The Product of the Proto-Oncogene c-cbl: A Negative Regulator of the Syk Tyrosine Kinase

AbstractCBL is a RING type E3 ubiquitin ligase that functions as a negative regulator of tyrosine kinase signaling and loss of CBL E3 function is implicated in several forms of leukemia. The Src-like adaptor proteins (SLAP/SLAP2) bind to CBL and are required for CBL-dependent downregulation of antigen receptor, cytokine receptor, and receptor tyrosine kinase signaling. Despite the established role of SLAP/SLAP2 in regulating CBL activity, the nature of the interaction and the mechanisms involved are not known. To understand the molecular basis of the interaction between SLAP/SLAP2 and CBL, we solved the crystal structure of CBL tyrosine kinase binding domain (TKBD) in complex with SLAP2. The carboxy-terminal region of SLAP2 adopts an α-helical structure which binds in a cleft between the 4H, EF-hand, and SH2 domains of the TKBD. This SLAP2 binding site is remote from the canonical TKBD phospho-tyrosine peptide binding site but overlaps with a region important for stabilizing CBL in its autoinhibited conformation. In addition, binding of SLAP2 to CBL in vitro activates the ubiquitin ligase function of autoinhibited CBL. Disruption of the CBL/SLAP2 interface through mutagenesis demonstrated a role for this protein-protein interaction in regulation of CBL E3 ligase activity in cells. Our results reveal that SLAP2 binding to a regulatory cleft of the TKBD provides an alternative mechanism for activation of CBL ubiquitin ligase function.

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The Caenorhabditis elegans locus lin-15, a negative regulator of a tyrosine kinase signaling pathway, encodes two different proteins.

Genetics ◽

10.1093/genetics/137.4.987 ◽

1994 ◽

Vol 137 (4) ◽

pp. 987-997 ◽

Cited By ~ 9

Author(s):

S G Clark ◽

X Lu ◽

H R Horvitz

Keyword(s):

Caenorhabditis Elegans ◽

Tyrosine Kinase ◽

Signaling Pathway ◽

Genomic Region ◽

Negative Regulator ◽

Ras Signaling ◽

Intercellular Signaling ◽

Tyrosine Kinase Signaling ◽

Genomic Regions ◽

Mrna Precursor

Abstract The Caenorhabditis elegans locus lin-15 negatively regulates an intercellular signaling process that induces formation of the hermaphrodite vulva. The lin-15 locus controls two separate genetic activities. Mutants that lack both activities have multiple, ectopic pseudo-vulvae resulting from the overproduction of vulval cells, whereas mutants defective in only one lin-15 activity appear wild-type. lin-15 acts non-cell-autonomously to prevent the activation of a receptor tyrosine kinase/ras signaling pathway. We report here the molecular characterization of the lin-15 locus. The two lin-15 activities are encoded by contiguous genomic regions and by two distinct, non-overlapping transcripts that may be processed from a single mRNA precursor by trans-splicing. Based on the DNA sequence, the 719- and 1,440-amino acid lin-15 proteins are not similar to each other or to known proteins. lin-15 multivulva mutants, which are defective in both lin-15 activities, contain deletions and insertions that affect the lin-15 genomic region.

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Amalgam regulates the receptor tyrosine kinase pathway through Sprouty in glial cell development in the Drosophila larval brain

Journal of Cell Science ◽

10.1242/jcs.250837 ◽

2020 ◽

Vol 133 (19) ◽

pp. jcs250837

Author(s):

Majd M. Ariss ◽

Alexander R. Terry ◽

Abul B. M. M. K. Islam ◽

Nissim Hay ◽

Maxim V. Frolov

Keyword(s):

Cell Proliferation ◽

Tyrosine Kinase ◽

Glial Cell ◽

Receptor Tyrosine Kinase ◽

Cell Development ◽

Negative Regulator ◽

Larval Brain ◽

Adhesion Protein ◽

Type Composition ◽

Signaling Activation

ABSTRACTThe receptor tyrosine kinase (RTK) pathway plays an essential role in development and disease by controlling cell proliferation and differentiation. Here, we profile the Drosophila larval brain by single-cell RNA-sequencing and identify Amalgam (Ama), which encodes a cell adhesion protein of the immunoglobulin IgLON family, as regulating the RTK pathway activity during glial cell development. Depletion of Ama reduces cell proliferation, affects glial cell type composition and disrupts the blood–brain barrier (BBB), which leads to hemocyte infiltration and neuronal death. We show that Ama depletion lowers RTK activity by upregulating Sprouty (Sty), a negative regulator of the RTK pathway. Knockdown of Ama blocks oncogenic RTK signaling activation in the Drosophila glioma model and halts malignant transformation. Finally, knockdown of a human ortholog of Ama, LSAMP, results in upregulation of SPROUTY2 in glioblastoma cell lines, suggesting that the relationship between Ama and Sty is conserved.

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Once Phosphorylated, Tyrosines in Carboxyl Terminus of Protein-tyrosine Kinase Syk Interact with Signaling Proteins, Including TULA-2, a Negative Regulator of Mast Cell Degranulation

Journal of Biological Chemistry ◽

10.1074/jbc.m111.326850 ◽

2012 ◽

Vol 287 (11) ◽

pp. 8194-8204 ◽

Cited By ~ 26

Author(s):

Rodrigo Orlandini de Castro ◽

Juan Zhang ◽

Jacqueline R. Groves ◽

Emilia Alina Barbu ◽

Reuben P. Siraganian

Keyword(s):

Mast Cell ◽

Tyrosine Kinase ◽

Protein Tyrosine Kinase ◽

Mast Cell Degranulation ◽

Negative Regulator ◽

Carboxyl Terminus ◽

Signaling Proteins ◽

Protein Tyrosine

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Loss of Tnk1/Kos1 in Mice Is Associated with B-Lymphomas Specifically DLBCL.

Blood ◽

10.1182/blood.v114.22.3972.3972 ◽

2009 ◽

Vol 114 (22) ◽

pp. 3972-3972

Author(s):

Kishalay Hoare ◽

Mary K Reinhard ◽

Sarasija Hoare ◽

Tammy Flagg ◽

W. Stratford May

Keyword(s):

Tyrosine Kinase ◽

B Cell ◽

Knockout Mice ◽

Cell Lymphoma ◽

B Cell Lymphoma ◽

Negative Regulator ◽

Allelic Loss ◽

B Cell Lymphomas ◽

Primary Tumors ◽

Cell Functions

Abstract Abstract 3972 Poster Board III-908 The ubiquitously expressed nonreceptor tyrosine kinase (NRPTK) Tnk1/Kos1 (Thirty-eight negative kinase/Kinase of the embryonic stem cell) functions as a negative regulator of growth in both murine and human cells by suppressing the Ras-Raf1-MapK growth pathway. Since Tnk1 requires its intrinsic protein tyrosine kinase activity to suppress Ras activity and cell growth, the kinase domain is critical for its function and deletion by targeted homologous recombination leads to spontaneous tumor development in mice. To date, Tnk1/Kos1 is the only reported NRPTK that functions as a tumor suppressor in vivo, while other tyrosine kinases may be oncogenic when mutated or activated. While Tnk1 knockout mice may develop primary tumors in different tissues/organs, mainly B-cell lymphomas develop in Tnk1-/- (80%, 47 of 60) and Tnk1+/- (57%, 31 of 54) mice with similar characteristics of Diffuse Large B-Cell Lymphoma (DLBCL) and Burkitt Lymphoma types. Typically in lymphomas from Tnk1+/- mice the intact wild type allele is epigenetically modified and silenced by promoter methylation. Importantly, the absence of Tnk1 occurs only in the tumor tissue but not in the adjacent uninvolved tissue. Now we find allelic loss with associated reduced expression of Tnk1 transcripts and protein in a cohort of human DLBCL patients. These data underscore the potential clinical relevance of Tnk1 in human hematological malignancies. Furthermore, the B-cell lymphomas that develop in the Tnk1 knockout mice express aberrantly high Ras activity indicating that unmutated Ras is a likely necessary effector of B-cell lymphoma development and survival. We also recently determined that the aberrantly high levels Ras activity in lymphoma (but not paired uninvolved lymphoid tissue) from mice results from a novel mechanism involving stabilization of the Grb2-Sos1 complex to maintain activated Ras in these tissues. Therefore, the Tnk1 knockout mouse provides a unique opportunity to test whether and how Tnk1 is involved in the development and/or maintenance of the B-cell lymphomas that develop in the absence of Ras mutation which may have clinical significance for patients with lymphoma. Disclosures: No relevant conflicts of interest to declare.

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CBL Deletion Mutant Found in AML Patients Cause Transformation of Receptor Tyrosine Kinase Class III Expressing Cells by Activation of the AKT Pathway

Blood ◽

10.1182/blood.v118.21.2447.2447 ◽

2011 ◽

Vol 118 (21) ◽

pp. 2447-2447

Author(s):

Harald Polzer ◽

Hanna Janke ◽

Wolfgang Hiddemann ◽

Dirk Eick ◽

Karsten Spiekermann

Keyword(s):

Tyrosine Kinase ◽

Receptor Tyrosine Kinase ◽

Deletion Mutant ◽

Negative Regulator ◽

Akt Pathway ◽

Deletion Mutants ◽

Class Iii ◽

Akt Phosphorylation ◽

Interaction Sites ◽

Ligand Stimulation

Abstract Abstract 2447 We examined the oncogenic potential of CBL deletion mutant found in AML patients in cytokine receptor and receptor tyrosine kinase (RTKs) expressing cells. In addition, we analyzed the interaction sites of FLT3/CBL and the critical pathways activated by CBL deletion mutants. RTK, CBL and AKT constructs were expressed in Ba/F3 cells via a retroviral expression vector. Stable protein expression after transduction and fluorescence-activated cell sorting (FACS) was confirmed by western blotting and cellsurface-marker expression of receptors by flow cytometry. Cell Proliferation and apoptosis assays were done in presence and absence of IL-3 or receptor-ligands. Coexpression of RTK III-WT (PDGFRA, PDGFRB, FLT3, KIT) and CBL deletion mutants cause IL-3 independent and ligand dependent growth of Ba/F3 cells. RTK III-WT/CBLΔexon8 cells show a more than 10 fold hyperproliferation in response to ligand stimulation. In contrast Non-class III receptor tyrosine kinases (EGFR, EPOR, MPL, IGF1R) and CSF1R show just a very weak hyperproliferation if coexpressed with the CBL deletion mutant. Selective protein tyrosine kinase inhibitors abrogate this proliferation. In cells coexpressing RTK-III receptor and CBLΔexon8 the receptor internalization is delayed and cells were protected from apoptosis after cytokine withdrawal. Ba/F3 cells after ligand stimulation and AML cell lines coexpressing CBL deletion mutants and FLT3 show an enhanced AKT phosphorylation. The PI3K inhibitor LY294002 and the AKT inhibitor MK2206 abolish the CBL mutant mediated hyperproliferation. Furthermore, a combined pharmacological inhibition of PI3K/AKT pathway and RTK shows an additive effect. The transforming potential of the CBL mutant is completely abolished by a mutated PTB domain of CBL (G306E) and decreased by mutation of tyrosines 589 and 591 in the juxtamembrane domain of FLT3. A constitutive active AKT mutant (E17K) recapitulates the CBL deletion mutant induced phenotype in Ba/F3 cells. CBL is a selective negative regulator of class III RTK receptors and the PI3K/AKT pathway is critical for the transforming potential of the CBL oncogene. An alternative mechanism for the constitutive activation of RTKs in tumors occurs through inactivation of a negative regulator. CBL mutants mirror the phenotype of oncogenic RTK and cause an enhanced AKT phosphorylation. Targeted inhibition of FLT3 and AKT might be of therapeutic value in AML patients carrying CBL deletion mutants.Figure:Hyperproliferation of Ba/F3 cells coexpressing indicated receptors and CBL deletion mutant is quoted as X-fold of CBL wildtype coexpressing cells.Figure:. Hyperproliferation of Ba/F3 cells coexpressing indicated receptors and CBL deletion mutant is quoted as X-fold of CBL wildtype coexpressing cells. Disclosures: No relevant conflicts of interest to declare.

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SHP1 tyrosine phosphatase negatively regulates NPM-ALK tyrosine kinase signaling

Blood ◽

10.1182/blood-2005-06-2421 ◽

2006 ◽

Vol 107 (10) ◽

pp. 4130-4138 ◽

Cited By ~ 35

Author(s):

Jean-François Honorat ◽

Ashraf Ragab ◽

Laurence Lamant ◽

Georges Delsol ◽

Jeannie Ragab-Thomas

Keyword(s):

Cell Proliferation ◽

Tyrosine Kinase ◽

Cell Lines ◽

Tyrosine Phosphatase ◽

Inverse Correlation ◽

Large Cell ◽

Tissue Microarrays ◽

Negative Regulator ◽

3T3 Fibroblasts ◽

Alk Positive

Anaplastic large-cell lymphoma (ALCL) is frequently associated with the 2;5 translocation and expresses the NPM-ALK fusion protein, which possesses a constitutive tyrosine kinase activity. We analyzed SHP1 tyrosine phosphatase expression and activity in 3 ALK-positive ALCL cell lines (Karpas 299, Cost, and SU-DHL1) and in lymph node biopsies (n = 40). We found an inverse correlation between the level of NPM-ALK phosphorylation and SHP1 phosphatase activity. Pull-down and coimmunoprecipitation experiments demonstrated a SHP1/NPM-ALK association. Furthermore, confocal microscopy performed on ALCL cell lines and biopsy specimens showed the colocalization of the 2 proteins in cytoplasmic bodies containing Y664-phosphorylated NPM-ALK. Dephosphorylation of NPM-ALK by SHP1 demonstrated that NPM-ALK was a SHP1 substrate. Downregulation of SHP1 expression by RNAi in Karpas cells led to hyperphosphorylation of NPM-ALK, STAT3 activation, and increase in cell proliferation. Furthermore, SHP1 overexpression in 3T3 fibroblasts stably expressing NPM-ALK led to the decrease of NPM-ALK phosphorylation, lower cell proliferation, and tumor progression in nude mice. These findings show that SHP1 is a negative regulator of NPM-ALK signaling. The use of tissue microarrays revealed that 50% of ALK-positive ALCLs were positive for SHP1. Our results suggest that SHP1 could be a critical enzyme in ALCL biology and a potential therapeutic target.

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