Active GTP-bound Conformation Research Articles

Quaternary organization of the human eEF1B complex reveals unique multi-GEF domain assembly.

Protein synthesis in eukaryotic cell is spatially and structurally compartmentalized that ensures high efficiency of this process. One of the distinctive features of higher eukaryotes is the existence of stable multi-protein complexes of aminoacyl-tRNA synthetases and translation elongation factors. Here, we report a quaternary organization of the human guanine-nucleotide exchange factor (GEF) complex, eEF1B, comprising α, β and γ subunits that specifically associate into a heterotrimeric form eEF1B(αβγ)3. As both the eEF1Bα and eEF1Bβ proteins have structurally conserved GEF domains, their total number within the complex is equal to six. Such, so far, unique structural assembly of the guanine-nucleotide exchange factors within a stable complex may be considered as a ‘GEF hub’ that ensures efficient maintenance of the translationally active GTP-bound conformation of eEF1A in higher eukaryotes.

Targeting Cancer by Using Nanoparticles to Modulate RHO GTPase Signaling.

Functionalized nanomaterials have recently been introduced as efficient vehicles for targeted delivery of drugs and other tailored molecules to cancer cells. They emerge as new opportunities for addressing particular challenging targets such as RHO guanosine triphosphatases (GTPases), a group of signaling molecules involved in the progression of a variety of tumor types. RHO GTPases comprise a subfamily of the Ras superfamily of small GTPases. They are best known for their role in cell migration through the remodeling of the actin cytoskeleton. However, they are also key regulators of a broad number of cellular functions, ranging from proliferation to cell adhesion and differentiation. Not surprisingly, their dysregulation has been implicated in the development and progression of many types of cancer. The RHO GTPase subfamily includes 20 members that can be further separated into typical and atypical RHO GTPases. The typical RHO family members include the classical RHOA, RAC1 and CDC42 proteins, which cycle between an active GTP-bound and inactive GDP-bound conformation, under the coordinated action of three types of regulators: GEFs, GAPs and GDIs. Atypical RHO family members have small changes in key residues that alter their regulatory mechanisms. Nevertheless, both typical and atypical RHO GTPases contribute to cancer progression but, in contrast to Ras proteins, very few mutations have been found in tumors. In most cancers, it is the expression level and/or activity of RHO GTPases that is dysregulated. RHO GTPase signaling has thus long been seen as an attractive target for cancer treatment but their ubiquity and the lack of isoform-specific drugs have posed significant obstacles to the development of viable therapeutic strategies. Based on the success of recent nanomedicine approaches, this chapter reviews representative studies of how functionalized nanoparticles can be designed to target tumor-specific molecules and directly or indirectly modulate the expression and/or activity of particular RHO GTPases in cancer cells.

Role of BAG3 in bovine Deltapapillomavirus-mediated autophagy.

Bovine Deltapapillomavirus genus (δPV), comprises four members that are highly pathogenic and are frequently associated with bladder tumors of adult cattle and water buffaloes. In particular, bovine δPV-2 and δPV-13 are commonly found in urinary bladder tumors in adult large ruminants reared fully or partially on hilly/mountain pasturelands rich in bracken fern (Pteridium spp.) as the urinary bladder of the herbivores is the specific target for bracken genotoxins such as ptaquiloside (PT). PT is a sesquiterpenoid responsible for alkylation of adenine of codon 61 of gene H-Ras, which results in Glutamine 61 substitution that is essential for guanosine triphosphate (GTP) hydrolysis. Glutamine substitution at position 61 impairs the intrinsic GTPase activity. Therefore, active GTP-bound conformations (Ras-GTP) accumulate in cells, thereby causing abnormal cell proliferation and differentiation. The aim of the present study is to stress how bovine δPVs upregulate different forms of selective autophagy, of which BAG3 is a key player. BAG3 plays a central role in autophagy and acts as a multifunctional hub for an interaction network at the cytosolic and mitochondrial level. BAG3 is a functional partner of bovine δPV E5 oncoprotein and forms a complex with molecular chaperones Hsc70/Hsp70/Hsp8B and with cochaperone CHIP. BAG3 interacts with Synpo2. It is believed that this interaction has a crucial role for autophagosome (mitophagosome) formation. Furthermore, in urothelial cells infected by bovine δPVs, BAG3 interacts with parkin and some receptors such as BNIP3/FUNDC1, which suggests that BAG3 is involved in both parkin-dependent and -independent mitophagy that appear upregulate in bladder carcinogenesis of cattle induced by bovine δPVs. Surprisingly, BAG3 interacts also with ERAS, a protein encoded by the ERas gene, a novel member of the RAS family. Unlike in humans, the ERas gene is a functional gene in the cells of adult cattle, and it appears to play a role in bovine BAG3-mediated selective autophagy, including mitophagy observed in urothelial cells spontaneously infected with bovine papillomavirus.

The RND1 Small GTPase: Main Functions and Emerging Role in Oncogenesis.

The Rho GTPase family can be classified into classic and atypical members. Classic members cycle between an inactive Guanosine DiPhosphate -bound state and an active Guanosine TriPhosphate-bound state. Atypical Rho GTPases, such as RND1, are predominantly in an active GTP-bound conformation. The role of classic members in oncogenesis has been the subject of numerous studies, while that of atypical members has been less explored. Besides the roles of RND1 in healthy tissues, recent data suggest that RND1 is involved in oncogenesis and response to cancer therapeutics. Here, we present the current knowledge on RND1 expression, subcellular localization, and functions in healthy tissues. Then, we review data showing that RND1 expression is dysregulated in tumors, the molecular mechanisms involved in this deregulation, and the role of RND1 in oncogenesis. For several aggressive tumors, RND1 presents the features of a tumor suppressor gene. In these tumors, low expression of RND1 is associated with a bad prognosis for the patients. Finally, we highlight that RND1 expression is induced by anticancer agents and modulates their response. Of note, RND1 mRNA levels in tumors could be used as a predictive marker of both patient prognosis and response to anticancer agents.

Regulation of RabGAPs involved in insulin action.

Rab (Ras-related proteins in brain) GTPases are key proteins responsible for a multiplicity of cellular trafficking processes. Belonging to the family of monomeric GTPases, they are regulated by cycling between their active GTP-bound and inactive GDP-bound conformations. Despite possessing a slow intrinsic GTP hydrolysis activity, Rab proteins rely on RabGAPs (Rab GTPase-activating proteins) that catalyze GTP hydrolysis and consequently inactivate the respective Rab GTPases. Two related RabGAPs, TBC1D1 and TBC1D4 (=AS160) have been described to be associated with obesity-related traits and type 2 diabetes in both mice and humans. Inactivating mutations of TBC1D1 and TBC1D4 lead to substantial changes in trafficking and subcellular distribution of the insulin-responsive glucose transporter GLUT4, and to subsequent alterations in energy substrate metabolism. The activity of the RabGAPs is controlled through complex phosphorylation events mediated by protein kinases including AKT and AMPK, and by putative regulatory interaction partners. However, the dynamics and downstream events following phosphorylation are not well understood. This review focuses on the specific role and regulation of TBC1D1 and TBC1D4 in insulin action.

RhoD localization and function is dependent on its GTP/GDP-bound state and unique N-terminal motif

The atypical Rho GTPase RhoD has previously been shown to have a major impact on the organization and function of the actin filament system. However, when first discovered, RhoD was found to regulate endosome trafficking and dynamics and we therefore sought to investigate this regulation in more detail. We found that exogenously expressed RhoD in human fibroblasts localized to vesicles and the plasma membrane and that the active GTP-bound conformation was required for the plasma membrane localization but not for vesicle localization. In contrast to the GTPase deficient atypical Rho GTPases, which have a stalled GTPase activity, RhoD has an elevated intrinsic GDP/GTP exchange activity, rendering the protein constitutively active. Importantly, RhoD can still hydrolyze GTP and we found that an intact GTPase activity was required for efficient fusion of RhoD-positive vesicles. RhoD has a unique N-terminal extension of 14 amino acid residues, which is not present in the classical Rho GTPases RhoA, Cdc42 and Rac1. Deletion of this N-terminal motif often lead to clustering of RhoD positive vesicles, which were found accumulated at the peripheral membrane border. In addition, the number of vesicles per cell was increased manifold, suggesting that the N-terminal motif has an important regulatory role in vesicle dynamics.

High-Throughput Dual Screening Method for Ras Activities and Inhibitors.

Ras GTPases act as "molecular switches", alternating between inactive GDP-bound and active GTP-bound conformation. Ras-oncogenes were discovered over three decades ago, but there are still no effective therapies for Ras-driven cancers. So far, drug discovery strategies have been unsuccessful, because of a lack of suitable screening methodologies and well-defined binding pockets on the Ras proteins. Here, we addressed the former by introducing a homogeneous quenching resonance energy transfer (QRET) technique-based screening strategy for Ras interfacial and competitive inhibitors. We demonstrate that using a unique GTP-specific antibody fragment to monitor GTPase cycling in the presence of a guanine nucleotide exchange factor (GEF) and a GTPase activating protein (GAP) is an efficient method for Ras inhibitor high-throughput screening. When compared to a conventional GEF-stimulated nucleotide exchange assay in a proof-of-concept screen, we identified an overlapping set of potential inhibitor compounds but also compounds found exclusively with the new GTP hydrolysis monitoring-based GTPase cycling assay.

Tools for live imaging of active Rho GTPases in Xenopus.

Rho family GTPases are signaling molecules that orchestrate cytoskeletal dynamics in a variety of cellular processes. Because they effect localized changes to the cytoskeleton only in their active (GTP-bound) conformation, the ability to monitor the active state of Rho GTPases in space and time is critical for understanding their function. Here, we summarize popular tools used for live imaging of active Rho GTPases, outlining advantages and drawbacks of these approaches. Additionally, we highlight key features of the Xenopus laevis embryo that make it well-suited for epithelial cell biology and discuss how application of Rho activity reporters in the Xenopus laevis embryo led to the discovery of a novel phenomenon, junctional Rho flares.

Protein prenylation in islet β-cell function in health and diabetes: Putting the pieces of the puzzle together

Post-translational prenylation involves incorporation of 15-(farnesyl) or 20-(geranylgeranyl) carbon derivatives of mevalonic acid into highly conserved C-terminal cysteines of proteins. The farnesyl transferase (FTase) and the geranylgeranyl transferase (GGTase) mediate incorporation of farnesyl and geranylgeranyl groups, respectively. At least 300 proteins are prenylated in the human genome; the majority of these are implicated in cellular processes including growth, differentiation, cytoskeletal function and vesicle trafficking. From a functional standpoint, isoprenylation is requisite for targeting of modified proteins to relevant cellular compartments for regulation of effector proteins. Pharmacological and molecular biological studies have provided compelling evidence for key roles of this signaling pathway in physiological insulin secretion in normal rodent and human islets. Recent evidence indicates that inhibition of prenylation results in mislocalization of unprenylated proteins, and surprisingly, they remain in active (GTP-bound) conformation. Sustained activation of G proteins has been reported in mice lacking GGTase, suggesting alternate mechanisms for the activation of non-prenylated G proteins. These findings further raise an interesting question if mislocalized, non-prenylated and functionally active G proteins cause cellular pathology since aberrant protein prenylation has been implicated in the onset of cardiovascular disease and diabetes. Herein, we overview the existing evidence to implicate prenylation in islet function and potential defects in this signaling pathways in the diabetic β-cell. We will also identify critical knowledge gaps that need to be addressed for the development of therapeutics to halt defects in these signaling steps in β cells in models of impaired insulin secretion, metabolic stress and diabetes.

Targeting of the Small GTPase Rab6A′ by the Legionella pneumophila Effector LidA

When the bacterium Legionella pneumophila, the causative agent of Legionnaires' disease, is phagocytosed by alveolar macrophages, it delivers a large number of effector proteins through its Dot/Icm type IV secretion system into the host cell cytosol. Among those proteins is LidA, an effector that interacts with several host GTPases of the Rab family, including Rab6A', a regulator of retrograde vesicle trafficking within eukaryotic cells. The effect of LidA on Rab6A' function and the role of Rab6A' for L. pneumophila growth within host cells has been unclear. Here, we show that LidA preferentially binds Rab6A' in the active GTP-bound conformation. Rab6 binding occurred through the central region of LidA and followed a stoichiometry for LidA and Rab6A' of 1:2. LidA maintained Rab6A' in the active conformation by efficiently blocking the hydrolysis of GTP by Rab6A', even in the presence of cellular GTPase-activating proteins, suggesting that the function of Rab6A' must be important for efficient intracellular replication of L. pneumophila. Accordingly, we found that production of constitutively inactive Rab6A'(T27N) but not constitutively active Rab6A'(Q72L) significantly reduced the ability of L. pneumophila to initiate intracellular replication in human macrophages. Thus, the presence of an active pool of Rab6 within host cells early during infection is required to support efficient intracellular growth of L. pneumophila.

Dissociation of Membrane-Anchored Heterotrimeric G-Protein Induced by GαSubunit Binding to GTP

Heterotrimeric G-proteins' activation on the intracellular side of the cell membrane is initiated by stimulation of the G-Protein Coupled Receptors (GPCRs) extra-cellular part. This two-step activation mechanism includes (1) an exchange between GDP and GTP molecules in the G(α) subunit and (2) a dissociation of the whole G(αβγ) complex into two membrane-anchored blocks, namely the isolated G(α) and G(βγ) subunits. Although X-ray data are available for both inactive G(αβγ):GDP and active G(α):GTP complexes, intermediate steps involved in the molecular mechanism of the dissociation have not yet been addressed at the molecular level. In this study, we first built a membrane-anchored intermediate G(iαβγ):GTP complex. This model was then equilibrated by molecular dynamics simulations before the Targeted Molecular Dynamics (TMD) technique was used to force the G(α) subunit to evolve from its inactive (GDP-bound) to its active (GTP-bound) conformations, as described by available X-ray data. The TMD constraint was applied only to the G(α) subunit so that the resulting global rearrangements acting on the whole G(αβγ):GTP heterotrimer could be analyzed. We showed how these mainly local conformational changes of G(α) could initiate large domain:domain motions of the whole complex, the G(βγ) behaving as an almost quasi-rigid block. This separation of the two G(α):GTP and G(βγ) subunits required the loss of several interactions at the G(α):G(βγ) interface that were reported. This study provided an atomistic view of the crucial intermediate step of the G-proteins activation, e.g., the dissociation, that could hardly be elucidated by the experiment.

Rnd3/RhoE Is down-regulated in hepatocellular carcinoma and controls cellular invasion

We performed a review of public microarray data that revealed a significant down-regulation of Rnd3 expression in hepatocellular carcinoma (HCC), as compared to nontumor liver. Rnd3/RhoE is an atypical RhoGTPase family member because it is always under its active GTP-bound conformation and not sensitive to classical regulators. Rnd3 down-regulation was validated by quantitative real-time polymerase chain reaction in 120 independent tumors. Moreover, Rnd3 down-expression was confirmed using immunohistochemistry on tumor sections and western blotting on human tumor and cell-line extracts. Rnd3 expression was significantly lower in invasive tumors with satellite nodules. Overexpression and silencing of Rnd3 in Hep3B cells led to decreased and increased three-dimensional cell motility, respectively. The short interfering RNA-mediated down-regulation of Rnd3 expression induced a loss of E-cadherin at cell-cell junctions that was linked to epithelial-mesenchymal transition through the up-regulation of the zinc finger E-box binding homeobox protein, ZEB2, and the down-regulation of miR-200b and miR-200c. Rnd3 knockdown mediated tumor hepatocyte invasion in a matrix-metalloproteinase-independent, and Rac1-dependent manner. Rnd3 down-regulation provides an invasive advantage to tumor hepatocytes, suggesting that RND3 might represent a metastasis suppressor gene in HCC.

Small inhibitors of ADP-ribosylation factor activation and function in mammalian cells

Small GTP-binding proteins of the ADP-ribosylation factor (Arf) family control various cell functional responses including protein transport and recycling between different cellular compartments, phagocytosis, proliferation, cytoskeletal remodelling, and migration. The activity of Arfs is tightly regulated. GTPase-activating proteins (GAPs) inactivate Arfs by stimulating GTP hydrolysis, and guanine nucleotide exchange factors (GEFs) stimulate the conversion of inactive GDP-bound Arf to the active GTP-bound conformation. There is increasing evidence that Arf small GTPases contribute to cancer growth and invasion. Increased expression of Arf6 and of Arf-GEPs, or deregulation Arf-GAP functions have been correlated with enhanced invasive capacity of tumor cells and metastasis. The spatiotemporal specificity of Arf activation is dictated by their GEFs that integrate various signals in stimulated cells. Brefeldin A (BFA), which inactivates a subset of Arf-GEFs, has been very useful for assessing the function of Golgi-localized Arfs. However, specific inhibitors to investigate the in dividual function of BFA-sensitive and insensitive ArfGEFs are lacking. In recent years, specific screens have been developed, and new inhibitors with improved selectivity and potency to study cell functional responses regulated by BFA-sensitive and BFA-insensitive Arf pathways have been identified. These inhibitors have been instrumental for our understanding of the spatiotemporal activation of Arf proteins in cells and demonstrate the feasibility of developing small molecules interfering with Arf activation to prevent tumor invasion and metastasis.

Off the beaten paths: alternative and crosstalk regulation of Rho GTPases

Rho proteins are small GTPases of the Ras superfamily that regulate a wide variety of biological processes, ranging from gene expression to cell migration. Mechanistically, the major Rho GTPases function as molecular switches cycling between an inactive GDP-bound and an active GTP-bound conformation, although several Rho proteins spontaneously exchange nucleotides or are simply devoid of GTPase activity. For over a decade, RhoGEFs and RhoGAPs have been established as the mainstream regulators of Rho proteins, respectively flipping the switch on or off. However, regulation by GEFs and GAPs leaves several fundamental questions on the operation of the Rho switch unanswered, indicating that the regulation of Rho proteins does not rely exclusively on RhoGEFs and RhoGAPs. Recent evidence indeed suggests that Rho GTPases are finely tuned by multiple alternative regulatory mechanisms, including post-translational modifications and protein degradation, as well as crosstalk mechanisms between Rho proteins. Here we review these alternative mechanisms and discuss how they alter Rho protein function and signaling. We also envision how the classic binary Rho switch may indeed function more like a switchboard with multiple switches and dials that can all contribute to the regulation of Rho protein function.

Potassium-activated GTPase Reaction in the G Protein-coupled Ferrous Iron Transporter B

FeoB is a prokaryotic membrane protein responsible for the import of ferrous iron (Fe(2+)). A defining feature of FeoB is that it includes an N-terminal 30-kDa soluble domain with GTPase activity, which is required for iron transport. However, the low intrinsic GTP hydrolysis rate of this domain appears to be too slow for FeoB either to function as a channel or to possess an active Fe(2+) membrane transport mechanism. Here, we present crystal structures of the soluble domain of FeoB from Streptococcus thermophilus in complex with GDP and with the GTP analogue derivative 2'-(or -3')-O-(N-methylanthraniloyl)-beta,gamma-imidoguanosine 5'-triphosphate (mant-GMPPNP). Unlike recent structures of the G protein domain, the mant-GMPPNP-bound structure shows clearly resolved, active conformations of the critical Switch motifs. Importantly, biochemical analyses demonstrate that the GTPase activity of FeoB is activated by K(+), which leads to a 20-fold acceleration in its hydrolysis rate. Analysis of the structure identified a conserved asparagine residue likely to be involved in K(+) coordination, and mutation of this residue abolished K(+)-dependent activation. We suggest that this, together with a second asparagine residue that we show is critical for the structure of the Switch I loop, allows the prediction of K(+)-dependent activation in G proteins. In addition, the accelerated hydrolysis rate opens up the possibility that FeoB might indeed function as an active transporter.

Establishing a Role for the GTPase Ypt1p at the Late Golgi

GTPases of the Rab family cycle between an inactive (GDP-bound) and active (GTP-bound) conformation. The active form of the Rab regulates a variety of cellular functions via multiple effectors. Guanine nucleotide exchange factors (GEFs) activate Rabs by accelerating the exchange of GDP for GTP, while GTPase activating proteins (GAPs) inactivate Rabs by stimulating the hydrolysis of GTP. The GTPase Ypt1p is required for endoplasmic reticulum (ER)-Golgi and intra-Golgi traffic in the yeast Saccharomyces cerevisiae. Recent findings, however, have shown that Ypt1p GEF, GAP and an effector are all required for traffic from the early endosome to the Golgi. Here we describe a screen for ypt1 mutants that block traffic from the early endosome to the late Golgi, but not general secretion. This screen has led to the identification of a collection of recessive and dominant mutants that block traffic from the early endosome. While it has long been known that Ypt1p regulates the flow of biosynthetic traffic into the cis side of the Golgi, these findings have established a role for Ypt1p in the regulation of early endosome-Golgi traffic. We propose that Ypt1p regulates the flow of traffic into the cis and trans side of the Golgi via multiple effectors.

Tuberous Sclerosis Tumor Suppressor Complex-like Complexes Act as GTPase-activating Proteins for Ral GTPases

The small GTPases RalA and RalB are multifunctional proteins regulating a variety of cellular processes. Like other GTPases, the activity of Ral is regulated by the opposing effects of guanine nucleotide exchange factors (GEFs) and GTPase-activating proteins (GAPs). Although several RalGEFs have been identified and characterized, the molecular identity of RalGAP remains unknown. Here, we report the first molecular identification of RalGAPs, which we have named RalGAP1 and RalGAP2. They are large heterodimeric complexes, each consisting of a catalytic alpha1 or alpha2 subunit and a common beta subunit. These RalGAP complexes share structural and catalytic similarities with the tuberous sclerosis tumor suppressor complex, which acts as a GAP for Rheb. In vitro GTPase assays revealed that recombinant RalGAP1 accelerates the GTP hydrolysis rate of RalA by 280,000-fold. Heterodimerization was required for this GAP activity. In PC12 cells, knockdown of the beta subunit led to sustained Ral activation upon epidermal growth factor stimulation, indicating that the RalGAPs identified here are critical for efficient termination of Ral activation induced by extracellular stimuli. Our identification of RalGAPs will enable further understanding of Ral signaling in many biological and pathological processes.

Evidence for two distinct Mg 2+ binding sites in G sα and G iα1 proteins

The function of guanine nucleotide binding (G) proteins is Mg 2+ dependent with guanine nucleotide exchange requiring higher metal ion concentration than guanosine 5′-triphosphate hydrolysis. It is unclear whether two Mg 2+ binding sites are present or if one Mg 2+ binding site exhibits different affinities for the inactive GDP-bound or the active GTP-bound conformations. We used furaptra, a Mg 2+-specific fluorophore, to investigate Mg 2+ binding to α subunits in both conformations of the stimulatory (G sα) and inhibitory (G iα1) regulators of adenylyl cyclase. Regardless of the conformation or α protein studied, we found that two distinct Mg 2+ sites were present with dissimilar affinities. With the exception of G sα in the active conformation, cooperativity between the two Mg 2+ sites was also observed. Whereas the high affinity Mg 2+ site corresponds to that observed in published X-ray structures of G proteins, the low affinity Mg 2+ site may involve coordination to the terminal phosphate of the nucleotide.

Intracellular Signals as Molecular Biomarkers for Therapeutic Responses in Kras Mutant Myeloid Cells.

Somatic RAS mutations are found in many human cancers, including lung, pancreas, and colon carcinomas, melanoma, and myeloid malignancies. Most of these diseases respond poorly to current treatments. Mutant RAS alleles found in leukemia and other cancers encode proteins that accumulate in a constitutively active GTP-bound conformation, and aberrantly activate downstream effectors such as Raf/MEK/ERK, PI-3K/Akt, and Ral-GDS pathways. To investigate the mechanisms by which hyperactive Ras deregulates myeloid growth, we generated Mx1-Cre , LSL-KrasG12D mice, which harbor a conditional oncogenic KrasG12D allele and uniformly develop a fatal myeloproliferative disease (MPD) after induction of KrasG12D expression (Braun et al., PNAS 10, 597, 2004). In contrast to acute myeloid leukemia (AML), which typically demonstrates multiple mutations, MPD are caused by fewer and even single mutations narrowing the window for targeted therapies. We are exploiting multiparametric FACS analysis as a general strategy to obtain a comprehensive biochemical understanding of mutant signaling networks and to identify and interrogate relevant populations mediating disease initiation and progression. This technique allows us to simultaneously measure intracellular phosphoproteins in phenotypically characterized subpopulations of cells. We have focused on comparing c-kit-positive, lineage-negative (c-kit+/lin-) cells in KrasG12D and normal marrow. This population, which comprises only 2–3% of total bone marrow cells, accounts for 85–90% of progenitors cells. Primary bone marrow from normal and KrasG12D mice with MPD were extracted, serum starved, and stimulated at different time points with granulocyte-macrophage colony stimulating factor (GM-CSF). Kras mutant cells exhibited hyper-phosphorylation not only of MAP kinase effectors (e.g. ERK and Mnk) but also of downstream components of the PI3 kinase cascades such as Akt and S6. We also detected elevated levels of phosphorylated STAT3 and 5 at baseline and after exposure to GM-CSF. In addition, KrasG12D mutant cells showed a more sustained activation of pERK and pS6 over time. Paradoxically, Kras mutant cells showed an intriguing attenuation of the same effectors upon SCF stimulation. Parallel studies showed that inhibiting PI3 kinase interferes with ERK activation, thus revealing crosstalk among pathways. These effects are most evident in defined subsets of stem/progenitor cells that exhibit aberrant proliferation and differentiation in vitro. Finally, we observe dramatic differences in Ras signaling networks with progression from MPD to AML. Our data suggest that oncogenic hyperactive Ras induces rewiring in cellular signaling networks that are not static, but are strongly modulated by secondary mutations. Multiparameter flow cytometry is a robust methodology for analyzing the biochemical profiles of primary cells that can be used to monitor biochemical responses to targeted therapeutics in disease-relevant populations of hematopoietic stem and progenitor cells.

Myelopoiesis Requires a Noncatalytic, Ras-Independent Function of SHP-2.

Activating mutations in PTPN11, which encodes the tyrosine phosphatase SHP-2, comprise the most frequent genetic lesion in juvenile myelomonocytic leukemia (JMML). Other etiologies of JMML include activating mutations in NRAS or KRAS2 and inactivation of the tumor suppressor NF1. These and other observations imply that PTPN11 functions in a common genetic pathway with RAS and NF1. Ras proteins are signal switch molecules that respond to extracellular stimuli by cycling between inactive GDP-bound and active GTP-bound conformations. Oncogenic alleles encode proteins that preferentially accumulate in the GTP-bound form. While NF1 encodes a GTPase activating protein for Ras that directly modulates Ras-GTP levels, the biochemical relationship between SHP-2 phosphatase activity and Ras signaling remains unclear. Most mammalian systems place SHP-2 upstream of Ras activation, but the mechanism is not known. Studies of Ptpn11 mutant embryos and of chimeric mice have shown that SHP-2 plays an essential role in hematopoietic development. We tested the hypothesis that the essential function of SHP-2 in primary hematopoietic cells is to activate Ras. To do this, we determined if Ras activation by expression of an oncogenic Kras2 allele could eliminate the requirement for SHP-2. We used conditional alleles of Kras2 (LSL-KrasG12D) and Ptpn11 (Ptpn11flox/flox) coupled with the inducible Mx1-Cre transgene. Juvenile mice were injected with polyI:polyC, resulting in expression of K-RasG12D and inactivation of Ptpn11. Although these mice uniformly developed fatal MPD similar to what we previously reported in Mx1-Cre, LSL-KrasG12D mice (Braun et al., PNAS 101(2):597–602), myeloid progenitors invariably retained an intact Ptpn11 allele despite uniform activation of the conditional KrasG12D allele. These data suggested that there was strong selective pressure to retain a functional Ptpn11 allele despite oncogenic K-Ras expression. To test this hypothesis directly, we enumerated myeloid progenitor colonies in methylcellulose medium immediately after inactivating Ptpn11 and activating KrasG12D via retroviral transduction. This confirmed a strong dependence on SHP-2 for formation of myeloid colonies either in the presence or absence of KrasG12D. Infecting Ptpn11flox/flox, LSL-KrasG12D cells with a Ptpn11-IRES-Cre virus fully restored the aberrant growth phenotype of KrasG12D mutant cells. Remarkably, alleles encoding phosphatase-deficient SHP-2 proteins also rescued CFU-GM growth. These data indicate that SHP-2 is required for growth of both normal and neoplastic myeloid progenitors in vivo and in vitro. Our data support a model in which SHP-2 has essential hematopoietic functions that are independent of Ras activation and do not require SHP-2 phosphatase activity. The presence of protein-protein interaction domains in SHP-2 suggests that it may have a noncatalytic adaptor function. Because transformation by leukemogenic Ptpn11 alleles requires catalytic activity, our data imply that inhibition of SHP-2 catalysis will selectively target neoplastic hematopoietic progenitors.