scholarly journals β-Chemokine Receptor CCR5 Signals Via the Novel Tyrosine Kinase RAFTK

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 791-797 ◽  
Author(s):  
Ramesh K. Ganju ◽  
Parmesh Dutt ◽  
Lijun Wu ◽  
Walter Newman ◽  
Hava Avraham ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Chemokine Receptors ◽  
Growth Regulation ◽  
Dominant Negative ◽  
Downstream Signaling ◽  
Inflammatory Protein ◽  
Ccr5 Receptor ◽  
Mitogen Activated Protein ◽  
Chemokine Receptor Ccr5 ◽  
Receptor Ccr5

Chemokine receptors are coupled to G-proteins and their activation results in prominent changes in cell migration and growth. The downstream signaling pathways that mediate these effects of chemokines are largely uncharacterized. Macrophage inflammatory protein 1β (MIP 1β) binding to its cognate receptor CCR5 resulted in activation of the related adhesion focal tyrosine kinase (RAFTK), with subsequent activation of the cytoskeletal protein paxillin and the downstream transcriptional activators, c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38 mitogen-activated protein (MAP) kinase. Inhibition of RAFTK by a dominant-negative kinase mutant markedly attenuated JNK/SAPK activity. Thus, RAFTK appears to provide a functional “bridge” for the transmission of CCR5 receptor signaling to the cytoskeleton and nucleus, primary sites of chemotaxis and growth regulation.

scholarly journals β-Chemokine Receptor CCR5 Signals Via the Novel Tyrosine Kinase RAFTK

Blood ◽  
1998 ◽  
Vol 91 (3) ◽  
pp. 791-797 ◽  
Author(s):  
Ramesh K. Ganju ◽  
Parmesh Dutt ◽  
Lijun Wu ◽  
Walter Newman ◽  
Hava Avraham ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Chemokine Receptors ◽  
Growth Regulation ◽  
Dominant Negative ◽  
Downstream Signaling ◽  
Inflammatory Protein ◽  
Ccr5 Receptor ◽  
Mitogen Activated Protein ◽  
Chemokine Receptor Ccr5 ◽  
Receptor Ccr5

Abstract Chemokine receptors are coupled to G-proteins and their activation results in prominent changes in cell migration and growth. The downstream signaling pathways that mediate these effects of chemokines are largely uncharacterized. Macrophage inflammatory protein 1β (MIP 1β) binding to its cognate receptor CCR5 resulted in activation of the related adhesion focal tyrosine kinase (RAFTK), with subsequent activation of the cytoskeletal protein paxillin and the downstream transcriptional activators, c-Jun N-terminal kinase (JNK)/stress-activated protein kinase (SAPK) and p38 mitogen-activated protein (MAP) kinase. Inhibition of RAFTK by a dominant-negative kinase mutant markedly attenuated JNK/SAPK activity. Thus, RAFTK appears to provide a functional “bridge” for the transmission of CCR5 receptor signaling to the cytoskeleton and nucleus, primary sites of chemotaxis and growth regulation.

PKC-ε regulation of extracellular signal-regulated kinase: a potential role in phenylephrine-induced cardiocyte growth

2004 ◽  
Vol 286 (6) ◽  
pp. H2195-H2203 ◽  
Author(s):  
Vijay U. Rao ◽  
Hirokazu Shiraishi ◽  
Paul J. McDermott
Keyword(s):  
Growth Regulation ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Neonatal Rat ◽  
Inhibitory Effects ◽  
Hypertrophic Growth ◽  
Extracellular Signal Regulated Kinase ◽  
Extracellular Signal ◽  
Mitogen Activated Protein ◽  
Acute Increase

Hypertrophic growth of cardiac muscle is dependent on activation of the PKC-ε isoform. To define the effectors of PKC-ε involved in growth regulation, recombinant adenoviruses were used to overexpress either wild-type PKC-ε (PKC-ε/WT) or dominant negative PKC-ε (PKC-ε/DN) in neonatal rat cardiocytes. PKC-ε/DN inhibited acute activation of PKC-ε produced in response to phorbol ester and reduced ERK1/2 activity as measured by the phosphorylation of p42 and p44 isoforms. The inhibitory effects were specific to PKC-ε because PKC-ε/DN did not prevent translocation of either PKC-α or PKC-δ. Overexpression of PKC-ε/DN blunted the acute increase in ERK1/2 phorphorylation induced by the α1-adrenergic agonist phenylephrine (PE ). Inhibition of PKC-δ with rottlerin potentiated the effects of PE on ERK1/2 phosphorylation. PKC-ε/DN adenovirus also blocked cardiocyte growth as measured after 48 h of PE treatment, although the multiplicity of infection was lower than that required to block acute ERK1/2 activation. PE activated p38 mitogen-activated protein kinase as measured by its phosphorylation, but the response was not blocked by PKC inhibitors or by overexpression of PKC-ε/DN. Taken together, these studies show that the hypertrophic agonist PE regulates ERK1/2 activity in cardiocytes by a pathway dependent on PKC-ε and that PE-induced growth is mediated by PKC-ε.

scholarly journals IL-1 receptor–associated kinase M is a central regulator of osteoclast differentiation and activation

2005 ◽  
Vol 201 (7) ◽  
pp. 1169-1177 ◽  
Author(s):  
Hongmei Li ◽  
Esteban Cuartas ◽  
Weiguo Cui ◽  
Yongwon Choi ◽  
Todd D. Crawford ◽  
...  
Keyword(s):  
Bone Loss ◽  
Bone Fractures ◽  
Bone Development ◽  
Osteoclast Differentiation ◽  
Dominant Negative ◽  
Mitogen Activated Protein Kinase ◽  
Downstream Signaling ◽  
Signaling Complex ◽  
Mitogen Activated Protein ◽  
Protein Kinase Signaling

Osteoporosis is a serious problem worldwide; it is characterized by bone fractures in response to relatively mild trauma. Osteoclasts originate from the fusion of macrophages and they play a central role in bone development and remodeling via the resorption of bone. Therefore, osteoclasts are important mediators of bone loss that leads, for example, to osteoporosis. Interleukin (IL)-1 receptor (IL-1R)–associated kinase M (IRAK-M) is only expressed in cells of the myeloid lineage and it inhibits signaling downstream of IL-1R and Toll-like receptors (TLRs). However, it lacks a functional catalytic site and, thus, cannot function as a kinase. IRAK-M associates with, and prevents the dissociation of, IRAK–IRAK-4–TNF receptor–associated factor 6 from the TLR signaling complex, with resultant disruption of downstream signaling. Thus, IRAK-M acts as a dominant negative IRAK. We show here that mice that lack IRAK-M develop severe osteoporosis, which is associated with the accelerated differentiation of osteoclasts, an increase in the half-life of osteoclasts, and their activation. Ligation of IL-1R or TLRs results in hyperactivation of NF-κB and mitogen-activated protein kinase signaling pathways, which are essential for osteoclast differentiation. Thus, IRAK-M is a key regulator of the bone loss that is due to osteoclastic resorption of bone.

PDGF-BB-mediated activation of p42MAPK is independent of PDGF β-receptor tyrosine phosphorylation

2001 ◽  
Vol 281 (4) ◽  
pp. L786-L798 ◽  
Author(s):  
Nicholas J. Cartel ◽  
Jason Liu ◽  
Jinxia Wang ◽  
Martin Post
Keyword(s):  
Tyrosine Phosphorylation ◽  
Dominant Negative ◽  
Janus Kinase ◽  
Mitogen Activated Protein Kinase ◽  
Phosphatidylinositol 3 Kinase ◽  
Downstream Signaling ◽  
Mitogen Activated Protein ◽  
Signaling Domain ◽  
Chemical Inhibition ◽  
Β Receptor

Herein, we investigated the activity of mitogen-activated protein kinase (MAPK), a key component of downstream signaling events, which is activated subsequent to platelet-derived growth factor (PDGF)-BB stimulation. Specifically, p42MAPK activity peaked 60 min after addition of PDGF-BB, declined thereafter, and was determined not to be a direct or necessary component of glycosaminoglycan (GAG) synthesis. PDGF-BB also activated MAPK kinase 2 (MAPKK2) but had no effect on MAPKK1 and Raf-1 activity. Chemical inhibition of Janus kinase, phosphatidylinositol 3-kinase, Src kinase, or tyrosine phosphorylation inhibition of the PDGF β-receptor (PDGFR-β) did not abrogate PDGF-BB-induced p42MAPK activation or its threonine or tyrosine phosphorylation. A dominant negative cytoplasmic receptor for hyaluronan-mediated motility variant 4 (RHAMMv4), a regulator of MAPKK-MAPK interaction and activation, did not inhibit PDGF-BB-induced p42MAPK activation nor did a construct expressing PDGFR-β with cytoplasmic tyrosines mutated to phenylalanine. However, overexpression of a dominant negative PDGFR-β lacking the cytoplasmic signaling domain abrogated p42MAPKactivity. These results suggest that PDGF-BB-mediated activation of p42MAPK requires the PDGFR-β but is independent of its tyrosine phosphorylation.

scholarly journals Identification of C-C Chemokine Receptor 1 (CCR1) as the Monocyte Hemofiltrate C-C Chemokine (HCC)-1 Receptor

1998 ◽  
Vol 188 (3) ◽  
pp. 603-608 ◽  
Author(s):  
Chia-Lin Tsou ◽  
Ron P. Gladue ◽  
Laurie A. Carroll ◽  
Tim Paradis ◽  
James G. Boyd ◽  
...  
Keyword(s):  
Chemokine Receptor ◽  
Chemokine Receptors ◽  
Human Monocytes ◽  
Normal Human Plasma ◽  
Transfected Cells ◽  
Downstream Signaling ◽  
Inflammatory Protein ◽  
Normal Human ◽  
High Concentrations ◽  
Macrophage Inflammatory Protein

Hemofiltrate C-C chemokine (HCC)-1 is a recently cloned C-C chemokine that is structurally similar to macrophage inflammatory protein (MIP)-1α. Unlike most chemokines, it is constitutively secreted by tissues and is present at high concentrations in normal human plasma. Also atypical for chemokines, HCC-1 is reported not to be chemotactic for leukocytes. In this paper, we have investigated the chemokine receptor usage and downstream signaling pathways of HCC-1. Cross-desensitization experiments using THP-1 cells suggested that HCC-1 and MIP-1α activated the same receptor. Experiments using a panel of cloned chemokine receptors revealed that HCC-1 specifically activated C-C chemokine receptor (CCR)1, but not closely related receptors, including CCR5. HCC-1 competed with MIP-1α for binding to CCR1-transfected cells, but with a markedly reduced affinity (IC50 = 93 nM versus 1.3 nM for MIP-1α). Similarly, HCC-1 was less potent than MIP-1α in inducing inhibition of adenylyl cyclase in CCR1-transfected cells. HCC-1 induced chemotaxis of freshly isolated human monocytes, THP-1 cells, and CCR1-transfected cells, and the optimal concentration for cell migration (100 nM) was ∼100-fold lower than that of MIP-1α (1 nM). These data demonstrate that HCC-1 is a chemoattractant and identify CCR1 as a functional HCC-1 receptor on human monocytes.

scholarly journals Novel FLT3 Mutation Shows Dominant Negative Effects on the Wild-Type FLT3 Receptor

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 5205-5205
Author(s):  
Julia Bauer ◽  
Nadine Sandhöfer ◽  
Wolfgang Hiddemann ◽  
Karsten Spiekermann
Keyword(s):  
Flow Cytometry ◽  
Tyrosine Kinase ◽  
Western Blotting ◽  
Dominant Negative ◽  
Stable Expression ◽  
Flt3 Ligand ◽  
Wild Type ◽  
Truncated Form ◽  
Downstream Signaling ◽  
Flt3 Mutation

Abstract Background The FMS-like tyrosine kinase-3 (FLT3) gene encodes for a receptor tyrosine kinase playing an important role in hematopoiesis. In acute leukemias it is one of the most frequently mutated genes. In this study we functionally characterized a novel frameshift deletion mutation of FLT3 found in a relapsed patient with acute myeloid leukemia (AML). The frameshift leads to a premature stop codon resulting in a truncated form of the receptor lacking most of the intracellular domains. Material and Methods FLT3 cDNA was expressed in the IL-3 dependent pro-B cell line Ba/F3 via a retroviral expression vector. The transduced cell lines were sorted by fluorescence-activated cell sorting (FACS). Stable expression of the receptor was confirmed on mRNA level by polymerase chain reaction (PCR) and on protein level by western blotting and cell surface expression of the receptor by flow cytometry. Cell proliferation assays were performed in presence or absence of IL-3 or FLT3-ligand. By western blotting receptor activation and its downstream signaling were analyzed. Ligand-binding of the receptor was shown via biotinylated and fluorescent FLT3-ligand-receptor-complexes by flow cytometry. Results and Discussion Stable expression of FLT3 Q569Vstop does not enable Ba/F3 cells to grow IL-3-independent. FLT3-ligand can still be bound by the mutated receptor but is not able to stimulate receptors´ signaling and growth of the cells. Furthermore coexpression of wild-type (WT) and mutant FLT3 receptor also abolishes the ability to stimulate the WT receptor with its ligand. This is confirmed by analyzing downstream signaling in the cells as MAPK is less phosphorylated in the FLT3-WT/Q569Vstop coexpressing cells than in FLT3-WT expressing cells alone. Conclusion Most of the FLT3 mutations are activating mutations leading to a constitutive activation of the receptor and ligand-independent growth. In our study we characterized a novel FLT3 mutation found in an AML patient which has not been described before. The resulting truncated receptor is still integrated in the plasma membrane and binds its ligand but its ability to be fully activated is completely lost. Furthermore coexpressed with a FLT3-WT receptor, it even prevents stimulation and activation of the WT receptor, thus acting in a dominant negative manner. How the truncated form of the receptor contributes to progression of acute leukemia is of great interest and will be further investigated. Disclosures No relevant conflicts of interest to declare.

Oncogenic CBL Mutants Have Transforming Potential in Cells Expressing the FLT3 Tyrosine Kinase.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1446-1446
Author(s):  
Carola Reindl ◽  
Sridhar Vempati ◽  
Hilmar Quentmeier ◽  
Gudrun Mellert ◽  
Wolfgang Hiddemann ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Kinases ◽  
Deletion Mutant ◽  
Ring Finger ◽  
Dominant Negative ◽  
Leukemic Cells ◽  
Regulatory Function ◽  
Lymphoid Leukemia ◽  
Acute Leukemias ◽  
Downstream Signaling

Abstract CBL is a E3-ubiquitin ligase, that negatively regulates many receptor tyrosine kinases (RTKs). Translocations (t(4;11); t(11;14) involving CBL in humans and two oncogenic CBL deletion mutants, CBL-70Z and v-CBL, inducing B-cell lymphomas in mice, were described. The RTK Fms-like tyrosine kinase 3 (FLT3) is expressed by the leukemic cells of 70–90% of patients with acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) and is highly implicated in the malignant transformation by harbouring activating mutations, aberrant autocrine stimulation or overexpression. We hypothesized that CBL could play a role in regulating FLT3 activity and if mutated, could cause aberrant FLT3 signaling in hematopoietic cells. To analyze if CBL mutants could lead to transformation of FLT3-expressing cells we stably transduced Ba/F3 cells with CBL-WT, CBL-70Z or v-CBL alone or together with FLT3-WT. Coexpression of FLT3-WT and a CBL mutant, CBL-70Z or v-CBL, but not CBL-WT or expression of a CBL-construct alone, conferred IL-3 independent growth to Ba/F3 cells. In presence of FLT3 ligand FLT3-WT/CBLmutant-expressing cells showed hyperproliferation. To determine whether the proliferative advantage of FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells is mediated by FLT3 we cultivated the cells in presence of selective FLT3 inhibitors, SU5614 and PKC412. Both inhibitors were able to abrogate the IL-3 independent and FL-stimulated growth of FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells. The FLT3-WT receptors and the downstream signaling pathway, STAT5, were constitutively active in FLT3-WT/CBLmutant-expressing cells in contrast to control cells. To clarify the mechanism of transformation we performed internalization assays. The rate and speed of internalization of the FLT3-WT receptors were not altered in FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells compared to FLT3-WT cells. However, we could show that CBL-WT promotes FLT3 degradation. To investigate if CBL mutations play a role in acute leukemias we screened the cDNA of 62 AML and 6 ALL cell lines and found a CBL deletion mutant in MOLM-13 and MOLM-14 cells lacking exon 8, which comprises parts of the linker and RING finger domain. Hence, this mutation targets the same functionally important regions of the CBL protein than the CBL-70Z deletion. In summary, CBL-70Z and v-CBL, but not CBL-WT, confer a transforming potential to cells expressing FLT3. The pro-proliferative phenotype of FLT3-WT/CBL-70Z and FLT3-WT/v-CBL cells is mediated by an increase in FLT3 tyrosine kinase activity. The CBL-70Z and v-CBL mutations were hypothesized to act in a dominant negative manner abrogating the negativ-regulatory function of CBL-WT. We identified a CBL deletion mutant in the MOLM-13 and MOLM-14 cells, that is reminiscent of CBL-70Z, indicating that CBL mutations might play a role in the pathogenesis of acute leukemias.

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