Gαq Binds Two Effectors Separately in Cells: Evidence for Predetermined Signaling Pathways

Abstract Constitutively activated tyrosine kinases associated with recurrent chromosomal abnormalities play an essential role in the pathogenesis and disease progression of a variety of hematopoietic malignancies. Selective tyrosine kinase inhibitors such as imatinib are effective in treating some forms of leukemia such as t(9;22) CML associated with expression of BCR-ABL fusion tyrosine kinase. However, they are not curative and clinical resistance may develop, prompting the design of alternate and/or complementary therapeutic strategies. To better understand the signaling properties of constitutively activated tyrosine kinases associated with different hematopoietic malignancies, we examined whether BCR-ABL, FLT3-ITD, NPM-ALK, TEL-PDGFbetaR, TEL-FGFR3 and ZNF198-FGFR1 activate the same set of signaling pathways. We found that they all activated AKT and MAPK signaling pathways. Activated AKT resulted in phosphorylation of FOXO3a at Thr-32 but not BAD at Ser-136, whereas activated MAPK led to phosphorylation of BAD at Ser-112. These phosphorylated residues subsequently sequestered the pro-apoptotic FOXO3a and BAD to 14-3-3, suggesting that 14-3-3 integrates pro-survival signals from AKT and MAPK pathways. We utilized a peptide-based 14-3-3 competitive antagonist, R18 to disrupt 14-3-3/ligand association. Expression of R18 effectively induced apoptosis in hematopoietic Ba/F3 cells transformed by these tyrosine kinases with significantly enhanced sensitivity compared to the control Ba/F3 cells. Moreover, doxycycline-induced expression of R18 significantly attenuated the disease latency and penetrance in mice induced by intravenous injection of representative ZNF198-FGFR1-transformed Ba/F3 cells. Co-immunoprecipitation experiments indicate that induced R18 expression disrupted interaction between 14-3-3 and FOXO3a, but not 14-3-3/BAD association. R18 induced apoptosis by rescuing the nuclear localization of FOXO3a and up-regulating FOXO3a transcription targets Bim and p27 in cells expressing ZNF198-FGFR1. Furthermore, fluorescent confocal microscopy revealed that expression of R18 generally resumed FOXO3a nuclear localization in cells transformed by the spectrum of diverse leukemogenic tyrosine kinases. Together, these data support a model that 14-3-3 functions as a general integrator of pro-survival signals in hematopoietic transformation induced by diverse leukemogenic fusion/mutant tyrosine kinases. Disrupting 14-3-3/ligand association may be a common and effective therapeutic strategy for hematopoietic neoplasms associated with these tyrosine kinases.

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Analysis of Changes in Endothelial Cell Protein Expression and Phosphorylation Induced by Cleaved High Molecular Weight Kininogen

Blood ◽

10.1182/blood.v120.21.5195.5195 ◽

2012 ◽

Vol 120 (21) ◽

pp. 5195-5195

Author(s):

Venkaiah Betapudi ◽

Keith R. McCrae

Keyword(s):

Molecular Weight ◽

Endothelial Cells ◽

Protein Expression ◽

Signaling Pathways ◽

High Molecular Weight ◽

Tissue Remodeling ◽

High Molecular Weight Kininogen ◽

Time Points ◽

Rapid Changes ◽

In Cells

Abstract Abstract 5195 Background and objective: High molecular weight kininogen (HK) is an abundant plasma protein that functions as a critical cofactor in the kallikrein-kinin system. HK normally circulates in the single chain form, but is cleaved by plasma kallikrein to release the nonapeptide bradykinin and form cleaved high molecular weight kininogen (HKa) that consists of a heavy and light chain linked by a single disulfide bond. Conformational changes occurring in cleaved kininogen result in increased exposure of histidine and glycine-rich regions within kininogen domain 5 that impart HKa with unique properties, including the ability to inhibit angiogenesis by causing selective apoptosis of proliferating endothelial cells. However, neither the receptors that mediate the antiangiogenic activity of HKa nor the signaling pathways that lead to apoptosis have been rigorously defined. In this study we attempted to define specific signaling pathways activated following exposure of proliferating endothelial cells to HKa using a high-throughput, unbiased, microarray approach (Kinexus, Vancouver BC). Results: Endothelial cells were cultured at low density and stimulated to proliferate using 20 ng/ml bFGF in the absence or presence of HKa (15 nM). At various time points (20, 60 and 300 minutes) total cell extracts were prepared and analyzed using the Kinexus antibody microarray that includes 530 pan-specific and 270 phospho-site specific antibodies. In cells exposed to HKa, the analysis revealed increased expression of 109, 141 and 162 proteins, and decreased expression of 117, 68 and 59 proteins at the 20 min, 60 min, and 300 minute time points, respectively. In cells exposed to HKa, the number of newly-phosphorylated proteins increased from 30 at 20 minutes to 61 at 300 minutes after HKa treatment. Segregation of proteins whose expression level and/or phosphorylation state changed following exposure of cells to HKa into families demonstrated that HKa primarily targets protein kinases (61–70% of all proteins affected at the various time points), transcription factors (8–11%), and phosphatases (4–5%). Increased expression of several proteins involved in apoptosis, such as caspases 4, 6 and 7 and DNA fragmentation factors 35 and 45, and increased phosphorylation of stress regulated activating transcription factor 2 (ATF2) and apoptosis signal regulating protein kinase1 (ASK1) were evident within 20 minutes of exposure of cells to HKa. Metacore and Ingenuity pathway analysis of proteins that exhibited rapid changes in expression or phosphorylation revealed activation of several major signaling pathways including apoptosis, DNA damage response, angiogenesis, inflammation, and tissue remodeling and wound repair. Conclusion: Exposure of proliferating endothelial cells to HKa led to rapid changes in protein expression and phosphorylation. Most remarkable was the increased expression of several caspases within 20 minutes of addition of HKa to cells. Patterns of protein expression were consistent with activation of several pathways related to apoptosis, inflammation and tissue remodeling. These findings support suspected physiological functions of HK/HKa in vivo, and suggest specific proteins that may be targeted to further dissect effects of HKa on discrete cellular functions. Disclosures: No relevant conflicts of interest to declare.

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Erratum: Many new down- and up-regulatory signaling pathways, from known cancer progression suppressors to matrix metalloproteinases, differ widely in cells of various cancers

Journal of Cellular Physiology ◽

10.1002/jcp.22351 ◽

2010 ◽

Vol 225 (3) ◽

pp. 916-916

Author(s):

Gregory S. DeLassus ◽

Hyojin Cho ◽

Stanley Hoang ◽

George L. Eliceiri

Keyword(s):

Matrix Metalloproteinases ◽

Signaling Pathways ◽

Cancer Progression ◽

In Cells

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ITD-Flt3 Enhances Migration of Hematopoietic Cells through Aberrant CXCR4 Signaling Pathways Overlapping but Functionally Distinct From SDF1/CXCR4 Signaling Axis in Normal Hematopoietic Cells.

Blood ◽

10.1182/blood.v114.22.2383.2383 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2383-2383

Author(s):

Seiji Fukuda ◽

Satomi Mori ◽

Mariko Abe ◽

Seiji Yamaguchi ◽

Louis M. Pelus

Keyword(s):

Cell Migration ◽

Signaling Pathways ◽

Cross Talk ◽

Hematopoietic Cells ◽

Leukemia Cells ◽

Wild Type ◽

Transcription Analysis ◽

Normal Cells ◽

Cxcr4 Signaling ◽

In Cells

Abstract Abstract 2383 Poster Board II-360 Elevated expression of CXCR4, a receptor for SDF1, with Internal Tandem Duplication of Flt3 (ITD-Flt3) is an indicator of poor prognosis in patients with acute myeloid leukemia. We previously showed that ITD-Flt3 enhances migration of hematopoietic cells to SDF1, suggesting that ITD-Flt3 may facilitate dissemination of leukemia cells by modulating SDF1/CXCR4 signaling and that blocking this functional cross-talk between ITD-Flt3 and CXCR4 pathways may have therapeutic benefit. While identification of selective pathways in cells transformed by ITD-Flt3, which are distinct from normal cells is crucial to develop therapeutic agents without hematopoietic toxicity, the mechanisms responsible for aberrant migration induced by ITD-Flt3 are not known. We now demonstrate the existence of CXCR4 signaling pathways regulated by ITD-Flt3 that are distinct from normal CXCR4 signaling using genome wide transcription analysis. Ectopic expression of ITD-Flt3 in Ba/F3 cells enhances random cell migration and modulates expression of 1,675 genes out of 41,174 genes (4.1%) examined compared to control cells lacking ITD-Flt3. ITD-Flt3 down-regulated CXCR4 mRNA by 55% compared to control and modulated 36 additional transcripts implicated in cell migration and localization (0.09%, P<0.05), further substantiating a role of ITD-Flt3 in cell migration and suggesting that enhanced migration was not dependent on CXCR4. Coordinately, ectopic ITD-Flt3 significantly enhanced cell migration to SDF1 compared to cells expressing wild-type Flt3 despite reduction in CXCR4. In contrast, in primary HPC, enhanced migration induced by ITD-Flt3 was inhibited by conditional deletion of CXCR4 in Cre-ERTM-CXCR4fl/fl mice cells or by incubation with the selective CXCR4 antagonist AMD3100. Analysis of gene expression in control and ITD-Flt3 expressing Ba/F3 cells migrating to SDF1 indicated that SDF1 modulates 1,647 (4.0%) of 41,174 genes analyzed in cells expressing ITD-Flt3, of which 1,190 genes (2.5%) were regulated by SDF1 exclusively in ITD-Flt3 cells, such as neurofibromatosis type 1 and p27kip1. SDF1 regulated 457 genes (1.1%) in both cells expressing wild-type Flt3 and ITD Flt3, whereas, 384 (0.5%) transcripts were regulated by SDF1 in wild-type Flt3 expressing cells but not in ITD-Flt3 cells. Genes associated with transcription, cytoskeleton, ubiquitination, kinase and iron transport were functionally clustered both in ITD-Flt3 and wild-type Flt3 cells migrating to SDF1. In contrast, genes involved in apoptosis, cell cycle, glycosylation and dephosphorylation were significantly enriched in ITD-Flt3 but not in wild-type Flt3 cells, suggesting that ITD-Flt3 generates CXCR4 signaling pathways that are qualitatively different from normal cells. In addition, 69 genes (0.17%) were down-regulated by SDF1 in ITD-Flt3 cells but up-regulated in control cells or vice versa, including Rho-associated coiled-coil forming kinase 1 (ROCK1), which has been linked with leukocyte transendothelial migration, cell motility and tumor cell invasion. While baseline expression of ROCK1 was not affected by the presence of ITD-Flt3, ROCK1 was up-regulated 251% by SDF1 in control cells but down-regulated by 67% in ITD-Flt3 cells. The ROCK antagonist Y27632 significantly decreased migration of control Ba/F3 cells lacking ITD-Flt3 to SDF1 (77 ± 6% inhibition, P<0.01), but had no effect on SDF1-induced migration of ITD-Flt3 cells, suggesting that an increase of ROCK1 is required for normal CXCR4 signaling, while enhanced migration to SDF1 induced by ITD-Flt3 is independent of ROCK system. Our data provide additional evidence for functional cross-talk between SDF1/CXCR4 and ITD-Flt3 signaling pathways. Inhibition of aberrant migration to SDF1 induced by ITD-Flt3 by blocking CXCR4 suggests that antagonizing CXCR4 may inhibit dissemination of hematopoietic cells expressing ITD-Flt3. ITD-Flt3 regulates overlapping but functionally distinct pathways down-stream of SDF1/CXCR4 compared to those in cells without ITD-Flt3. Genes differentially regulated by SDF1 specifically in ITD-Flt3 cells may represent key targets regulating aberrant migration by ITD-Flt3 in response to SDF1 to prevent unnecessary dissemination and invasion of ITD-Flt3+ acute leukemia cells without affecting normal hematopoiesis. Disclosures: No relevant conflicts of interest to declare.

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