Flow-Cytometric Phosphoprotein Analysis Reveals Agonist and Temporal Differences in Responses of Murine Hematopoietic Stem/Progenitor Cells

Hematopoietic stem cells (HSCs) are probably the best-studied adult tissue-restricted stem cells. Although methods for flow cytometric detection of phosphoproteins in hematopoeitic progenitors and mature cells are available, analogous protocols for HSC are lacking. We present a robust method to study intracellular signaling in immunophenotypically-defined murine HSC/progenitor cell (HPC)-enriched populations. Using this method, we uncover differences in the response dynamics of several phosphoproteins representative of the Ras/MAP-Kinase(K), PI3K, mTOR and Jak/STAT pathways in HSC/HPCs stimulated by Scf, Thpo, as well as several other important HSC/HPC agonists.     

Phosphoproteomics identifies oncogenic Ras signaling targets and their involvement in lung adenocarcinomas

Background

Ras is frequently mutated in a variety of human cancers, including lung cancer, leading to constitutive activation of MAPK signaling. Despite decades of research focused on the Ras oncogene, Ras-targeted phosphorylation events and signaling pathways have not been described on a proteome-wide scale.

Methodology/Principal Findings

By functional phosphoproteomics, we studied the molecular mechanics of oncogenic Ras signaling using a pathway-based approach. We identified Ras-regulated phosphorylation events (n = 77) using label-free comparative proteomics analysis of immortalized human bronchial epithelial cells with and without the expression of oncogenic Ras. Many were newly identified as potential targets of the Ras signaling pathway. A majority (∼60%) of the Ras-targeted events consisted of a [pSer/Thr]-Pro motif, indicating the involvement of proline-directed kinases. By integrating the phosphorylated signatures into the Pathway Interaction Database, we further inferred Ras-regulated pathways, including MAPK signaling and other novel cascades, in governing diverse functions such as gene expression, apoptosis, cell growth, and RNA processing. Comparisons of Ras-regulated phosphorylation events, pathways, and related kinases in lung cancer-derived cells supported a role of oncogenic Ras signaling in lung adenocarcinoma A549 and H322 cells, but not in large cell carcinoma H1299 cells.

Conclusions/Significance

This study reveals phosphorylation events, signaling networks, and molecular functions that are regulated by oncogenic Ras. The results observed in this study may aid to extend our knowledge on Ras signaling in lung cancer.     

Serine 338 phosphorylation is dispensable for activation of c-Raf1

Numerous extracellular agonists induce consecutive stimulation of Ras guanine nucleotide exchange factors, Ras and c-Raf1, as the starting point of the intracellular mitogen-activated protein kinase cascade. Recent data point to a more complex reaction pattern of this simple sequence. This study was aimed at elucidating the activation process of endogenous c-Raf1 in U937 cells. Treatment of permeabilized U937 cells with the nonhydrolyzable nucleotide guanosine 5'-3-O-(thio)triphosphate (GTPgammaS) induced prolonged stimulation of Ras and c-Raf1 activity. Intriguingly, both signaling proteins expressed differential responses toward specific inhibitors of phosphoinositide 3-kinases and tyrosine kinases, which indicates diverse signaling reactions feeding into Ras and cRaf-1. Phosphorylation of c-Raf1 serine 338 by p21-activated kinase has been recently reported to contribute to phosphoinositide 3-kinase-dependent activation of c-Raf1. However, in U937 cells stimulation of c-Raf1 activity by GTPgammaS did not correlate with p21-activated kinase activity and Ser-338 phosphorylation. Thus Ser-338 phosphorylation appears dispensable for c-Raf1 activation under the conditions used. Together these data deny an essential role for serine 338 phosphorylation in c-Raf1 activation and disclose divergent signaling connections of Ras and c-Raf1 in U937 cells.     

Ras signaling contributes to survival of human T-cell leukemia/lymphoma virus type 1 (HTLV-1) Tax-positive T-cells

Ras signaling pathways play an important role in cellular proliferation and survival, and inappropriate activation of Ras frequently results in cell transformation and cancer. Human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is the etiological agent of the adult T-cell leukemia/lymphoma (ATLL), a severe malignancy that has a poor prognosis and exhibits resistance to conventional chemotherapy. Although the mechanisms involved in cell transformation by HTLV-1 have not been completely clarified, it is generally thought that Tax plays a pivotal role in the process. We have previously proposed that a functionally active Ras protein is needed for efficient anti-apoptotic activity of Tax. In this study we report data indicating that the apoptotic resistance of cells expressing Tax, constitutively or transiently, is linked to the intracellular levels of Ras-GTP. Indeed, we found that Tax-positive cells have a high content of active Ras, and that inhibition of Ras signaling, using the antagonist farnesyl thyosalicylic acid (FTS), increases their sensitivity to apoptosis. FTS treatment was also accompanied by a decrease in ERK, but not Akt, phosphorylation. Thus, all together our data suggest that the interaction between Tax and Ras could be important to ATLL pathogenesis, and indicate Ras as a possible target for therapeutic intervention in ATLL patients.     

Regulation of Hxt3 and Hxt7 Turnover Converges on the Vid30 Complex and Requires Inactivation of the Ras/cAMP/PKA Pathway in Saccharomyces cerevisiae

Eukaryotic cells adjust their intracellular protein complement as a mechanism to adapt to changing environmental signals. In Saccharomyces cerevisiae the hexose transporters Hxt3 and Hxt7 are expressed and function on the plasma membrane in high and low glucose abundance, respectively. By contrast, Hxt3 is endocytosed and degraded in the vacuole when cells are starved of glucose and Hxt7 in response to rapamycin treatment or when nitrogen is limiting. Yeast uses several signaling pathways, including the TORC1 and Ras/cAMP/Protein Kinase A (PKA) pathways, to adapt to nutrient changes in the environment. The multi-protein Vid30 complex (Vid30c), an E3 ubiquitin ligase required for the degradation of FBPase, assists in this adaptation process in a mechanism that is poorly understood. Here we show the endocytosis and the subsequent degradation of both Hxt3 and Hxt7, in response to different nutrient signals, is dependent on components of the Vid30c. Additionally, we define the signaling events required for the turnover of Hxt3 and Hxt7 by showing that Hxt3 turnover requires Ras2 and PKA inactivation, whereas Hxt7 turnover requires TORC1 and Ras2 inactivation. Further investigation led us to identify Rim15, a kinase that is inhibited by both the TORC1 and Ras/cAMP/PKA pathways, as a key downstream effector in signaling both turnover events. Finally, we show that the turnover of both Hxt3 and Hxt7 is dependent on the essential E3 ubiquitin ligase, Rsp5, indicating that the role of the Vid30c might be indirect of Hxt ubiquitylation.     

The Ras‐ERK pathway: Understanding site‐specific signaling provides hope of new anti‐tumor therapies

Recent discoveries have suggested the concept that intracellular signals are the sum of multiple, site‐specified subsignals, rather than single, homogeneous entities. In the context of cancer, searching for compounds that selectively block subsignals essential for tumor progression, but not those regulating “house‐keeping” functions, could help in producing drugs with reduced side effects compared to compounds that block signaling completely. The Ras‐ERK pathway has become a paradigm of how space can differentially shape signaling. Today, we know that Ras proteins are found in different plasma membrane microdomains and endomembranes. At these localizations, Ras is subject to site‐specific regulatory mechanisms, distinctively engaging effector pathways and switching‐on diverse genetic programs to generate different biological responses. The Ras effector pathway leading to ERKs activation is also under strict, space‐related regulatory processes. These findings may open a gate for aiming at the Ras‐ERK pathway in a spatially restricted fashion, in our quest for new anti‐tumor therapies.     

A rit GTPase-p38 mitogen-activated protein kinase survival pathway confers resistance to cellular stress

Cells mobilize diverse signaling cascades to protect against stress-mediated injury. Ras family GTPases play a pivotal role in cell fate determination, serving as molecular switches to control the integration of multiple signaling pathways. p38 mitogen-activated protein kinase (MAPK) signaling serves as a critical fulcrum in this process, regulating networks that stimulate cellular apoptosis but also have the capacity to promote cell survival. However, relatively little is known concerning this functional dichotomy, particularly the regulation of p38-dependent survival pathways. Here, we demonstrate that the Rit GTPase promotes cell survival by directing an unexpected p38 MAPK-dependent AKT survival pathway. Following stress exposure, Rit small hairpin RNA interference (shRNAi)-treated cells display increased apoptosis and selective disruption of p38 MAPK signaling, while expression of constitutively activated Rit promotes p38-AKT-dependent cell survival. Rit, but not Ras or Rap GTPases, can associate with, and is critical for, stress-mediated activation of the scaffolded p38-MK2-HSP27-AKT prosurvival signaling complex. Together, our studies establish Rit as a central regulator of a p38 MAPK-dependent signaling cascade that functions as a critical cellular survival mechanism in response to stress.     

The voyage of stem cell toward terminal differentiation: a brief overview

Presently, worldwide attempts are being made to apply stem cells and stem cell-derived products to a wide range of clinical applications and for the development of cell-based therapies. In order to harness stem cells and manipulate them for therapeutic application, it is very important to understand the basic biology of stem cells and identify the factors that govern the dynamics of these cells in the body. Several signaling pathways have emerged as key regulators of stem cells. Some of these signaling pathways regulate the stem cell's proliferative capacity and therefore act as direct regulators of the stem cell, whereas others are involved in shaping and maintaining the stem cell niche and therefore act as indirect regulators of the stem cell. It is difficult to identify which signaling pathways critically affect the stem cell's behavior and which are important for maintaining the quiescent population. A stem cell receives different extrinsic signals compared with the bulk population and responds to them differently. In order to manipulate these adult cells for therapeutic approaches it is crucial to identify how signaling pathways regulate stem cells either directly by regulating proliferative status or indirectly by influencing the niche. The main challenge is to identify whether different factors provide diverse extrinsic signals to the stem cell and its daughter cell population, or whether there are intrinsic differences in stem cell and daughter cell populations that is reflected in their behavior. In this study, we will focus on the various aspects of stem cell biology and differentiation, as well as exploring the potential strategies to intervene the differentiation process in order to obtain the desired yield of cells applicable in regenerative medicine.     

Thyrotropin-induced mitogenesis is Ras dependent but appears to bypass the Raf-dependent cytoplasmic kinase cascade.

Cellular growth control requires the coordination and integration of multiple signaling pathways which are likely to be activated concomitantly. Mitogenic signaling initiated by thyrotropin (TSH) in thyroid cells seems to require two distinct signaling pathways, a cyclic AMP (cAMP)-dependent signaling pathway and a Ras-dependent pathway. This is a paradox, since activated cAMP-dependent protein kinase disrupts Ras-dependent signaling induced by growth factors such as epidermal growth factor and platelet-derived growth factor. This inhibition may occur by preventing Raf-1 protein kinase from binding to Ras, an event thought to be necessary for the activation of Raf-1 and the subsequent activation of the mitogen-activated protein (MAP)/extracellular signal-regulated kinase (ERK) kinases (MEKs) and MAP kinase (MAPK)/ERKs. Here we report that serum-stimulated hyperphosphorylation of Raf-1 was inhibited by TSH treatment of Wistar rat thyroid cells, indicating that in this cell line, as in other cell types, increases in intracellular cAMP levels inhibit activation of downstream kinases targeted by Ras. Ras-stimulated expression of genes containing AP-1 promoter elements was similarly inhibited by TSH. On the other hand, stimulation of thyroid cells with TSH resulted in stimulation of DNA synthesis which was Ras dependent but both Raf-1 and MEK independent. We also show that Ras-stimulated DNA synthesis required the use of this kinase cascade in untreated quiescent cells but not in TSH-treated cells. These data suggest that in TSH-treated thyroid cells, Ras might be able to signal through effectors other than the well-studied cytoplasmic kinase cascade.     

Fibroblast growth factor receptor 2 phosphorylation on serine 779 couples to 14-3-3 and regulates cell survival and proliferation

The fibroblast growth factors (FGFs) exert their diverse (or pleiotropic) biological responses through the binding and activation of specific cell surface receptors (FGFRs). While FGFRs are known to initiate intracellular signaling through receptor tyrosine phosphorylation, the precise mechanisms by which the FGFRs regulate pleiotropic biological responses remain unclear. We now identify a new mechanism by which FGFR2 is able to regulate intracellular signaling and cellular responses. We show that FGFR2 is phosphorylated on serine 779 (S779) in response to FGF2. S779, which lies adjacent to the phospholipase Cgamma binding site at Y766, provides a docking site for the 14-3-3 phosphoserine-binding proteins and is essential for the full activation of the phosphatidylinositol 3-kinase and Ras/mitogen-activated protein kinase pathways. Furthermore, S779 signaling is essential for promoting cell survival and proliferation in both Ba/F3 cells and BALB/c 3T3 fibroblasts. This new mode of FGFR2 phosphoserine signaling via the 14-3-3 proteins may provide an increased repertoire of signaling outputs to allow the regulation of pleiotropic biological responses. In this regard, we have identified conserved putative phosphotyrosine/phosphoserine motifs in the cytoplasmic domains of diverse cell surface receptors, suggesting that they may perform important functional roles beyond the FGFRs.     

Modulation of intracellular signaling pathways to induce apoptosis in prostate cancer cells

An understanding of the molecular pathways defining the susceptibility of prostate cancer, especially refractory prostate cancer, to apoptosis is the key for developing a cure for this disease. We previously demonstrated that up-regulating Ras signaling, together with suppression of protein kinase C (PKC), induces apoptosis. Dysregulation of various intracellular signaling pathways, including those governed by Ras, is the important element in the development of prostate cancer. In this study, we tested whether it is possible to modulate the activities of these pathways and induce an apoptotic crash among them in prostate cancer cells. Our data showed that DU145 cells express a high amount of JNK1 that is phosphorylated after endogenous PKC is suppressed, which initiates caspase 8 cleavage and cytochrome c release, leading to apoptosis. PC3 and LNCaP cells contain an activated Akt. The inhibition of PKC further augments Akt activity, which in turn induces ROS production and the accumulation of unfolded proteins in the endoplasmic reticulum, resulting in cell death. However, the concurrent activation of JNK1 and Akt, under the condition of PKC abrogation, dramatically augment the magnitude of apoptosis in the cells. Thus, our study suggests that Akt, JNK1, and PKC act in concert to signal the intracellular apoptotic machinery for a full execution of apoptosis in prostate cancer cells.     

Vasoactive intestinal peptide-induced neurite remodeling in human neuroblastoma SH-SY5Y cells implicates the Cdc42 GTPase and is independent of Ras-ERK pathway

Vasoactive intestinal peptide (VIP) is known to regulate proliferation or differentiation in normal and tumoral cells. SH-SY5Y is a differentiated cell subclone derived from the SK-N-SH human neuroblastoma cell line and possess all the components for an autocrine action of VIP. In the present study, we investigated the morphological changes and intracellular signaling pathways occurring upon VIP treatment of SH-SY5Y cells. VIP induced an early remodeling of cell projections: a branched neurite network spread out and prominent varicosities developed along neurites. Although activated by VIP, the Ras/ERK pathway was not required for the remodeling process. In contrast, pull-down experiments revealed a strong Cdc42 activation by VIP while expression of a dominant-negative Cdc42 prevented the VIP-induced neurite changes, suggesting an important role for this small GTPase in the process. These data provide the first evidence for a regulation of the activity of Rho family GTPases by VIP and bring new insights in the signaling pathways implicated in neurite remodeling process induced by VIP in neuroblastoma cells.     

The Ras/Raf/MEK/extracellular signal-regulated kinase pathway induces autocrine-paracrine growth inhibition via the leukemia inhibitory factor/JAK/STAT pathway

Sustained activation of the Ras/Raf/MEK/extracellular signal-regulated kinase (ERK) pathway can lead to cell cycle arrest in many cell types. We have found, with human medullary thyroid cancer (MTC) cells, that activated Ras or c-Raf-1 can induce growth arrest by producing and secreting an autocrine-paracrine factor. This protein was purified from cell culture medium conditioned by Raf-activated MTC cells and was identified by mass spectrometry as leukemia inhibitory factor (LIF). LIF expression upon Raf activation and subsequent activation of JAK-STAT3 was also observed in small cell lung carcinoma cells, suggesting that this autocrine-paracrine signaling may be a common response to Ras/Raf activation. LIF was sufficient to induce growth arrest and differentiation of MTC cells. This effect was mediated through the gp130/JAK/STAT3 pathway, since anti-gp130 blocking antibody or dominant-negative STAT3 blocked the effects of LIF. Thus, LIF expression provides a novel mechanism allowing Ras/Raf signaling to activate the JAK-STAT3 pathway. In addition to this cell-extrinsic growth inhibitory pathway, we find that the Ras/Raf/MEK/ERK pathway induces an intracellular growth inhibitory signal, independent of the LIF/JAK/STAT3 pathway. Therefore, activation of the Ras/Raf/MEK/ERK pathway can lead to growth arrest and differentiation via at least two different signaling pathways. This use of multiple pathways may be important for "fail-safe" induction and maintenance of cell cycle arrest.     

The Rap1 GTPase functions as a regulator of morphogenesis in vivo.

The Ras-related Rap GTPases are highly conserved across diverse species but their normal biological function is not well understood. Initial studies in mammalian cells suggested a role for Rap as a Ras antagonist. More recent experiments indicate functions in calcium- and cAMP-mediated signaling and it has been proposed that protein kinase A-mediated phosphorylation activates Rap in vivo. We show that Ras1-mediated signaling pathways in Drosophila are not influenced by Rap1 levels, suggesting that Ras1 and Rap1 function via distinct pathways. Moreover, a mutation that abolishes the putative cAMP-dependent kinase phosphorylation site of Drosophila Rap1 can still rescue the Rap1 mutant phenotype. Our experiments show that Rap1 is not needed for cell proliferation and cell-fate specification but demonstrate a critical function for Rap1 in regulating normal morphogenesis in the eye disk, the ovary and the embryo. Rap1 mutations also disrupt cell migrations and cause abnormalities in cell shape. These findings indicate a role for Rap proteins as regulators of morphogenesis in vivo.     

Src tyrosine kinase-induced loss of luteinizing hormone responsiveness is via a Ras-dependent,phosphatidylinositol-3-kinase independent pathway

Gonadotropins stimulate gonadal cell steroid secretion primarily through activation of a cAMP-protein kinase A signal transduction pathway. Various growth factors have been shown to inhibit gonadotropin-stimulated steroidogenesis, however, the intracellular signaling cascades involved in growth factor inhibition have not been characterized. The present study investigated whether Src tyrosine kinase, a nonreceptor tyrosine kinase activated in response to growth factor stimulation and previously shown to inhibit LH-stimulated progesterone secretion, acts via activation of Ras stimulated pathways, phosphatidylinositol-3-kinase (PI3-kinase) stimulated pathways, or both in MA10 Leydig cells. Direct activation of Src in MA10 cells that express a temperature sensitive Src was associated with an increase in GTP-bound Ras, indicating increased Ras activity in response to Src activation. Direct activation of Ras by way of expression of a constitutively active Ras (Ras+) was associated with a decrease in LH responsiveness. Coexpression of a dominant negative Src, which by itself increases LH responsiveness in MA10 cells, had no effect on Ras+ inhibition on LH responsiveness, further demonstrating that Src is upstream of Ras. In addition, MA10(Ras+) cells were relatively unresponsive to cholera toxin or 8-bromo cAMP, indicating the effects of Ras are independent of cAMP generation. Wortmannin, a PI3-kinase inhibitor, did not restore LH responsiveness to cells expressing activated Src or constitutively active Ras. These results demonstrate that Src activates a Ras pathway in MA10 Leydig cells, and that activation of Ras is associated with a loss of LH responsiveness that is independent of PI3-kinase.