert region does not result in impaired effector binding. Instead, this insert deletion mutant (RhoDeltaRas, in which the insert helix has been replaced with loop 8 of Ras) acted in a dominant inhibitory fashion to block RhoA-induced transformation. Since RhoDeltaRas failed to promote stress fiber formation, we examined the ability of this mutant to bind to and subsequently activate Rho kinase. Surprisingly, RhoDeltaRas-GTP coprecipitated with Rho kinase but failed to activate it in vivo. These data suggested that the insert domain is not required for Rho kinase binding but plays a role in its activation. The constitutively active catalytic domain of Rho kinase did not promote focus formation alone or in the presence of Raf(340D) but cooperated with RhoDeltaRas to induce cellular transformation. This suggests that Rho kinase needs to cooperate with additional Rho effectors to promote transformation. Further, the Rho kinase catalytic domain reversed the inhibitory effect of RhoDeltaRas on Rho-induced transformation, suggesting that one of the downstream targets of Rho-induced transformation abrogated by RhoDeltaRas is indeed Rho kinase. In conclusion, we have demonstrated that the insert region of RhoA is required for Rho kinase activation but not for binding and that this kinase activity is required to induce morphologic transformation of NIH 3T3 cells.

Several genetic conditions can lead to left ventricular hypertrophy (LVH). Among them, hypertrophic cardiomyopathy (HCM), caused by mutations in sarcomere genes, is the most common inherited cardiac disease. Instead, RASopathies, a rare class of disorders characterized by neuro-cardio-facial-cutaneous abnormalities and sometimes presenting with LVH, are caused by mutations in the RAS-MAPK pathway. We report on a 62-years-old male who presented isolated severe obstructive LVH but did not carry the sarcomere mutation previously identified in his affected relatives. By exome sequencing, we detected a novel mutation in HRAS gene (NM_005343.2:p.Arg68Trp), present also in the proband's daughter, who showed mild LVH and severe intellectual disability. The cardiac phenotype was indistinguishable between family members carrying either mutation. In silico studies suggested that the mutated HRAS protein is constitutionally activated. Consistently, functional characterization in vitro confirmed elevated HRAS-GTP accumulation and downstream RAS-MAPK pathway activation that are known to drive cell proliferation in LVH. Our study emphasizes the role of RAS signaling in cardiac hypertrophy and highlights the complexity in differential diagnosis of RASopathies. In fact, the mild features of RASopathy and the recurrence of sarcomeric HCM in this family delayed the correct diagnosis until comprehensive genetic testing was performed.

Although the Ras subfamily of GTPases consists of ∼20 members, only a limited number of guanine nucleotide exchange factors (GEFs) that couple extracellular stimuli to Ras protein activation have been identified. Furthermore, no novel downstream effectors have been identified for the M-Ras/R-Ras3 GTPase. Here we report the identification and characterization of three Ras family GEFs that are most abundantly expressed in brain. Two of these GEFs, MR-GEF (M-Ras-regulated GEF, KIAA0277) and PDZ-GEF (KIAA0313) bound specifically to nucleotide-free Rap1 and Rap1/Rap2, respectively. Both proteins functioned as Rap1 GEFs in vivo. A third GEF, GRP3 (KIAA0846), activated both Ras and Rap1 and shared significant sequence homology with the calcium- and diacylglycerol-activated GEFs, GRP1 and GRP2. Similarly to previously identified Rap GEFs, C3G and Smg GDS, each of the newly identified exchange factors promoted the activation of Elk-1 in the LNCaP prostate tumor cell line where B-Raf can couple Rap1 to the extracellular receptor-activated kinase cascade. MR-GEF and PDZ-GEF both contain a region immediately N-terminal to their catalytic domains that share sequence homology with Ras-associating orRalGDS/AF6 homology (RA) domains. By searching for in vitro interaction with Ras-GTP proteins, PDZ-GEF specifically bound to Rap1A- and Rap2B-GTP, whereas MR-GEF bound to M-Ras-GTP. C-terminally truncated MR-GEF, lacking the GEF catalytic domain, retained its ability to bind M-Ras-GTP, suggesting that the RA domain is important for this interaction. Co-immunoprecipitation studies confirmed the interaction of M-Ras-GTP with MR-GEF in vivo. In addition, a constitutively active M-Ras(71L) mutant inhibited the ability of MR-GEF to promote Rap1A activation in a dose-dependent manner. These data suggest that M-Ras may inhibit Rap1 in order to elicit its biological effects.

Guanine nucleotide exchange factors (GEFs) are responsible for coupling cell surface receptors to Ras protein activation. Here we describe the characterization of a novel family of differentially expressed GEFs, identified by database sequence homology searching. These molecules share the core catalytic domain of other Ras family GEFs but lack the catalytic non-conserved (conserved non-catalytic/Ras exchange motif/structurally conserved region 0) domain that is believed to contribute to Sos1 integrity. In vitro binding and in vivo nucleotide exchange assays indicate that these GEFs specifically catalyze the GTP loading of the Ral GTPase when overexpressed in 293T cells. A central proline-rich motif associated with the Src homology (SH)2/SH3-containing adapter proteins Grb2 and Nck in vivo, whereas a pleckstrin homology (PH) domain was located at the GEF C terminus. We refer to these GEFs as RalGPS 1A, 1B, and 2 (Ral GEFs with PH domain and SH3 binding motif). The PH domain was required for in vivo GEF activity and could be functionally replaced by the Ki-Ras C terminus, suggesting a role in membrane targeting. In the absence of the PH domain RalGPS 1B cooperated with Grb2 to promote Ral activation, indicating that SH3 domain interaction also contributes to RalGPS regulation. In contrast to the Ral guanine nucleotide dissociation stimulator family of Ral GEFs, the RalGPS proteins do not possess a Ras-GTP-binding domain, suggesting that they are activated in a Ras-independent manner.

In response to different cellular stresses, a family of protein kinases regulates translation by phosphorylation of the α subunit of eukaryotic initiation factor-2 (eIF-2α). Recently, we identified a new family member, pancreatic eIF-2α kinase (PEK) from rat pancreas. PEK, also referred to as RNA-dependent protein kinase (PKR)-like endoplasmic reticulum (ER) kinase (PERK) is a transmembrane protein implicated in translational control in response to stresses that impair protein folding in the ER. In this study, we identified and characterized PEK homologues from humans, Drosophila melanogaster and Caenorhabditis elegans. Expression of human PEK mRNA was found in over 50 different tissues examined, with highest levels in secretory tissues. In mammalian cells subjected to ER stress, we found that elevated eIF-2α phosphorylation was coincident with increased PEK autophosphorylation and eIF-2α kinase activity. Activation of PEK was abolished by deletion of PEK N-terminal sequences located in the ER lumen. To address the role of C. elegans PEK in translational control, we expressed this kinase in yeast and found that it inhibits growth by hyperphosphorylation of eIF-2α and inhibition of eIF-2B. Furthermore, we found that vaccinia virus K3L protein, an inhibitor of the eIF-2α kinase PKR involved in an anti-viral defence pathway, also reduced PEK activity. These results suggest that decreased translation initiation by PEK during ER stress may provide the cell with an opportunity to remedy the folding problem prior to introducing newly synthesized proteins into the secretory pathway.

Like Ras, constitutively activated mutants of the Ras-related protein R-Ras cause tumorigenic transformation of NIH3T3 cells. However, since R-Ras causes a transformed phenotype distinct from that induced by Ras, it is likely that R-Ras controls signaling pathways and cellular processes distinct from those regulated by Ras. To address this possibility, we determined if R-Ras is regulated by activators and effectors distinct from those that regulate Ras function. We observed that Ras guanine nucleotide exchange factors failed to activate R-Ras in vivo, indicating that R-Ras is activated by distinct GEFs. Consistent with this, mutants of R-Ras with mutations analogous to the Ras(15A)/(17N) dominant negative proteins did not antagonize Ras GEF function and lacked the growth inhibitory activity seen with these mutant Ras proteins. Thus, R-Ras, but not Ras, is dispensable for the viability of NIH3T3 cells. Finally, whereas constitutively activated Ras can overcome the growth inhibitory action of the Ras(17N) dominant negative protein via Raf-dependent and -independent activities, transforming mutants of R-Ras failed to do so. This inability was consistent with our observation that Ras-, but not R-Ras-transformed, NIH3T3 cells possessed constitutively upregulated Raf kinase activities. Thus, R-Ras and Ras are regulators of distinct signaling pathways and cellular processes.

Loss of barrier integrity precedes the development of pathologies such as metastasis, inflammatory disorders, and blood-retinal barrier breakdown present in neovascular age-related macular degeneration. Rap1 GTPase is involved in regulating both endothelial and epithelial cell junctions; the specific role of Rap1A vs. Rap1B isoforms is less clear. Compromise of retinal pigment epithelium barrier function is a contributing factor to the development of AMD. We utilized shRNA of Rap1 isoforms in cultured human retinal pigment epithelial cells, along with knockout mouse models to test the role of Rap1 on promoting RPE barrier properties, with emphasis on the dynamic junctional regulation that is triggered when the adhesion between cells is challenged. In vitro, Rap1A shRNA reduced steady-state barrier integrity, whereas Rap1B shRNA affected dynamic junctional responses. In a laser-induced choroidal neovascularization (CNV) model of macular degeneration, Rap1b(-/-) mice exhibited larger CNV volumes compared to wild-type or Rap1a(-/-) . In vivo, intravitreal injection of a cAMP analog (8CPT-2'-O-Me-cAMP) that is a known Rap1 activator significantly reduced laser-induced CNV volume, which correlated with the inhibition of CEC transmigration across 8CPT-2'O-Me-cAMP-treated RPE monolayers in vitro. Rap1 activation by 8CPT-2'-O-Me-cAMP treatment increased recruitment of junctional proteins and F-actin to cell-cell contacts, increasing both the linearity of junctions in vitro and in cells surrounding laser-induced lesions in vivo. We conclude that in vitro, Rap1A may be important for steady state barrier integrity, while Rap1B is involved more in dynamic junctional responses such as resistance to junctional disassembly induced by EGTA and reassembly of cell junctions following disruption. Furthermore, activation of Rap1 in vivo inhibited development of choroidal neovascular lesions in a laser-injury model. Our data suggest that targeting Rap1 isoforms in vivo with 8CPT-2'-O-Me-cAMP may be a viable pharmacological means to strengthen the RPE barrier against the pathological choroidal endothelial cell invasion that occurs in macular degeneration.

Many factors, including duration and intensity of the unfolded protein response (UPR), dictate whether cells will adapt to endoplasmic reticulum stress or undergo apoptosis. In tuberous sclerosis (TSC), elevation of mammalian target of rapamycin complex 1 (mTORC1) activity has been proposed to compound the induction of UPR transcription factors ATF4 and CHOP, suggesting that the UPR could be targeted to eradicate TSC1/2-null cells during patient therapy. Here we report that control of c-MYC translation by mTORC1 plays a key role in determining whether TSC2-null Elt3 rat leiomyoma cells apoptose in response to UPR induction by the proteasome inhibitor bortezomib. Although bortezomib induces eukaryotic initiating factor 2α phosphorylation, mTORC1 activity was also required for downstream induction of the UPR transcription factors ATF4 and CHOP by a mechanism involving increased expression of c-MYC. Although bortezomib-induced c-MYC transcription was resistant to rapamycin treatment, mTORC1 activity was required for efficient c-MYC translation. c-MYC subsequently bound to the ATF4 promoter, suggesting direct involvement of an mTORC1/c-MYC-driven signaling pathway in the activation of the UPR. Consistent with this notion, exogenously expressed c-MYC reversed the ability of rapamycin to prevent bortezomib-induced CHOP and ATF4 expression as well as apoptosis. These findings indicate that the induction of ATF4/CHOP expression occurs via mTORC1 regulation of c-MYC and that this signaling pathway is a major determinant in the ability of bortezomib to induce apoptosis.

The NCK adapter protein is comprised of three consecutive Src homology 3 (SH3) protein-protein interaction domains and a C-terminal SH2 domain. Although the association of NCK with activated receptor protein-tyrosine kinases, via its SH2 domain, implicates NCK as a mediator of growth factor-induced signal transduction, little is known about the pathway(s) downstream of NCK recruitment. To identify potential downstream effectors of NCK we screened a bacterial expression library to isolate proteins that bind its SH3 domains. Two molecules were isolated, the Wiskott-Aldrich syndrome protein (WASP, a putative CDC42 effector) and a serine/threonine protein kinase (PRK2, closely related to the putative Rho effector PKN). Using interspecific backcross analysis the Prk2 gene was mapped to mouse chromosome 3. Unlike WASP, which bound the SH3 domains of several signaling proteins, PRK2 specifically bound to the middle SH3 domain of NCK and (weakly) that of phospholipase Cgamma. PRK2 also specifically bound to Rho in a GTP-dependent manner and cooperated with Rho family proteins to induce transcriptional activation via the serum response factor. These data suggest that PRK2 may coordinately mediate signal transduction from activated receptor protein-tyrosine kinases and Rho and that NCK may function as an adapter to connect receptor-mediated events to Rho protein signaling.

Caveolae are plasma membrane specializations that have been implicated in signal transduction. Caveolin, a 21-24-kDa integral membrane protein, is a principal structural component of caveolae membranes in vivo. G protein α subunits are concentrated in purified preparations of caveolae membranes, and caveolin interacts directly with multiple G protein α subunits, including Gs, Go, and Gi2. Mutational or pharmacologic activation of Gα subunits prevents the interaction of caveolin with G proteins, indicating that inactive Gα subunits preferentially interact with caveolin. Here, we show that caveolin interacts with another well characterized signal transducer, Ras. Using a detergent-free procedure for purification of caveolin-rich membrane domains and a polyhistidine tagged form of caveolin, we find that Ras and other classes of lipid-modified signaling molecules co-fractionate and co-elute with caveolin. The association of Ras with caveolin was further evaluated using two distinct in vitro binding assays. Wild-type H-Ras interacted with glutathione S-transferase (GST)-caveolin fusion proteins but not with GST alone. Using a battery of GST fusion proteins encoding distinct regions of caveolin, Ras binding activity was localized to a 41amino acid membrane proximal region of the cytosolic N-terminal domain of caveolin. In addition, reconstituted caveolin-rich membranes (prepared with purified recombinant caveolin and purified lipids) interacted with a soluble form of wild-type H-Ras but failed to interact with mutationally activated soluble H-Ras (G12V). Thus, a single amino acid change (G12V) that constitutively activates Ras prevents or destabilizes this interaction. These results clearly indicate that (i) caveolin is sufficient to recruit soluble Ras onto lipid membranes and (ii) membrane-bound caveolin preferentially interacts with inactive Ras proteins. In direct support of these in vitro studies, we also show that recombinant overexpression of caveolin in intact cells is sufficient to functionally recruit a nonfarnesylated mutant of Ras (C186S) onto membranes, overcoming the normal requirement for lipid modification of Ras. Taken together, these observations suggest that caveolin may function as a scaffolding protein to localize or sequester certain caveolin-interacting proteins, such as wild-type Ras, within caveolin-rich microdomains of the plasma membrane.

To understand the mechanism of Axl signaling, we have initiated studies to delineate downstream components in interleukin-3-dependent 32D cells by using a chimeric receptor containing the recombinant epidermal growth factor (EGF) receptor extracellular and transmembrane domains and the Axl kinase domain (EAK [for EGF receptor-Axl kinase]). We have previously shown that upon exogenous EGF stimulation, 32D-EAK cells are capable of proliferation in the absence of interleukin-3. With this system, we determined that EAK-induced cell survival and mitogenesis are dependent upon the Ras/extracellular-signal-regulated protein kinase (ERK) cascade. Although the phosphatidylinositol-3 kinase pathway is activated upon EAK signaling, it appears to be dispensable for the biological actions of the Axl kinase. Furthermore, we demonstrated that different threshold levels of Ras/ERK activation are needed to induce a block to apoptosis or proliferation in 32D cells. Recently, we have identified an Axl ligand, GAS6. Surprisingly, GAS6-stimulated 32D-Axl cells exhibited no blockage to apoptosis or mitogenic response which is correlated with the absence of Ras/ERK activation. Taken together, these data suggest that different extracellular domains dramatically alter the intracellular response of the Axl kinase. Furthermore, our data suggest that the GAS6-Axl interaction does not induce mitogenesis and that its exact role remains to be determined.

Tumor inflammation, the recruitment of myeloid lineage cells into the tumor microenvironment, promotes angiogenesis, immunosuppression and metastasis. CD11b+Gr1lo monocytic lineage cells and CD11b+Gr1hi granulocytic lineage cells are recruited from the circulation by tumor-derived chemoattractants, which stimulate PI3-kinase γ (PI3Kγ)-mediated integrin α4 activation and extravasation. We show here that PI3Kγ activates PLCγ, leading to RasGrp/CalDAG-GEF-I&II mediated, Rap1a-dependent activation of integrin α4β1, extravasation of monocytes and granulocytes, and inflammation-associated tumor progression. Genetic depletion of PLCγ, CalDAG-GEFI or II, Rap1a, or the Rap1 effector RIAM was sufficient to prevent integrin α4 activation by chemoattractants or activated PI3Kγ (p110γCAAX), while activated Rap (RapV12) promoted constitutive integrin activation and cell adhesion that could only be blocked by inhibition of RIAM or integrin α4β1. Similar to blockade of PI3Kγ or integrin α4β1, blockade of Rap1a suppressed both the recruitment of monocytes and granulocytes to tumors and tumor progression. These results demonstrate critical roles for a PI3Kγ-Rap1a-dependent pathway in integrin activation during tumor inflammation and suggest novel avenues for cancer therapy.

Tuberous sclerosis complex (TSC), an inherited tumor predisposition syndrome associated with mutations in TSC1 or TSC2, affects ∼1 in 6,000 individuals. Eighty percent of TSC patients develop renal angiomyolipomas, and renal involvement is a major contributor to patient morbidity and mortality. Recent work has shown that mammalian target of rapamycin complex 1 (mTORC1) inhibition caused angiomyolipoma shrinkage but that this treatment may cause cytostatic not a cytotoxic effect. Endoplasmic reticulum (ER) stress can develop in TSC-associated cells due to mTORC1-driven protein translation. We hypothesized that renal angiomyolipoma cells experience ER stress that can be leveraged to result in targeted cytotoxicity. We used immortalized human angiomyolipoma cells stably transfected with empty vector or TSC2 (encoding tuberin). Using cell number quantification and cell death assays, we found that mTORC1 inhibition with RAD001 suppressed angiomyolipoma cell proliferation in a cytostatic manner. Angiomyolipoma cells exhibited enhanced sensitivity to proteasome inhibitor-induced ER stress compared with TSC2-rescued cells. After proteasome inhibition with MG-132, Western blot analyses showed greater induction of C/EBP-homologous protein (CHOP) and more poly (ADP-ribose) polymerase (PARP) and caspase-3 cleavage, supporting ER stress-induced apoptosis. Live cell numbers also were decreased and cell death increased by MG-132 in angiomyolipoma cells compared with TSC2 rescued. Intriguingly, while pretreatment of angiomyolipoma cells with RAD001 attenuated CHOP and BiP induction, apoptotic markers cleaved PARP and caspase-3 and eukaryotic translation initiation factor 2α phosphorylation were increased, along with evidence of increased autophagy. These results suggest that human angiomyolipoma cells are uniquely susceptible to agents that exacerbate ER stress and that additional synergy may be achievable with targeted combination therapy.

The liver kinase B1 (LKB1) tumor suppressor inhibits cell growth through its regulation of cellular metabolism and apical-basal polarity. The best understood mechanism whereby LKB1 limits cell growth is through activation of the AMP-activated-protein-kinase/mammalian-target-of-rapamycin (AMPK/mTOR) pathway to control metabolism. As LKB1 is also required for polarized epithelial cells to resist hyperplasia, it is anticipated to function through additional mechanisms. Recently, Yes-associated protein (Yap) has emerged as a transcriptional co-activator that modulates tissue homeostasis in response to cell-cell contact. Thus this study examined a possible connection between Yap and LKB1. Restoration of LKB1 expression in HeLa cells, which lack this tumor suppressor, or short-hairpin RNA knockdown of LKB1 in NTERT immortalized keratinocytes, demonstrated that LKB1 promotes Yap phosphorylation, nuclear exclusion and proteasomal degradation. The ability of phosphorylation-defective Yap mutants to rescue LKB1 phenotypes, such as reduced cell proliferation and cell size, suggest that Yap inhibition contributes to LKB1 tumor suppressor function(s). However, failure of Lats1/2 knockdown to suppress LKB1-mediated Yap regulation suggested that LKB1 signals to Yap via a non-canonical pathway. Additionally, LKB1 inhibited Yap independently of either AMPK or mTOR activation. These findings reveal a novel mechanism whereby LKB1 may restrict cancer cell growth via the inhibition of Yap.