Identification of Critical Phosporylation Sites within NPM-ALK That Regulate JUNB mRNA Translation.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3571-3571
Author(s):  
Claudia Fuchs ◽  
Paul Vesely ◽  
Isabella Bambach ◽  
Silvia Schauer ◽  
Werner Linkesch ◽  
...  
Keyword(s):  
Tyrosine Kinases ◽  
Translational Regulation ◽  
Chromosomal Translocation ◽  
Mrna Translation ◽  
Large Cell ◽  
Wild Type ◽  
Tyrosine Residues ◽  
Anaplastic Lymphoma ◽  
Rps6 Phosphorylation ◽  
Mutant Cells

Abstract Anaplastic large cell lymphoma (ALCL) accounts for approximately 30% of childhood lymphomas and 3% of adult non-Hodgkin lymphomas. The nucleophosmin - anaplastic lymphoma kinase (NPM-ALK) fusion which is the product of a t(2;5)(p23;q35) chromosomal translocation is present in about half of nodal ALCL. Expression of this fusion kinase results in induction of the AP-1 transcription factor JunB and IL-3 independent outgrow of murine hematopoietic Ba/F3 cells. We demonstrated that wild type NPM-ALK increases the amount of ribosomes bound to JUNB mRNA resulting in its more effective translation in large polysomes. The NPM-ALK fusion tyrosine kinase has 20 potential tyrosine residues available for autophosphorylation and phosphorylation by other protein tyrosine kinases. Here we used series of Y-to-F-substituted mutants of NPM-ALK to identify tyrosine residues that are required to regulate the segregation of JUNB mRNAs between polysomes and monosomes as well as ribonucleic particles (RNPs). Neither JUNB transcription nor JunB translation was altered in Ba/F3 cells expressing NPM-ALK mutants Y17F/Y29F/Y67F Y138F/Y152F Y156F/Y191F/Y299F Y378F/Y418F/Y445F and Y646F/Y664F compared to NPM-ALK wild type. Conversely, in NPM-ALK Y567F/Y461F/Y644F mutant cells proliferation was markedly decreased. These cells demonstrated active MEK-ERK pathway, while AKT, mTOR, and rpS6 phosphorylation was impaired. Moreover a shift of JUNB mRNA from the polysomic to the monosomic/mRNP fraction could be observed. In conclusion, we identified specific NPM-ALK phosphorylation sites required to mediate the effect of NPM-ALK on the JUNB translational regulation and therefore provide further insights in the transforming mechanisms of the oncoprotein NPM-ALK.

scholarly journals Dependency on the TYK2/STAT1/MCL1 axis in anaplastic large cell lymphoma

Leukemia ◽  
2018 ◽  
Vol 33 (3) ◽  
pp. 696-709 ◽  
Author(s):  
Nicole Prutsch ◽  
Elisabeth Gurnhofer ◽  
Tobias Suske ◽  
Huan Chang Liang ◽  
Michaela Schlederer ◽  
...  
Keyword(s):  
Cell Survival ◽  
Tyrosine Kinases ◽  
Drug Target ◽  
Cell Lymphoma ◽  
Chromosomal Translocation ◽  
Large Cell ◽  
Bcl2 Family ◽  
Anaplastic Lymphoma ◽  
Stat3 Phosphorylation ◽  
Specific Receptors

Abstract TYK2 is a member of the JAK family of tyrosine kinases that is involved in chromosomal translocation-induced fusion proteins found in anaplastic large cell lymphomas (ALCL) that lack rearrangements activating the anaplastic lymphoma kinase (ALK). Here we demonstrate that TYK2 is highly expressed in all cases of human ALCL, and that in a mouse model of NPM-ALK-induced lymphoma, genetic disruption of Tyk2 delays the onset of tumors and prolongs survival of the mice. Lymphomas in this model lacking Tyk2 have reduced STAT1 and STAT3 phosphorylation and reduced expression of Mcl1, a pro-survival member of the BCL2 family. These findings in mice are mirrored in human ALCL cell lines, in which TYK2 is activated by autocrine production of IL-10 and IL-22 and by interaction with specific receptors expressed by the cells. Activated TYK2 leads to STAT1 and STAT3 phosphorylation, activated expression of MCL1 and aberrant ALCL cell survival. Moreover, TYK2 inhibitors are able to induce apoptosis in ALCL cells, regardless of the presence or absence of an ALK-fusion. Thus, TYK2 is a dependency that is required for ALCL cell survival through activation of MCL1 expression. TYK2 represents an attractive drug target due to its essential enzymatic domain, and TYK2-specific inhibitors show promise as novel targeted inhibitors for ALCL.

scholarly journals NPM-ALK: The Prototypic Member of a Family of Oncogenic Fusion Tyrosine Kinases

2012 ◽  
Vol 2012 ◽  
pp. 1-14 ◽  
Author(s):  
Joel D. Pearson ◽  
Jason K. H. Lee ◽  
Julinor T. C. Bacani ◽  
Raymond Lai ◽  
Robert J. Ingham
Keyword(s):  
Tyrosine Kinases ◽  
Fusion Proteins ◽  
Cell Lymphoma ◽  
Critical Role ◽  
Chromosomal Translocation ◽  
Large Cell ◽  
Signalling Pathways ◽  
Gene Encoding ◽  
Anaplastic Lymphoma ◽  
Constitutively Active

Anaplastic lymphoma kinase (ALK) was first identified in 1994 with the discovery that the gene encoding for this kinase was involved in the t(2;5)(p23;q35) chromosomal translocation observed in a subset of anaplastic large cell lymphoma (ALCL). The NPM-ALK fusion protein generated by this translocation is a constitutively active tyrosine kinase, and much research has focused on characterizing the signalling pathways and cellular activities this oncoprotein regulates in ALCL. We now know about the existence of nearly 20 distinct ALK translocation partners, and the fusion proteins resulting from these translocations play a critical role in the pathogenesis of a variety of cancers including subsets of large B-cell lymphomas, nonsmall cell lung carcinomas, and inflammatory myofibroblastic tumours. Moreover, the inhibition of ALK has been shown to be an effective treatment strategy in some of these malignancies. In this paper we will highlight malignancies where ALK translocations have been identified and discuss why ALK fusion proteins are constitutively active tyrosine kinases. Finally, using ALCL as an example, we will examine three key signalling pathways activated by NPM-ALK that contribute to proliferation and survival in ALCL.

NPM-ALK Converts JUNB from a Tumor Suppressor to an Oncogene.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1448-1448
Author(s):  
Paul Vesely ◽  
Philipp B. Staber ◽  
Rene Ott ◽  
Montserrat Pinent ◽  
Werner Linkesch ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Tumor Necrosis Factor Receptor ◽  
Large Cell ◽  
Cell Types ◽  
Wild Type ◽  
Anaplastic Lymphoma ◽  
Supershift Assay ◽  
Factor C ◽  
Electro Mobility ◽  
Opposing Effects

Abstract High expression of the tumor necrosis factor receptor CD30 and the AP-1 transcription factor JunB are the hallmark of anaplastic large cell lymphoma (ALCL). In contrast to the prototypic AP-1 factor c-Jun, JunB exerts an antioncogenic function in most cell types. Its functional role in ALCL remains uncertain. In about 50% of nodal ALCL the balanced chromosomal rearrangement t(2;5)(p23;q35), generating the fusion protein nucleophosmin-anaplastic lymphoma kinase (NPM-ALK), can be detected. Expression of this fusion kinase induces malignancy in mice and also leads to IL-3 independent outgrowth of the murine hematopoietic cell line Ba/F3. Using NPM-ALK transduced Ba/F3 cells, we show that NPM-ALK induces JunB expression and activation as verified by quantitative RT-PCR, immunoblot and electro-mobility supershift assay. Interestingly, NPM-ALK transduced Ba/F3 cells also express CD30, which is undetectable in the corresponding wild type cells. Since NPM-ALK induces JunB and CD30 and also leads to growth factor independent proliferation in Ba/F3 cells, these cells mimic conditions present in ALCL. Knock down of JUNB in NPM-ALK expressing cells using RNA interference leads to downregulation of CD30. Moreover, this partial loss of JunB induces upregulation of p16INK4a and downregulation of CCND1, which directly affect the cell cycle at the G1/S transition. These observations indicate that JunB is an essential factor for CD30 regulation and for neoplastic transformation. To test if JunB by itself is sufficient to induce CD30 expression and IL-3 independence, we stably transduced Ba/F3 cells with JUNB. In Ba/F3 wild type (WT) cells, JunB expression leads to reverse effects compared to that observed in NPM-ALK transduced Ba/F3. Ba/F3 WT cells do not become IL-3 independent. In addition, compared to vector control, JUNB-transduced Ba/F3 cells show a decrease in proliferation. Furthermore, an induction of p16INK4a and a decrease of CCND1 expression are observed. Moreover, aberrant JunB expression does not trigger CD30 expression in this system. Taken together, we show that both NPM-ALK and JunB are essential to induce CD30 expression. Furthermore the opposing effects of JunB on p16INK4a and CCND1 in the presence or absence of NPM-ALK indicate that NPM-ALK converts JUNB from a tumor suppressor to an oncogene.

Detection of Normal and Chimeric Nucleophosmin in Human Cells

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 632-642 ◽  
Author(s):  
Jacqueline L. Cordell ◽  
Karen A.F. Pulford ◽  
Barbara Bigerna ◽  
Giovanna Roncador ◽  
Alison Banham ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fusion Gene ◽  
Chromosomal Translocation ◽  
Large Cell ◽  
Chromosomal Anomalies ◽  
Wild Type ◽  
Terminal Portion ◽  
Cell Nuclei ◽  
Tissue Samples ◽  
Alk Positive

Abstract In anaplastic large-cell lymphoma (ALCL), the (2;5) chromosomal translocation creates a fusion gene encoding the 80-kD NPM-ALK hybrid protein. This report describes three new monoclonal antibodies, two of which recognize, by Western blotting, the N-terminal portion of NPM present in the NPM-ALK fusion protein and also in two other NPM fusion proteins (NPM-RAR and NPM-MLF1). The third antibody recognizes the C-terminal portion (deleted in NPM-ALK) and reacts only with wild-type NPM. The three antibodies immunostain wild-type NPM (in paraffin-embedded normal tissue samples) in cell nuclei and in the cytoplasm of mitotic cells. Cerebral neurones, exceptionally, show diffuse cytoplasmic labeling. In contrast to normal tissues, the two antibodies against the N-terminal portion of NPM labeled the cytoplasm of neoplastic cells, in four ALK-positive ALCL, reflecting their reactivity with NPM-ALK fusion protein, whereas the antibody to the C-terminal NPM epitope labeled only cell nuclei. Immunocytochemical labeling with these antibodies can therefore confirm that an ALK-positive lymphoma expresses NPM-ALK (rather than a variant ALK-fusion protein) and may also provide evidence for chromosomal anomalies involving the NPM gene other than the classical (2;5) translocation.

Detection of Normal and Chimeric Nucleophosmin in Human Cells

Blood ◽  
1999 ◽  
Vol 93 (2) ◽  
pp. 632-642 ◽  
Author(s):  
Jacqueline L. Cordell ◽  
Karen A.F. Pulford ◽  
Barbara Bigerna ◽  
Giovanna Roncador ◽  
Alison Banham ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Fusion Gene ◽  
Chromosomal Translocation ◽  
Large Cell ◽  
Chromosomal Anomalies ◽  
Wild Type ◽  
Terminal Portion ◽  
Cell Nuclei ◽  
Tissue Samples ◽  
Alk Positive

In anaplastic large-cell lymphoma (ALCL), the (2;5) chromosomal translocation creates a fusion gene encoding the 80-kD NPM-ALK hybrid protein. This report describes three new monoclonal antibodies, two of which recognize, by Western blotting, the N-terminal portion of NPM present in the NPM-ALK fusion protein and also in two other NPM fusion proteins (NPM-RAR and NPM-MLF1). The third antibody recognizes the C-terminal portion (deleted in NPM-ALK) and reacts only with wild-type NPM. The three antibodies immunostain wild-type NPM (in paraffin-embedded normal tissue samples) in cell nuclei and in the cytoplasm of mitotic cells. Cerebral neurones, exceptionally, show diffuse cytoplasmic labeling. In contrast to normal tissues, the two antibodies against the N-terminal portion of NPM labeled the cytoplasm of neoplastic cells, in four ALK-positive ALCL, reflecting their reactivity with NPM-ALK fusion protein, whereas the antibody to the C-terminal NPM epitope labeled only cell nuclei. Immunocytochemical labeling with these antibodies can therefore confirm that an ALK-positive lymphoma expresses NPM-ALK (rather than a variant ALK-fusion protein) and may also provide evidence for chromosomal anomalies involving the NPM gene other than the classical (2;5) translocation.

scholarly journals Platelet-derived growth factor (PDGF)-induced activation of signal transducer and activator of transcription (Stat) 5 is mediated by PDGF β-receptor and is not dependent on c-Src, Fyn, Jak1 or Jak2 kinases

2000 ◽  
Vol 345 (3) ◽  
pp. 759-766 ◽  
Author(s):  
Kirsi PAUKKU ◽  
Sigrídur VALGEIRSDÓTTIR ◽  
Pipsa SAHARINEN ◽  
Mathias BERGMAN ◽  
Carl-Henrik HELDIN ◽  
...  
Keyword(s):  
Growth Factor ◽  
Tyrosine Kinases ◽  
Mammalian Cells ◽  
Platelet Derived Growth Factor ◽  
Wild Type ◽  
Signal Transducers ◽  
Tyrosine Residues ◽  
Type C ◽  
Β Receptor

Several growth factors activate signal transducers and activators of transcription (Stats) but the mechanism of Stat activation in receptor tyrosine kinase signalling has remained elusive. In the present study we have analysed the roles of different platelet-derived growth factor (PDGF)-induced tyrosine kinases in the activation of Stat5. Co-expression experiments in insect and mammalian cells demonstrated that both PDGF β-receptor (PDGF β-R) and Jak1, but not c-Src, induced the activation of Stat5. Furthermore, immune-complex-purified PDGF β-R was able to phosphorylate Stat5 directly. The role of the cytoplasmic tyrosine kinases in the PDGF-induced activation of Stat5 was further investigated by overexpressing kinase-negative (KN) and wild-type Jak and c-Src kinases. Jak1-KN or Jak2-KN had no effect but both Src-KN and wild-type c-Src similarly decreased the PDGF-β-R-induced activation of Stat5. The activation of both Src and Stat5 is dependent on the same tyrosine residues Tyr579 and Tyr581 in PDGF β-R; thus the observed inhibition by Src might result from competition for binding of Stat5 to the receptor. Finally, fibroblasts derived from Src-/- and Fyn-/- mice showed normal pattern of PDGF-induced tyrosine phosphorylation of Stat5. Taken together, these results indicate that Stat5 is a direct substrate for PDGF β-R and that the activation does not require Jak1, Jak2, c-Src or Fyn tyrosine kinases.

scholarly journals The enzymatic activity of 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/IMP cyclohydrolase is enhanced by NPM-ALK: new insights in ALK-mediated pathogenesis and the treatment of ALCL

Blood ◽  
2009 ◽  
Vol 113 (12) ◽  
pp. 2776-2790 ◽  
Author(s):  
Francesco E. Boccalatte ◽  
Claudia Voena ◽  
Chiara Riganti ◽  
Amalia Bosia ◽  
Lucia D'Amico ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Cell Lines ◽  
Tyrosine Kinases ◽  
Cellular Transformation ◽  
Large Cell ◽  
Cytoskeletal Proteins ◽  
Primary Tumors ◽  
Anaplastic Lymphoma ◽  
Alk Positive ◽  
Inosine Monophosphate Cyclohydrolase

AbstractAnaplastic large cell lymphoma represents a subset of neoplasms caused by translocations that juxtapose the anaplastic lymphoma kinase (ALK) to dimerization partners. The constitutive activation of ALK fusion proteins leads to cellular transformation through a complex signaling network. To elucidate the ALK pathways sustaining lymphomagenesis and tumor maintenance, we analyzed the tyrosine-kinase protein profiles of ALK-positive cell lines using 2 complementary proteomic-based approaches, taking advantage of a specific ALK RNA interference (RNAi) or cell-permeable inhibitors. A well-defined set of ALK-associated tyrosine phosphopeptides, including metabolic enzymes, kinases, ribosomal and cytoskeletal proteins, was identified. Validation studies confirmed that vasodilator-stimulated phosphoprotein and 5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase (ATIC) associated with nucleophosmin (NPM)–ALK, and their phosphorylation required ALK activity. ATIC phosphorylation was documented in cell lines and primary tumors carrying ALK proteins and other tyrosine kinases, including TPR-Met and wild type c-Met. Functional analyses revealed that ALK-mediated ATIC phosphorylation enhanced its enzymatic activity, dampening the methotrexate-mediated transformylase activity inhibition. These findings demonstrate that proteomic approaches in well-controlled experimental settings allow the definition of informative proteomic profiles and the discovery of novel ALK downstream players that contribute to the maintenance of the neoplastic phenotype. Prediction of tumor responses to methotrexate may justify specific molecular-based chemotherapy.

scholarly journals Anaplastic lymphoma kinase in human cancer

2011 ◽  
Vol 47 (1) ◽  
pp. R11-R23 ◽  
Author(s):  
Antonella Barreca ◽  
Elena Lasorsa ◽  
Ludovica Riera ◽  
Rodolfo Machiorlatti ◽  
Roberto Piva ◽  
...  
Keyword(s):  
Anaplastic Lymphoma Kinase ◽  
Tyrosine Kinases ◽  
Fusion Proteins ◽  
Molecular Mechanisms ◽  
Cell Lymphoma ◽  
Human Cancer ◽  
Critical Role ◽  
Large Cell ◽  
Anaplastic Lymphoma ◽  
Alk Positive

The receptor tyrosine kinases (RTKs) play a critical role, controlling cell proliferation, survival, and differentiation of normal cells. Their pivotal function has been firmly established in the pathogenesis of many cancers as well. The anaplastic lymphoma kinase (ALK), a transmembrane RTK, originally identified in the nucleophosmin (NPM)–ALK chimera of anaplastic large cell lymphoma, has emerged as a novel tumorigenic player in several human cancers. In this review, we describe the expression of the ALK–RTK, its related fusion proteins, and their molecular mechanisms of activation. Novel tailored strategies are briefly illustrated for the treatment of ALK-positive neoplasms.

Impaired integrin-mediated signal transduction, altered cytoskeletal structure and reduced motility in Hck/Fgr deficient macrophages

1999 ◽  
Vol 112 (22) ◽  
pp. 4067-4078 ◽  
Author(s):  
P.W. Suen ◽  
D. Ilic ◽  
E. Caveggion ◽  
G. Berton ◽  
C.H. Damsky ◽  
...  
Keyword(s):  
Signal Transduction ◽  
Subcellular Localization ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Double Mutant ◽  
Src Family Kinases ◽  
Wild Type ◽  
Associated Proteins ◽  
Coated Surfaces ◽  
Mutant Cells

Integrin-mediated adhesion of monocytes and macrophages initiates a signal transduction pathway that leads to actin cytoskeletal reorganization, cell migration and immunologic activation. This signaling pathway is critically dependent on tyrosine kinases. To investigate the role of the Src-family of tyrosine kinases in integrin signal transduction, we have examined the adhesive properties of macrophages isolated from hck-/-fgr-/- double knockout mice which lack two of the three predominant Src-family kinases expressed in myeloid cells. Previous examination of polymorphonuclear leukocytes from these animals indicated that these kinases were critical in initiating the actin cytoskeletal rearrangements that lead to respiratory burst and granule secretion following integrin ligation. Double mutant peritoneal exudate macrophages demonstrated markedly reduced tyrosine phosphorylation responses compared to wild-type cells following plating on fibronectin, collagen or vitronectin-coated surfaces. Tyrosine phosphorylation of several actin-associated proteins (cortactin, paxillin, and tensin), as well as the Syk and Pyk2 tyrosine kinases, were all significantly reduced in double mutant cells. The subcellular localization of focal-adhesion associated proteins was also dramatically altered in mutant macrophages cultured on fibronectin-coated surfaces. In wild-type cells, filamentous actin, paxillin, and talin were concentrated along leading edges of the plasma membrane, suggesting that these proteins contribute to cellular polarization during migration in culture. Double mutant cells failed to show the polarized subcellular localization of these proteins. Likewise, double mutant macrophages failed to form normal filopodia under standard culture conditions. Together, these signaling and cytoskeletal defects may contribute to the reduced motility observed in in vitro assays. These data provide biochemical and morphological evidence that the Src-family kinases Hck and Fgr are required for normal integrin-mediated signal transduction in murine macrophages.

Oncogenic Anaplastic Lymphoma Kinase Rearrangements in Lymphoma

2009 ◽  
Vol 03 (01) ◽  
pp. 50
Author(s):  
Katrien Van Roosbroeck ◽  
Iwona Wlodarska ◽  
◽  
Keyword(s):  
Anaplastic Lymphoma Kinase ◽  
Tyrosine Kinases ◽  
Cell Lymphoma ◽  
B Cell Lymphoma ◽  
Large Cell ◽  
Null Cell ◽  
Anaplastic Lymphoma ◽  
Large B Cell Lymphoma ◽  
Alk Positive ◽  
Inappropriate Expression

Lymphomas expressing anaplastic lymphoma kinase (ALK) represent two distinct lymphoma entities: ALK-positive T-/null-cell anaplastic large cell lymphoma (ALK+ ALCL) and ALK-positive large B-cell lymphoma (ALK+ LBCL). In both subtypes, the inappropriate expression of ALK is driven by 2p23/ALK-involving chromosomal translocations found to target several partner genes. These translocations lead to constitutively activated and oncogenic ALK fusions, of which nucleophosmin (NPM1)-ALK associated with t(2;5)(p23;q35) is the most common. Recently, various ALK fusions, including those previously described in lymphomas, have been identified in several types of nonhaematological malignancy. Identification of further types of ALK+ tumours is clinically important because in future these patients may benefit from targeted therapy, already applied in neoplasms driven by, for example, the mutated ABL1, KIT and PDGFRA/B tyrosine kinases. In this article, we will focus mainly on oncogenic ALK rearrangements in lymphomas and their molecular consequences.

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