scholarly journals Unusual Interplay of Two Types of Ras Activators, RasGRP and SOS, Establishes Sensitive and Robust Ras Activation in Lymphocytes

2007 ◽  
Vol 27 (7) ◽  
pp. 2732-2745 ◽  
Author(s):  
Jeroen P. Roose ◽  
Marianne Mollenauer ◽  
Mary Ho ◽  
Tomohiro Kurosaki ◽  
Arthur Weiss
Keyword(s):  
B Lymphocytes ◽  
Feedback Loop ◽  
Binding Pocket ◽  
Lymphocyte Development ◽  
Ras Signaling ◽  
Receptor Stimulation ◽  
Gtp Binding ◽  
Ras Activation ◽  
Low Levels

ABSTRACT Ras activation is crucial for lymphocyte development and effector function. Both T and B lymphocytes contain two types of Ras activators: ubiquitously expressed SOS and specifically expressed Ras guanyl nucleotide-releasing protein (RasGRP). The need for two activators is enigmatic since both are activated following antigen receptor stimulation. In addition, RasGRP1 appears to be dominant over SOS in an unknown manner. The crystal structure of SOS provides a clue: an unusual allosteric Ras-GTP binding pocket. Here, we demonstrate that RasGRP orchestrates Ras signaling in two ways: (i) by activating Ras directly and (ii) by facilitating priming of SOS with RasGTP that binds the allosteric pocket. Priming enhances SOS' in vivo activity and creates a positive RasGTP-SOS feedback loop that functions as a rheostat for Ras activity. Without RasGRP1, initiation of this loop is impaired because SOS' catalyst is its own product (RasGTP)—hence the dominance of RasGRP1. Introduction of an active Ras-like molecule (RasV12C40) in T- and B-cell lines can substitute for RasGRP function and enhance SOS' activity via its allosteric pocket. The unusual RasGRP-SOS interplay results in sensitive and robust Ras activation that cannot be achieved with either activator alone. We hypothesize that this mechanism enables lymphocytes to maximally respond to physiologically low levels of stimulation.

scholarly journals Isoform-specific Ras signaling is growth factor dependent

2019 ◽  
Vol 30 (9) ◽  
pp. 1108-1117 ◽  
Author(s):  
Fiona E. Hood ◽  
Bertram Klinger ◽  
Anna U. Newlaczyl ◽  
Anja Sieber ◽  
Mathurin Dorel ◽  
...  
Keyword(s):  
Growth Factor ◽  
Cell Lines ◽  
Ectopic Expression ◽  
In Vivo Studies ◽  
Ras Signaling ◽  
Oncogenic Ras ◽  
Ras Activation ◽  
Pathway Activation ◽  
Ras Isoforms

HRAS, NRAS, and KRAS isoforms are almost identical proteins that are ubiquitously expressed and activate a common set of effectors. In vivo studies have revealed that they are not biologically redundant; however, the isoform specificity of Ras signaling remains poorly understood. Using a novel panel of isogenic SW48 cell lines endogenously expressing wild-type or G12V-mutated activated Ras isoforms, we have performed a detailed characterization of endogenous isoform-specific mutant Ras signaling. We find that despite displaying significant Ras activation, the downstream outputs of oncogenic Ras mutants are minimal in the absence of growth factor inputs. The lack of mutant KRAS-induced effector activation observed in SW48 cells appears to be representative of a broad panel of colon cancer cell lines harboring mutant KRAS. For MAP kinase pathway activation in KRAS-mutant cells, the requirement for coincident growth factor stimulation occurs at an early point in the Raf activation cycle. Finally, we find that Ras isoform-specific signaling was highly context dependent and did not conform to the dogma derived from ectopic expression studies.

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

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 635-635
Author(s):  
Benjamin S. Braun ◽  
Joehleen A. Archard ◽  
Wentian Yang ◽  
Gordon Chan ◽  
Benjamin G. Neel ◽  
...  
Keyword(s):  
Phosphatase Activity ◽  
Tyrosine Phosphatase ◽  
Strong Dependence ◽  
Juvenile Myelomonocytic Leukemia ◽  
Ras Signaling ◽  
Activating Mutations ◽  
Ras Activation ◽  
Myeloid Progenitors

Abstract 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.

Regulation of immunoglobulin D synthesis in murine neonatal B lymphocytes

1986 ◽  
Vol 6 (4) ◽  
pp. 1015-1022
Author(s):  
D Yuan
Keyword(s):  
B Lymphocytes ◽  
Polypeptide Chain ◽  
Large Fraction ◽  
State Level ◽  
Nascent Transcript ◽  
Translation Rate ◽  
Immunoglobulin D ◽  
Low Levels ◽  
Lower Expression

We analyzed the regulatory basis for the lower expression of immunoglobulin D (IgD) in lymphocytes from neonatal mice of various ages. The results indicate that the relative transcriptional rate of RNA for delta chains is similar to adult levels even in cells which have not started to express IgD. These results suggest that very early after the initiation of mu gene transcription, a defined fraction of polymerases is programmed to progress through the termination site to the delta exons regardless of the developmental stage of the cell. Similar results were obtained from adult CBA/N mice whose spleens contain a large fraction of cells expressing low levels of IgD. On the other hand, the relative steady state level of mRNA in neonatal lymphocytes is approximately half of that in adults, suggesting that there may be differences in the processing or stability of the nascent transcript. In addition, measurements of the in vivo translation rate show that an inefficient delta polypeptide chain processing machinery in neonatal lymphocytes is also an important factor contributing to the reduced expression of IgD.

scholarly journals Regulation of immunoglobulin D synthesis in murine neonatal B lymphocytes.

1986 ◽  
Vol 6 (4) ◽  
pp. 1015-1022 ◽  
Author(s):  
D Yuan
Keyword(s):  
B Lymphocytes ◽  
Polypeptide Chain ◽  
Large Fraction ◽  
State Level ◽  
Nascent Transcript ◽  
Translation Rate ◽  
Immunoglobulin D ◽  
Low Levels ◽  
Lower Expression

We analyzed the regulatory basis for the lower expression of immunoglobulin D (IgD) in lymphocytes from neonatal mice of various ages. The results indicate that the relative transcriptional rate of RNA for delta chains is similar to adult levels even in cells which have not started to express IgD. These results suggest that very early after the initiation of mu gene transcription, a defined fraction of polymerases is programmed to progress through the termination site to the delta exons regardless of the developmental stage of the cell. Similar results were obtained from adult CBA/N mice whose spleens contain a large fraction of cells expressing low levels of IgD. On the other hand, the relative steady state level of mRNA in neonatal lymphocytes is approximately half of that in adults, suggesting that there may be differences in the processing or stability of the nascent transcript. In addition, measurements of the in vivo translation rate show that an inefficient delta polypeptide chain processing machinery in neonatal lymphocytes is also an important factor contributing to the reduced expression of IgD.

scholarly journals The RASSF1A Tumor Suppressor Binds the RasGAP DAB2IP and Modulates RAS Activation in Lung Cancer

Cancers ◽  
2020 ◽  
Vol 12 (12) ◽  
pp. 3807
Author(s):  
Desmond R. Harrell Stewart ◽  
M. Lee Schmidt ◽  
Howard Donninger ◽  
Geoffrey J. Clark
Keyword(s):  
Lung Cancer ◽  
Tumor Suppressor ◽  
Promoter Hypermethylation ◽  
Negative Regulator ◽  
Ras Signaling ◽  
Mitogenic Signaling ◽  
Protein Levels ◽  
Ras Activation

Lung cancer is the leading cause of cancer-related death worldwide. Lung cancer is commonly driven by mutations in the RAS oncogenes, the most frequently activated oncogene family in human disease. RAS-induced tumorigenesis is inhibited by the tumor suppressor RASSF1A, which induces apoptosis in response to hyperactivation of RAS. RASSF1A expression is suppressed in cancer at high rates, primarily owing to promoter hypermethylation. Recent reports have shown that loss of RASSF1A expression uncouples RAS from apoptotic signaling in vivo, thereby enhancing tumor aggressiveness. Moreover, a concomitant upregulation of RAS mitogenic signaling upon RASSF1A loss has been observed, suggesting RASSF1A may directly regulate RAS activation. Here, we present the first mechanistic evidence for control of RAS activation by RASSF1A. We present a novel interaction between RASSF1A and the Ras GTPase Activating Protein (RasGAP) DAB2IP, an important negative regulator of RAS. Using shRNA-mediated knockdown and stable overexpression approaches, we demonstrate that RASSF1A upregulates DAB2IP protein levels in NSCLC cells. Suppression of RASSF1A and subsequent downregulation of DAB2IP enhances GTP loading onto RAS, thus increasing RAS mitogenic signaling in both mutant- and wildtype-RAS cells. Moreover, co-suppression of RASSF1A and DAB2IP significantly enhances in vitro and in vivo growth of wildtype-RAS cells. Tumors expressing wildtype RAS, therefore, may still suffer from hyperactive RAS signaling when RASSF1A is downregulated. This may render them susceptible to the targeted RAS inhibitors currently in development.

Farnesyl transferase inhibitors: the next targeted therapies for breast cancer?

2004 ◽  
Vol 11 (2) ◽  
pp. 191-205 ◽  
Author(s):  
R M O'Regan ◽  
F R Khuri
Keyword(s):  
Breast Cancer ◽  
Point Mutations ◽  
Receptor Activation ◽  
Ras Signaling ◽  
Farnesyl Transferase ◽  
Ras Mutations ◽  
Ras Activation ◽  
Farnesyl Transferase Inhibitors

The ras family of proto-oncogenes are upstream mediators of several essential cellular signal transduction pathways involved in cell proliferation and survival. Point mutations of ras oncogenes result in constitutively active Ras and have been shown to be oncogenic. However, ras activation can occur in the absence of ras mutations secondary to upstream receptor activation. The first important step in Ras activation is farnesylation by farnesyl transferase, and inhibitors of this enzyme have been demonstrated to inhibit Ras signaling, and have anti-tumor effects. However, it is now clear that farnesyl transferase inhibitors (FTIs) have activity independent of Ras, most likely due to effects on prenylated proteins downstream of Ras, which explains their activity in several malignancies, including breast cancer, where ras mutations are rare. Several FTIs are in clinical development for the treatment of solid tumors. Preclinical evidence suggests that FTIs can inhibit breast cancers in vitro and in vivo, and a phase II trial of the FTI, R115777, in patients with advanced breast cancer produced encouraging results. Based on prior successful outcomes with agents targeting the estrogen and epidermal growth factor receptor pathways in breast cancer, the FTIs, used alone or more likely with other agents, may be the next exciting targeted therapy in breast cancer.

scholarly journals EGFRAP encodes a new negative regulator of the EGFR acting in both normal and oncogenic EGFR/Ras-driven tissue morphogenesis

PLoS Genetics ◽  
2021 ◽  
Vol 17 (8) ◽  
pp. e1009738
Author(s):  
Jennifer Soler Beatty ◽  
Cristina Molnar ◽  
Carlos M. Luque ◽  
Jose F. de Celis ◽  
María D. Martín-Bermudo
Keyword(s):  
Imaginal Disc ◽  
Feedback Loop ◽  
Notch Pathway ◽  
Sh2 Domain ◽  
Negative Regulator ◽  
Ras Signaling ◽  
Ras Pathway ◽  
Phosphorylated Form ◽  
Ras Activation ◽  
Surveillance Mechanism

Activation of Ras signaling occurs in ~30% of human cancers. However, activated Ras alone is insufficient to produce malignancy. Thus, it is imperative to identify those genes cooperating with activated Ras in driving tumoral growth. In this work, we have identified a novel EGFR inhibitor, which we have named EGFRAP, for EGFR adaptor protein. Elimination of EGFRAP potentiates activated Ras-induced overgrowth in the Drosophila wing imaginal disc. We show that EGFRAP interacts physically with the phosphorylated form of EGFR via its SH2 domain. EGFRAP is expressed at high levels in regions of maximal EGFR/Ras pathway activity, such as at the presumptive wing margin. In addition, EGFRAP expression is up-regulated in conditions of oncogenic EGFR/Ras activation. Normal and oncogenic EGFR/Ras-mediated upregulation of EGRAP levels depend on the Notch pathway. We also find that elimination of EGFRAP does not affect overall organogenesis or viability. However, simultaneous downregulation of EGFRAP and its ortholog PVRAP results in defects associated with increased EGFR function. Based on these results, we propose that EGFRAP is a new negative regulator of the EGFR/Ras pathway, which, while being required redundantly for normal morphogenesis, behaves as an important modulator of EGFR/Ras-driven tissue hyperplasia. We suggest that the ability of EGFRAP to functionally inhibit the EGFR pathway in oncogenic cells results from the activation of a feedback loop leading to increase EGFRAP expression. This could act as a surveillance mechanism to prevent excessive EGFR activity and uncontrolled cell growth.

scholarly journals Impaired V(D)J Recombination and Lymphocyte Development in Core RAG1-expressing Mice

2003 ◽  
Vol 198 (9) ◽  
pp. 1439-1450 ◽  
Author(s):  
Darryll D. Dudley ◽  
JoAnn Sekiguchi ◽  
Chengming Zhu ◽  
Moshe J. Sadofsky ◽  
Scott Whitlow ◽  
...  
Keyword(s):  
T Cell ◽  
B Cells ◽  
B Lymphocytes ◽  
Lymphocyte Development ◽  
Cell Numbers ◽  
Developmental Progression ◽  
Ordered Assembly ◽  
B And T Lymphocytes

RAG1 and RAG2 are the lymphocyte-specific components of the V(D)J recombinase. In vitro analyses of RAG function have relied on soluble, highly truncated “core” RAG proteins. To identify potential functions for noncore regions and assess functionality of core RAG1 in vivo, we generated core RAG1 knockin (RAG1c/c) mice. Significant B and T cell numbers are generated in RAG1c/c mice, showing that core RAG1, despite missing ∼40% of the RAG1 sequence, retains significant in vivo function. However, lymphocyte development and the overall level of V(D)J recombination are impaired at the progenitor stage in RAG1c/c mice. Correspondingly, there are reduced numbers of peripheral RAG1c/c B and T lymphocytes. Whereas normal B lymphocytes undergo rearrangement of both JH loci, substantial levels of germline JH loci persist in mature B cells of RAG1c/c mice, demonstrating that DJH rearrangement on both IgH alleles is not required for developmental progression to the stage of VH to DJH recombination. Whereas VH to DJH rearrangements occur, albeit at reduced levels, on the nonselected alleles of RAG1c/c B cells that have undergone D to JH rearrangements, we do not detect VH to DH rearrangements in RAG1c/c B cells that retain germline JH alleles. We discuss the potential implications of these findings for noncore RAG1 functions and for the ordered assembly of VH, DH, and JH segments.

Enhanced Increases in Cytosolic Ca2+ in ADP-Stimulated Platelets from Patients with Delta-Storage Pool Deficiency – A Possible Indicator of Interactions between Granule-Bound ADP and the Membrane ADP Receptor

1997 ◽  
Vol 77 (02) ◽  
pp. 376-382 ◽  
Author(s):  
Bruce Lages ◽  
Harvey J Weiss
Keyword(s):  
Platelet Rich Plasma ◽  
Limited Range ◽  
Dense Granule ◽  
Receptor Desensitization ◽  
Fura 2 ◽  
Storage Pool ◽  
Low Levels ◽  
The Right

SummaryThe possible involvement of secreted platelet substances in agonist- induced [Ca2+]i increases was investigated by comparing these increases in aspirin-treated, fura-2-loaded normal platelets and platelets from patients with storage pool deficiencies (SPD). In the presence and absence of extracellular calcium, the [Ca2+]i response induced by 10 µM ADP, but not those induced by 0.1 unit/ml thrombin, 3.3 µM U46619, or 20 µM serotonin, was significantly greater in SPD platelets than in normal platelets, and was increased to the greatest extent in SPD patients with Hermansky-Pudlak syndrome (HPS), in whom the dense granule deficiencies are the most severe. Pre-incubation of SPD-HPS and normal platelets with 0.005-5 µM ADP produced a dose-dependent inhibition of the [Ca2+]i response induced by 10 µ M ADP, but did not alter the [Ca2+]i increases induced by thrombin or U46619. Within a limited range of ADP concentrations, the dose-inhibition curve of the [Ca2+]i response to 10 µM ADP was significantly shifted to the right in SPD-HPS platelets, indicating that pre-incubation with greater amounts of ADP were required to achieve the same extent of inhibition as in normal platelets. These results are consistent with a hypothesis that the smaller ADP-induced [Ca2+]i increases seen in normal platelets may result from prior interactions of dense granule ADP, released via leakage or low levels of activation, with membrane ADP receptors, causing receptor desensitization. Addition of apyrase to platelet-rich plasma prior to fura-2 loading increased the ADP-induced [Ca2+]i response in both normal and SPD-HPS platelets, suggesting that some release of ADP derived from both dense granule and non-granular sources occurs during in vitro fura-2 loading and platelet washing procedures. However, this [Ca2+]i response was also greater in SPD-HPS platelets when blood was collected with minimal manipulation directly into anticoagulant containing apyrase, raising the possibility that release of dense granule ADP resulting in receptor desensitization may also occur in vivo. Thus, in addition to enhancing platelet activation, dense granule ADP could also act to limit the ADP-mediated reactivity of platelets exposed in vivo to low levels of stimulation.

scholarly journals Real-time observation of tetrapyrrole binding to an engineered bacterial phytochrome

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Yusaku Hontani ◽  
Mikhail Baloban ◽  
Francisco Velazquez Escobar ◽  
Swetta A. Jansen ◽  
Daria M. Shcherbakova ◽  
...  
Keyword(s):  
Real Time ◽  
Rational Design ◽  
Near Infrared ◽  
Fluorescent Proteins ◽  
Covalent Bond ◽  
Binding Pocket ◽  
Tissue Imaging ◽  
Covalent Attachment ◽  
Time Resolved

AbstractNear-infrared fluorescent proteins (NIR FPs) engineered from bacterial phytochromes are widely used for structural and functional deep-tissue imaging in vivo. To fluoresce, NIR FPs covalently bind a chromophore, such as biliverdin IXa tetrapyrrole. The efficiency of biliverdin binding directly affects the fluorescence properties, rendering understanding of its molecular mechanism of major importance. miRFP proteins constitute a family of bright monomeric NIR FPs that comprise a Per-ARNT-Sim (PAS) and cGMP-specific phosphodiesterases - Adenylyl cyclases - FhlA (GAF) domain. Here, we structurally analyze biliverdin binding to miRFPs in real time using time-resolved stimulated Raman spectroscopy and quantum mechanics/molecular mechanics (QM/MM) calculations. Biliverdin undergoes isomerization, localization to its binding pocket, and pyrrolenine nitrogen protonation in <1 min, followed by hydrogen bond rearrangement in ~2 min. The covalent attachment to a cysteine in the GAF domain was detected in 4.3 min and 19 min in miRFP670 and its C20A mutant, respectively. In miRFP670, a second C–S covalent bond formation to a cysteine in the PAS domain occurred in 14 min, providing a rigid tetrapyrrole structure with high brightness. Our findings provide insights for the rational design of NIR FPs and a novel method to assess cofactor binding to light-sensitive proteins.

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