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

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.

scholarly journals Patched1–ArhGAP36–PKA–Inversin axis determines the ciliary translocation of Smoothened for Sonic Hedgehog pathway activation

2018 ◽  
Vol 116 (3) ◽  
pp. 874-879 ◽  
Author(s):  
Boyan Zhang ◽  
Tenghan Zhuang ◽  
Qiaoyu Lin ◽  
Biying Yang ◽  
Xiaowei Xu ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Sonic Hedgehog ◽  
Initial Step ◽  
Negative Regulator ◽  
Individual Development ◽  
Sonic Hedgehog Pathway ◽  
Shh Pathway ◽  
Pathway Activation ◽  
Mother Centriole ◽  
Signaling Activation

The Sonic Hedgehog (Shh) pathway conducts primarily in the primary cilium and plays important roles in cell proliferation, individual development, and tumorigenesis. Shh ligand binding with its ciliary membrane-localized transmembrane receptor Patched1 results in the removal of Patched1 from and the translocation of the transmembrane oncoprotein Smoothened into the cilium, leading to Shh signaling activation. However, how these processes are coupled remains unknown. Here, we show that the Patched1–ArhGAP36–PKA–Inversin axis determines the ciliary translocation of Smoothened. We find that Patched1 interacts with and stabilizes the PKA negative regulator ArhGAP36 to the centrosome. Activating the Shh pathway results in the removal of ArhGAP36 from the mother centriole and the centrosomal PKA accumulation. This PKA then phosphorylates Inversin and promotes its interaction with and the ciliary translocation of Smoothened. Knockdown of Inversin disrupts the ciliary translocation of Smoothened and Shh pathway activation. These findings reveal a regulatory molecular mechanism for the initial step of Shh pathway activation.

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

Blood ◽  
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.

SHP1 tyrosine phosphatase negatively regulates NPM-ALK tyrosine kinase signaling

Blood ◽  
2006 ◽  
Vol 107 (10) ◽  
pp. 4130-4138 ◽  
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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