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Jeffrey Settleman - One of the best experts on this subject based on the ideXlab platform.
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Loss of the Rho GTPase activating Protein P190-B enhances hematopoietic stem cell engraftment potential
Blood, 2009Co-Authors: Satyam Eleswarapu, Jeffrey Settleman, Kathleen Szczur, Hartmut Geiger, Yi Zheng, Edward F. Srour, David A. Williams, Deidre Daria, Marie-dominique FilippiAbstract:Hematopoietic stem cell (HSC) engraftment is a multistep process involving HSC homing to bone marrow, self-renewal, proliferation, and differentiation to mature blood cells. Here, we show that loss of P190-B RhoGTPase activating Protein, a negative regulator of Rho GTPases, results in enhanced long-term engraftment during serial transplantation. This effect is associated with maintenance of functional HSC-enriched cells. Furthermore, loss of P190-B led to marked improvement of HSC in vivo repopulation capacity during ex vivo culture without altering proliferation and multilineage differentiation of HSC and progeny. Transcriptional analysis revealed that P190-B deficiency represses the up-regulation of p16Ink4a in HSCs in primary and secondary transplantation recipients, providing a possible mechanism of P190-B–mediated HSC functions. Our study defines P190-B as a critical transducer element of HSC self-renewal activity and long-term engraftment, thus suggesting that P190-B is a target for HSC-based therapies requiring maintenance of engraftment phenotype.
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Absence of the Rho GTPase Activating Protein P190-B Enhances Long Term Hematopoietic Stem Cell Engraftment
Blood, 2008Co-Authors: Hartmut Geiger, Jeffrey Settleman, Kathleen Szczur, Yi Zheng, Edward F. Srour, David A. Williams, Deidra Deira, Marie-dominique FilippiAbstract:Hematopoietic stem cell (HSC) engraftment is a multistep process involving HSC homing to bone marrow (BM), self-renewal, proliferation and differentiation to mature blood cells. However, the molecular regulation of HSC engraftment is still poorly defined. Small Rho GTPases are critical regulator of cell migration, proliferation and differentiation in multiple cell types. While their role in HSC functions has begun to be understood, the role of their regulator in vivo has been understudied. P190-B GTPase Activating Protein (GAP), a negative regulator of Rho activity, has been implicated in regulating cell size and adipogenesis-myogenesis cell fate determination during fetal development (Sordella, Dev Cell, 2002; Cell 2003). Here, we investigated the role of P190-B in HSC/P engraftment. Since mice lacking P190-B die before birth, serial competitive repopulation assay was performed using fetal liver (FL) tissues from day E14.5 WT and P190-B −/− embryos. WT and P190-B −/− FL cells exhibited similar levels of engraftment in primary recipients. However, the level of contribution of P190-B −/− cells to peripheral blood and bone marrow was maintained between the primary and secondary recipients and still easily detectable in tertiary recipients, while the level of contribution of FL WT cells dramatically decreased with successive serial transplantion and was barely detectable in tertiary recipients. The contribution to T cell, B cell and myeloid cell reconstitution was similar between the genotypes. A pool of HSC was maintained in serially transplanted P190-B −/− animals, since Lin neg Sca pos Kit pos (LSK) cells were still present in the BM of P190-B −/− secondary engrafted mice while this population disappeared in WT controls. Importantly, this enhanced long term engraftment was due to a difference in the functional capacity of P190-B −/− HSC compared to WT HSC since highly enriched P190-B −/− HSC (LSK) demonstrated similar enhanced serial transplantation potential. Because previous studies have suggested that the loss of long term function of HSC during serial transplantation can depend, at least in part, on the upregulation of the cyclin dependent kinase inhibitor p16 Ink4a (Ito et al, Nat Med 2006), the expression of p16 Ink4a was examined during serial transplantation. While expression of p16 Ink4a increased in WT HSC in primary and secondary recipients, p16 Ink4a remained low in P190-B −/− HSC, which indicated that P190-B-deficiency represses the upregulation of p16 Ink4a in HSC in primary and secondary transplant recipients. This provides a possible mechanism of P190-B-mediated HSC functions. We next examined whether P190-B-deficiency may preserve the repopulating capacity of HSC/P during ex vivo cytokine-induced culture. While freshly isolated LSK cells from WT and P190-B −/− mice exhibited comparable intrinsic clonogenic capacity, the frequency of colony-forming unit after 7 days in culture was 2 fold-higher in P190-B −/− compared with WT cultures, resulting in a net CFU expansion. Furthermore, competitive repopulation assays showed significantly higher repopulating activity in mice that received P190-B −/− cultured cells compared with WT cells equivalent to a 4.4-fold increase in the estimated frequency of repopulating units. Interestingly, P190-deficiency did not alter cell cycling rate or survival both in vivo and in vitro. Therefore, P190-B-deficiency maintains key HSC functions either in vivo or in ex vivo culture without altering cycling rate and survival of these cells. These findings define P190-B as a critical regulator of HSC functions regulating self renewal activity while maintaining a balance between proliferation and differentiation.
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Loss of the Rho GTPase Activating Protein P190-B Enhances Hematopoietic Stem Cell Engraftment Potential.
Blood, 2007Co-Authors: Harmut Geiger, Jeffrey Settleman, Kathleen Szczur, Yi Zheng, Deidra Deira, David Williams, Marie-dominique FilippiAbstract:Abstract Hematopoietic stem cell (HSC) engraftment is a multistep process involving HSC homing to bone marrow (BM), self-renewal, proliferation and differentiation to mature blood cells. However, the molecular regulation of HSC engraftment is still poorly defined. Small Rho GTPases are critical regulator of cell migration, proliferation and differentiation in multiple cell types. P190-B GTPase Activating Protein (GAP) which regulates RhoA activity has been implicated in IGF-1 signaling via cAmp-response element binding Proteins (CREB) activity regulating cell size during fetal development (Sordella, Dev Cell, 2002). Since IGF-1 and CREB play important roles in hematopoiesis, we hypothesize that P190-B is critical for HSC functions. Mice lacking P190-B die before birth. P190-B−/− embryos at 14.5 day post coitum exhibited fetal liver (FL) hypocellularity associated with profound anemia. Surprisingly, P190-B−/− FL showed normal frequency of myeloid colonies and a two-fold increase in day 28 and 35 CAFC frequency compared to WT. Overall, the number of progenitors per P190-B−/− liver was decreased while the number of HSC remained unchanged compared to WT FL, suggesting maintenance of the HSC pool in P190-B−/− FL despite the anemia. The repopulating capacity of P190-B−/− FL was assessed by serial competitive repopulation assay. Short-term repopulating ability of P190-B−/− FL was modestly increased compared to WT as assessed by peripheral blood (PB) chimerism 3 months post-transplant (58.3 ± 3.5% vs 45.5±5.4%, p<0.05, respectively). Remarkably, in secondary and tertiary recipients, P190-B−/− FL exhibited dramatically enhanced engraftment compared to WT (65.9±6.4% vs 29.3±6%, p<0.01; 21.7±7.9% vs 1.5±0.5%, p<0.05, respectively). The contribution to T cell, B cell and myeloid cell reconstitution was similar between the genotypes. BM analysis of secondary recipients revealed loss of the HSC pool in WT engrafted animals, while HSC frequency was near normal in P190-B−/− engrafted mice (LinnegScaposKitpos frequency: 0.034±0.019% vs 0.219±0.110%, p<0.05, respectively). Importantly, this enhanced long term engraftment was due to a difference in the functional capacity of P190-B−/− HSC compared to WT HSC since highly enriched P190-B−/− HSC (LSKMac1low) demonstrated similar enhanced engraftment in the BM 4 months post transplant. P190-B-mediated enhanced HSC engraftment was associated with normal proliferation and differentiation of P190-B−/− progenitors. Interestingly, a pool of quiescent HSC was maintained in serially transplanted P190-B−/− animals, since Pyronin Yneg LSK cells were still present in the BM of P190-B−/− engrafted mice while this population disappeared in WT controls. P190-B−/− FL cells also exhibited increased colony-forming unit homing to BM compared to WT controls. Together, these results suggest that loss of P190-B is associated with HSC quiescence and more efficient homing of HSCs that may contribute to long term regeneration of the hematopoietic stem cell pool after transplantation.
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P190-B RhoGAP Is Critical for Hematopoietic Stem/Progenitor Cell Homing and Short Term Engraftment.
Blood, 2006Co-Authors: Kathleen Szczur, Jeffrey Settleman, Yi Zheng, David A. Williams, Marie-dominique FilippiAbstract:Hematopoietic stem/progenitor cell (HSC/P) engraftment is a complex process which requires HSC/P to migrate across endothelium barrier from the blood towards the bone marrow (BM) cavities. The cellular and molecular mechanisms that regulate HSC/P engraftment are still poorly understood. Rho GTPases, Rac1, Rac2, CDC42 and RhoA, are major regulators of cell adhesion and migration via cytoskeleton rearrangement. While the roles of Rac and CDC42 in HSC/P functions have begun to be understood (Gu, Science 2003; Wang, Blood 2006), the role of RhoA has yet to be examined in physiological settings. To examine the role of RhoA in HSC/P engraftment, we used mice genetically deficient in the Rho-inhibitory Protein P190-B RhoGAP (P190-B), which represent gain of RhoA activity (Sordella, Dev Cell, 2000). P190-B-deficient c-Kit + cells demonstrated increased short-term engraftment in non-obese diabetic/severe combined-immunodeficiency (NOD/SCID) mice compared to WT cells. In addition, P190-B −/− was associated with increased colony forming-unit (CFU) homing to BM compared with WT (7.5%±1.8% vs 4.2%±0.6%, p −/− FL cells showed significantly increased CFU adhesion to fibronectin (FN) compared to WT FL cells (11.2%±1.2% vs 8.5%±1.02%, p −/− cells exhibited significantly increased CFU migration towards SDF-1α in the presence of FN (44.9%±1.3% vs 25.9%±4.2%, p + WT cells polarized with a single F-actin lamellipodia at the leading edge and a short uropod. In contrast, P190-B −/− showed multiple membrane protrusions of F-actin at the leading edge associated with abnormally elongated uropod in a significantly number of cells (16% vs 1%, p −/− cells F-actin was assembled in a ring structure at the leading edge. Thus, RhoA may regulate HSC/P migration and homing by stimulating membrane protrusions via integrin signaling. HSC/P migration into BM cavities is restricted to the cells in the G 0 /G 1 -phase of the cell cycle. This restriction may be dependent on adhesion properties of these cells (Giet, Blood 1997; Orchell-Traycoff, Blood 2000). RhoA has been implicated in the coordination of cell cycle transit and migration in non hematopoietic cells. To test whether the increased CFU migration of P190-B-deficient cells was due to the ability of cells in cycle to migrate, c-Kit + cells from WT and P190-B +/− BM were fractionated using the DNA dye Hoechst which allows separation of cells in G 0 /G 1 versus S/G 2 +M. Remarkably, FN-induced migration of S/G 2 +M c-Kit + P190-B +/− cells was significantly higher than that of WT cells. Therefore, this study suggests that the regulation of RhoA activity via P190-B is important for HSC/P short-term engraftment and homing maybe by coordinating migration and cell cycle transit and that rearrangement of the cytoskeleton likely plays an essential role during this process.
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A memory GAP
Trends in neurosciences, 2003Co-Authors: Jeffrey SettlemanAbstract:Pavlovian conditioning of the response to fear has provided an important model for memory formation in mammals. Several studies have begun to identify the key signal-transduction components that provide the molecular basis for this complex neural process. A recent report has revealed that the Rho GTPase and its regulator P190 Rho-GTPase-activating Protein (P190 RhoGAP) also play an important regulatory role in fear memory formation.
Nandigama Krishnamachary - One of the best experts on this subject based on the ideXlab platform.
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Phosphorylation of the multidrug resistance associated Protein gene encoded Protein P190.
Biochemistry, 1995Co-Authors: Nandigama KrishnamacharyAbstract:Recent studies suggest that multidrug resistance of HL60/ADR cells is related to an overexpression of the MRP (multidrug resistance associated Protein) gene which encodes a 190-kDa ATP-binding membrane glycoProtein. In the present study we have further characterized P190 and have examined phosphorylation properties of the Protein. The results demonstrate that P190 is highly phosphorylated and that the phosphate groups are metabolically active and undergo cycles of phosphorylation and dephosphorylation in the cell. Serine is the single amino acid phosphorylated in P190 and the phosphate groups are contained in nine tryptic peptides. Experiments have also been conducted to analyze the effect of various Protein kinase inhibitors on phosphorylation levels of P190. The results show that H-7, staurosporine, and chelerythrine can reduce the phosphorylation of this Protein. In the presence of both H-7 (200 microM) and staurosporine (200 nM) the phosphorylation of P190 is completely blocked. It has also been found that in the presence of these agents there is a major increase in drug accumulation and concomitant inhibition in drug efflux of resistant cells. These results therefore suggest the possibility that certain phosphate groups of Protein P190 play an important role in modulating drug accumulation in resistant cells.
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Analysis of MRP gene expression and function in HL60 cells isolated for resistance to adriamycin
Oncology research, 1994Co-Authors: Nandigama Krishnamachary, Ling Zheng, Ahmad R. SafaAbstract:In an effort to define clearly the basis of non-P-glycoProtein multidrug resistance in HL60/ADR cells, we have analyzed expression of MRP mRNA levels and the MRP-encoded Protein in resistant cells and also in resistant cells that have undergone a reversion to drug sensitivity. The results demonstrate that an MRP cDNA containing 5'-end coding sequences reacts with a 6-kb RNA, which is overexpressed in the resistant isolate. As resistant cells revert to drug sensitivity there is essentially a complete loss of the 6-kb RNA. Southern blot analysis indicates that the MRP gene is amplified compared to the copy number found in sensitive cells. Revertant cells no longer contain amplified MRP sequences. Western blot analysis has been conducted using an antibody prepared against the carboxyl terminus (15 amino acids) of the deduced sequence of the MRP-encoded Protein. The antibody is reactive with a 190-kDa Protein (P190) and with two closely migrating Proteins of 65 and 70 kDa (P70), which are overexpressed in plasma membranes and endoplasmic reticulum of resistant cells. Both Proteins are greatly reduced in revertant cells. Growth of cells in the presence of tunicamycin demonstrates that both P190 and P70 are glycosylated, with the deglycosylated forms migrating in polyacrylamide gels as Proteins of 165 kDa and 45 kDa, respectively. Additional antisera have also been prepared against sequence domains contained in the C-terminal region of P190. These antisera are reactive with both P190 and P70. Antisera directed against sequences of the amino terminal region of P190 do not react with P70.(ABSTRACT TRUNCATED AT 250 WORDS)
Robert A. Weinberg - One of the best experts on this subject based on the ideXlab platform.
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Association between GTPase activators for Rho and Ras families
Nature, 1992Co-Authors: Jeffrey Settleman, Charles F. Albright, Lauren C. Foster, Robert A. WeinbergAbstract:THE ras -related low-molecular-mass GTPases participate in signal transduction involving a variety of cellular functions, including cell-cycle progression, cellular differentiation, cytoskeletal organization, Protein transport and secretion^1,2. The cycling of these Proteins between GTP-bound and GDP-bound states is partially controlled by GTPase activating Proteins (GAPs) which stimulate the intrinsic GTP-hydrolysing activity of specific GTPases^1–6. The ras GTPase-activating Protein (Ras-GAP) forms a complex with a second Protein, P190 ( M _r 190,000), in growth-factor stimulated and tyrosine-kinase transformed cells^7,8. At its carboxy-terminal end, P190 contains a region that is conserved in the breakpoint cluster region, n -chimaerin, and Rho-GAP^9. Each of these three Proteins exhibits GAP activity for at least one member of the rho family of small GTPases^10. We have tested recombinant P190 Protein for GAP activity on GTPases of the ras , rho and rab families, and show here that P190 can function as a GAP specifically for members of the rho family. Consequently, the formation of a complex between Ras-GAP and P190 in growth-factor stimulated cells may allow the coupling of signalling pathways that involve ras and rho GTPases.
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molecular cloning of cdnas encoding the gap associated Protein P190 implications for a signaling pathway from ras to the nucleus
Cell, 1992Co-Authors: Jeffrey Settleman, Lauren C. Foster, Vikram Narasimhan, Robert A. WeinbergAbstract:Abstract In mitogenically stimulated and tyrosine kinase—transformed cells, a substantial fraction of the ras GTPase-activating Protein (GAP) forms a complex with a Protein termed P190. We have cloned several cDNAs encoding the P190 Protein. Analysis of the predicted Protein sequence reveals three distinct domains with homology to previously described sequences. An N-terminal domain of P190 contains sequence motifs that are found in all of the known GTPases. At the C-terminus of the Protein is a domain that contains sequences very similar to those found in the breakpoint cluster region gene product, n-chimerin, and rho GAP, all of which have been shown to possess intrinsic GAP activity on small GTPases. Finally, a 778 aa segment in the middle of P190 is nearly identical in sequence to a recently described transcriptional repressor. This raises the possibility that P190, acting via GAP, can transduce signals from p21 ras to the nucleus, perhaps affecting expression of specific cellular genes.
Maria Lorena Abate - One of the best experts on this subject based on the ideXlab platform.
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ABL Proteins in Philadelphia-positive acute leukaemias and chronic myelogenous leukaemia blast crises.
British journal of haematology, 1990Co-Authors: Carola Ponzetto, Angelo Guerrasio, C. Rosso, Giancarlo Avanzi, Angela Tassinari, Alfonso Zaccaria, Francesco Lo-coco, Robin Foà, Giuseppe Basso, Maria Lorena AbateAbstract:Summary. The Philadelphia chromosome (Ph1) is present in 95% of chronic myelogenous leukaemias (CML) and 15% of acute lymphoblastic leukaemias (ALL). This cytogenetic marker is due to a t(9;22) translocation, which causes a rearrangement of the ABL oncogene. In order to better define the relationship between type of genomic rearrangement, variant ABL Protein expressed and haematological phenotype, a series of Phl-positive acute leukaemias, both myeloblastic (AML) and lymphoblastic, and several CML lymphoid blast crises have been analysed at the DNA and Protein level. The results confirm the presence of the ABL Protein P210 in all cases of CML, ALL and AML positive for rearrangement in the bcr region of chromosome 22, and, surprisingly, in one AML case apparently negative for bcr rearrangement. The ABL Protein P190 was found to be present only in cases of ALL negative for bcr rearrangement. Polymerase chain reaction (PCR) analysis of the types of 9/22 junctions present in the mRNA of CML lymphoid blast crises showed no evidence of‘ALL-type’transcripts.
Rosemary Foster - One of the best experts on this subject based on the ideXlab platform.
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[13] Purification and GTPase-activating Protein activity of baculovirus expressed P190
Methods in enzymology, 1995Co-Authors: Jeffrey Settleman, Rosemary FosterAbstract:Publisher Summary GTPase-activating Proteins (GAPs) are important regulators of the nucleotide state of GTP-binding Proteins and may additionally serve as effector targets of activated GTPases. In mitogenically stimulated and tyrosine kinase-transformed cells, the major Ras-specific GAP, p120 Ras–GAP, forms an abundant complex with the cellular Protein P190. In a study discussed in the chapter, P190 was purified by virtue of its association with p120 Ras–GAP, and corresponding cDNA clones were isolated and sequenced. The expression and purification of the full-length P190 Protein in the baculovirus–insect cell system have provided a useful reagent for addressing the biochemical properties of P190. The complete coding sequence of P190 was subcloned into a baculovirus expression vector (pEV55). To detect P190 Protein, 2μl of each fraction is spotted onto a nitrocellulose sheet that has been pregridded in pencil with 1-cm squares. The nitrocellulose is air dried and then blocked for 30 minutes in 5% of dry milk in tris-buffered saline and tween 20 (TBST) at room temperature. Excellent purification of P190 can also be achieved by replacing the Mono Q column step with a gel-filtration step using a Superose 12 column.
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P190 RhoGAP, the major RasGAP-associated Protein, binds GTP directly.
Molecular and cellular biology, 1994Co-Authors: Rosemary Foster, D. A. Shaywitz, Jeffrey SettlemanAbstract:In mitogenically stimulated cells, a specific complex forms between the Ras GTPase-activating Protein (RasGAP) and the cellular Protein P190. We have previously reported that P190 contains a carboxy-terminal domain that functions as a GAP for the Rho family GTPases. Thus, the RasGAP-P190 complex may serve to couple Ras- and Rho-mediated signalling pathways. In addition to its RhoGAP domain, P190 contains an amino-terminal domain that contains sequence motifs found in all known GTPases. Here, we report that P190 binds GTP and GDP through this conserved domain and that the structural requirements for binding are similar to those seen with other GTPases. While the purified Protein is unable to hydrolyze GTP, we detect an activity in cell lysates that can promote GTP hydrolysis by P190. A mutated form of P190 that fails to bind nucleotide retains its RasGAP binding and RhoGAP activities, indicating that GTP binding by P190 is not required for these functions. The sequence of P190 in the GTP-binding domain, which shares structural features with both the Ras-like small GTPases and the larger G Proteins, suggests that this Protein defines a novel class of guanine nucleotide-binding Proteins.
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P190RhoGAP,theMajorRasGAP-Associated Protein, BindsGTPDirectly
1994Co-Authors: Rosemary FosterAbstract:Protein (RasGAP) andthecellular Protein P190. We havepreviously reported thatP190contains a carboxy-terminal domainthatfunctions as a GAPfortheRhofamily GTPases. Thus, theRasGAP-P190 complex may serveto couple Ras-andRho-mediated signalling pathways. Inaddition toitsRhoGAPdomain, P190contains an amino-terminal domainthatcontains sequencemotifs foundinallknownGTPases. Here, we report thatP190 bindsGTPandGDP through thisconserved domainandthatthestructural requirements forbinding are similar tothose seenwithother GTPases. Whilethepurified Protein isunable tohydrolyze GTP,we detect an activity incelllysates that can promote GTP hydrolysis byP190. A mutated formofP190thatfails tobind nucleotide retains itsRasGAPbinding andRhoGAPactivities, indicating thatGTP binding byP190isnot required forthese functions. ThesequenceofP190intheGTP-binding domain, whichsharesstructural features withboththeRas-like smallGTPasesandthelarger G Proteins, suggests thatthisProtein defines a novel class ofguanine nucleotide-binding Proteins. Theability ofnumerous Proteins tobindandhydrolyze GTP hasbeenmaintained throughout evolution asa switching mechanism foravariety ofcellular functions (16). Processes as diverse astranslational elongation, Protein sorting, andsignal
