scholarly journals Regulation of Protein Tyrosine Kinase Signaling by Substrate Degradation during Brain Development

2003 ◽  
Vol 23 (24) ◽  
pp. 9293-9302 ◽  
Author(s):  
Lionel Arnaud ◽  
Bryan A. Ballif ◽  
Jonathan A. Cooper
Keyword(s):  
Brain Development ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Adaptor Protein ◽  
Degradation Pathway ◽  
Mammalian Brain ◽  
Normal Brain ◽  
Protein Levels

ABSTRACT Disabled-1 (Dab1) is a cytoplasmic adaptor protein that regulates neuronal migrations during mammalian brain development. Dab1 function in vivo depends on tyrosine phosphorylation, which is stimulated by extracellular Reelin and requires Src family kinases. Reelin signaling also negatively regulates Dab1 protein levels in vivo, and reduced Dab1 levels may be part of the mechanism that regulates neuronal migration. We have made use of mouse embryo cortical neuron cultures in which Reelin induces Dab1 tyrosine phosphorylation and Src family kinase activation. We have found that Dab1 is normally stable, but in response to Reelin it becomes polyubiquitinated and degraded via the proteasome pathway. We have established that tyrosine phosphorylation of Dab1 is required for its degradation. Dab1 molecules lacking phosphotyrosine are not degraded in neurons in which the Dab1 degradation pathway is active. The requirements for Reelin-induced degradation of Dab1 in vitro correctly predict Dab1 protein levels in vivo in different mutant mice. We also provide evidence that Dab1 serine/threonine phosphorylation may be important for Dab1 tyrosine phosphorylation. Our data provide the first evidence for how Reelin down-regulates Dab1 protein expression in vivo. Dab1 degradation may be important for ensuring a transient Reelin response and may play a role in normal brain development.

scholarly journals Fibronectin-stimulated signaling from a focal adhesion kinase-c-Src complex: involvement of the Grb2, p130cas, and Nck adaptor proteins.

1997 ◽  
Vol 17 (3) ◽  
pp. 1702-1713 ◽  
Author(s):  
D D Schlaepfer ◽  
M A Broome ◽  
T Hunter
Keyword(s):  
Focal Adhesion Kinase ◽  
Focal Adhesion ◽  
Intracellular Signaling ◽  
Protein Tyrosine Kinase ◽  
Adaptor Protein ◽  
Adaptor Proteins ◽  
Sh2 Domain ◽  
Mitogen Activated Protein Kinase

The focal adhesion kinase (FAK), a protein-tyrosine kinase (PTK), associates with integrin receptors and is activated by cell binding to extracellular matrix proteins, such as fibronectin (FN). FAK autophosphorylation at Tyr-397 promotes Src homology 2 (SH2) domain binding of Src family PTKs, and c-Src phosphorylation of FAK at Tyr-925 creates an SH2 binding site for the Grb2 SH2-SH3 adaptor protein. FN-stimulated Grb2 binding to FAK may facilitate intracellular signaling to targets such as ERK2-mitogen-activated protein kinase. We examined FN-stimulated signaling to ERK2 and found that ERK2 activation was reduced 10-fold in Src- fibroblasts, compared to that of Src- fibroblasts stably reexpressing wild-type c-Src. FN-stimulated FAK phosphotyrosine (P.Tyr) and Grb2 binding to FAK were reduced, whereas the tyrosine phosphorylation of another signaling protein, p130cas, was not detected in the Src- cells. Stable expression of residues 1 to 298 of Src (Src 1-298, which encompass the SH3 and SH2 domains of c-Src) in the Src- cells blocked Grb2 binding to FAK; but surprisingly, Src 1-298 expression also resulted in elevated p130cas P.Tyr levels and a two- to threefold increase in FN-stimulated ERK2 activity compared to levels in Src- cells. Src 1-298 bound to both FAK and p130cas and promoted FAK association with p130cas in vivo. FAK was observed to phosphorylate p130cas in vitro and could thus phosphorylate p130cas upon FN stimulation of the Src 1-298-expressing cells. FAK-induced phosphorylation of p130cas in the Src 1-298 cells promoted the SH2 domain-dependent binding of the Nck adaptor protein to p130cas, which may facilitate signaling to ERK2. These results show that there are additional FN-stimulated pathways to ERK2 that do not involve Grb2 binding to FAK.

scholarly journals The Novel Adaptor Protein Tks4 (SH3PXD2B) Is Required for Functional Podosome Formation

2009 ◽  
Vol 20 (5) ◽  
pp. 1302-1311 ◽  
Author(s):  
Matthew D. Buschman ◽  
Paul A. Bromann ◽  
Pilar Cejudo-Martin ◽  
Fang Wen ◽  
Ian Pass ◽  
...  
Keyword(s):  
Protein Tyrosine Kinase ◽  
Metastatic Cancer ◽  
Adaptor Protein ◽  
Membrane Structures ◽  
Kinase Substrate ◽  
Amino Terminal ◽  
Ecm Degradation ◽  
Membrane Type

Metastatic cancer cells have the ability to both degrade and migrate through the extracellular matrix (ECM). Invasiveness can be correlated with the presence of dynamic actin-rich membrane structures called podosomes or invadopodia. We showed previously that the adaptor protein tyrosine kinase substrate with five Src homology 3 domains (Tks5)/Fish is required for podosome/invadopodia formation, degradation of ECM, and cancer cell invasion in vivo and in vitro. Here, we describe Tks4, a novel protein that is closely related to Tks5. This protein contains an amino-terminal Phox homology domain, four SH3 domains, and several proline-rich motifs. In Src-transformed fibroblasts, Tks4 is tyrosine phosphorylated and predominantly localized to rosettes of podosomes. We used both short hairpin RNA knockdown and mouse embryo fibroblasts lacking Tks4 to investigate its role in podosome formation. We found that lack of Tks4 resulted in incomplete podosome formation and inhibited ECM degradation. Both phenotypes were rescued by reintroduction of Tks4, whereas only podosome formation, but not ECM degradation, was rescued by overexpression of Tks5. The tyrosine phosphorylation sites of Tks4 were required for efficient rescue. Furthermore, in the absence of Tks4, membrane type-1 matrix metalloproteinase (MT1-MMP) was not recruited to the incomplete podosomes. These findings suggest that Tks4 and Tks5 have overlapping, but not identical, functions, and implicate Tks4 in MT1-MMP recruitment and ECM degradation.

Regulation of angiopoietin expression by bacterial lipopolysaccharide

2008 ◽  
Vol 294 (5) ◽  
pp. L955-L963 ◽  
Author(s):  
Mahroo Mofarrahi ◽  
Thamir Nouh ◽  
Salman Qureshi ◽  
Loic Guillot ◽  
Dominique Mayaki ◽  
...  
Keyword(s):  
Tyrosine Phosphorylation ◽  
Inflammatory Responses ◽  
Cell Types ◽  
Vascular Leakage ◽  
Saline Control ◽  
E Coli ◽  
Protein Levels ◽  
Angiopoietin 2

Angiopoietins are ligands for Tie-2 receptors and play important roles in angiogenesis and inflammation. While angiopoietin-1 (Ang-1) inhibits inflammatory responses, angiopoietin-2 (Ang-2) promotes cytokine production and vascular leakage. In this study, we evaluated in vivo and in vitro effects of Escherichia coli lipopolysaccharides (LPS) on angiopoietin expression. Wild-type C57/BL6 mice were injected with saline (control) or E. coli LPS (20 mg/ml ip) and killed 6, 12, and 24 h later. The diaphragm, lung, and liver were excised and assayed for mRNA and protein expression of Ang-1, Ang-2, and Tie-2 protein and tyrosine phosphorylation. LPS injection elicited a severalfold rise in Ang-2 mRNA and protein levels in the three organs. By comparison, both Ang-1 and Tie-2 levels in the diaphragm, liver, and lung were significantly attenuated by LPS administration. In addition, Tie-2 tyrosine phosphorylation in the lung was significantly reduced in response to LPS injection. In vitro exposure to E. coli LPS elicited cell-specific changes in Ang-1 expression, with significant induction in Ang-1 expression being observed in cultured human epithelial cells, whereas significant attenuation of Ang-1 expression was observed in response to E. coli LPS exposure in primary human skeletal myoblasts. In both cell types, E. coli LPS elicited substantial induction of Ang-2 mRNA, a response that was mediated in part through NF-κB. We conclude that in vivo endotoxemia triggers functional inhibition of the Ang-1/Tie-2 receptor pathway by reducing Ang-1 and Tie-2 expression and inducing Ang-2 levels and that this response may contribute to enhanced vascular leakage in sepsis.

scholarly journals Tyrosine phosphorylation of CRKL in Philadelphia+ leukemia

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1731-1736 ◽  
Author(s):  
J ten Hoeve ◽  
RB Arlinghaus ◽  
JQ Guo ◽  
N Heisterkamp ◽  
J Groffen
Keyword(s):  
Tyrosine Phosphorylation ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Catalytic Domain ◽  
Lymphoblastic Leukemia ◽  
Adaptor Protein ◽  
Myelogenous Leukemia ◽  
Peripheral Blood Cells

Abstract The chimeric BCR/ABL protein is characteristic of Philadelphia (Ph)+ leukemia because it is the direct product of the Ph translocation and it has been shown to play a causal role in the genesis of leukemia. The BCR/ABL protein exhibits a deregulated tyrosine-kinase activity capable of phosphorylating different cellular substrates in vivo and in vitro. CRKL, an adaptor protein consisting of SH2 and SH3 domains in the absence of a catalytic domain, is one potential in vivo substrate of BCR/ABL. Previous experiments have shown that CRKL is phosphorylated on tyrosine in the chronic myelogenous leukemia (CML) cell line K562 and that CRKL is a substrate for ABL and for BCR/ABL in COS-1 cells. In the current study, we show that in peripheral blood cells a direct correlation exists between the presence of BCR/ABL and the phosphorylation status of CRKL. In Ph- peripheral blood cells, CRKL is present only in the nonphosphorylated form. In contrast, all BCR/ABL+ CML and acute lymphoblastic leukemia patient samples examined showed clear tyrosine-phosphorylation of CRKL. This result strongly suggests that CRKL is a biologically significant substrate for BCR/ABL and is likely to play a major role in the development of Ph+ leukemia.

scholarly journals Tyrosine phosphorylation of CRKL in Philadelphia+ leukemia

Blood ◽  
1994 ◽  
Vol 84 (6) ◽  
pp. 1731-1736 ◽  
Author(s):  
J ten Hoeve ◽  
RB Arlinghaus ◽  
JQ Guo ◽  
N Heisterkamp ◽  
J Groffen
Keyword(s):  
Tyrosine Phosphorylation ◽  
Peripheral Blood ◽  
Blood Cells ◽  
Catalytic Domain ◽  
Lymphoblastic Leukemia ◽  
Adaptor Protein ◽  
Myelogenous Leukemia ◽  
Peripheral Blood Cells

The chimeric BCR/ABL protein is characteristic of Philadelphia (Ph)+ leukemia because it is the direct product of the Ph translocation and it has been shown to play a causal role in the genesis of leukemia. The BCR/ABL protein exhibits a deregulated tyrosine-kinase activity capable of phosphorylating different cellular substrates in vivo and in vitro. CRKL, an adaptor protein consisting of SH2 and SH3 domains in the absence of a catalytic domain, is one potential in vivo substrate of BCR/ABL. Previous experiments have shown that CRKL is phosphorylated on tyrosine in the chronic myelogenous leukemia (CML) cell line K562 and that CRKL is a substrate for ABL and for BCR/ABL in COS-1 cells. In the current study, we show that in peripheral blood cells a direct correlation exists between the presence of BCR/ABL and the phosphorylation status of CRKL. In Ph- peripheral blood cells, CRKL is present only in the nonphosphorylated form. In contrast, all BCR/ABL+ CML and acute lymphoblastic leukemia patient samples examined showed clear tyrosine-phosphorylation of CRKL. This result strongly suggests that CRKL is a biologically significant substrate for BCR/ABL and is likely to play a major role in the development of Ph+ leukemia.

scholarly journals SH3KBP1 Promotes Glioblastoma Tumorigenesis by Activating EGFR Signaling

2021 ◽  
Vol 10 ◽  
Author(s):  
Hai Song ◽  
Yanpei Wang ◽  
Chaojia Shi ◽  
Jianxiang Lu ◽  
Tian Yuan ◽  
...  
Keyword(s):  
Growth Factor Receptor ◽  
Adaptor Protein ◽  
Egfr Signaling ◽  
Potential Therapeutic Target ◽  
Downstream Signaling ◽  
Protein Levels ◽  
Egfr Activation ◽  
Epidermal Growth

Glioblastoma (GBM) is the most common and aggressive brain tumor in adults. Overexpression or activation of epidermal growth factor receptor (EGFR) occurs commonly in multiple human cancers and promotes tumorigenesis. However, the underlying molecular mechanism of EGFR aberrant activation and the downstream signaling pathways remains largely unknown. In this study, we report that both SH3-domain kinase binding protein 1 (SH3KBP1) mRNA and protein levels are highly expressed in GBM and its high expression is associated with worse survival of glioma patients. In addition, we provide evidence that SH3KBP1 is prominently expressed in GBM stem cells (GSCs) and have potential to serve as a novel GSCs marker. Moreover, silencing SH3KBP1 dramatically impairs GBM cell proliferation, migration and GSCs self-renewal ability in vitro and xenograft tumors growth in vivo. Most importantly, we found that SH3KBP1 directly interacts with EGFR and may act as an adaptor protein to transduce EGFR signaling. Together, our work uncovers SH3KBP1 as a novel regulator of oncogenic EGFR signaling and also as a potential therapeutic target for GBM patients with EGFR activation.

scholarly journals Identification of Csk tyrosine phosphorylation sites and a tyrosine residue important for kinase domain structure

1997 ◽  
Vol 322 (3) ◽  
pp. 927-935 ◽  
Author(s):  
Vladimir JOUKOV ◽  
Mauno VIHINEN ◽  
Satu VAINIKKA ◽  
Janusz M. SOWADSKI ◽  
Kari ALITALO ◽  
...  
Keyword(s):  
Tyrosine Kinase ◽  
Tyrosine Phosphorylation ◽  
Protein Tyrosine Kinase ◽  
Three Dimensional ◽  
Kinase Domain ◽  
Sh2 Domain ◽  
Binding Studies ◽  
Inactive Form

The lack of a conserved tyrosine autophosphorylation site is a unique feature of the C-terminal Src-kinase, Csk, although this protein tyrosine kinase can be autophosphorylated on tyrosine residues in vitro and in bacteria. Here we show that human Csk is tyrosine phosphorylated in HeLa cells treated with sodium pervanadate. Phosphorylation in vivo occurs mainly at Tyr-184 and in vitro mainly at Tyr-304. A Y304F mutation strongly decreased Csk phosphorylation in vitro, and a Y184F mutation abolished tyrosine phosphorylation in vivo. A catalytically inactive form of Csk was also phosphorylated on Tyr-184 in vivo, suggesting that this is not a site of autophosphorylation. The kinase activity of the Y184F protein was not changed, while the Y304F protein showed one-third of wild-type activity. Three-dimensional modelling of the Csk kinase domain indicated that the Y304F mutation abolishes one of two conserved hydrogen bonds between the upper and the lower lobes in the open conformation of the kinase domain. Phosphopeptide binding studies suggested that phosphorylation of Tyr-184 creates a binding site for low-molecular-mass proteins. Cellular Csk was associated with several phosphoproteins, some of which were interacting with the Csk SH2 domain. Taken together these results indicate that Csk can be phosphorylated in vivo at Tyr-184 by an as yet unknown tyrosine kinase, and that autophosphorylation of Tyr-304 occurs only at abnormally high Csk concentrations in vitro. Furthermore, Tyr-304 is required for the maintenance of the structure of the Csk kinase domain.

scholarly journals The TRIM26-SOX2 Ubiquitin Signaling Axis Regulates the Maintenance and Tumorigenicity of Glioblastoma Stem-like Cells

Neurosurgery ◽  
2019 ◽  
Vol 66 (Supplement_1) ◽  
Author(s):  
Tatenda Mahlokozera ◽  
Mounica Paturu ◽  
Daniel Hafez ◽  
Diane Mao ◽  
Albert H Kim
Keyword(s):  
Protein Interactions ◽  
Normal Brain ◽  
Mrna Levels ◽  
Major Question ◽  
Self Renewal ◽  
Protein Levels ◽  
Signaling Axis ◽  
Related Transcription Factor

Abstract INTRODUCTION Glioblastomas harbor inter and intratumoral genetic diversity, posing a challenge for targeted therapies. A major question is whether shared mechanisms might control the malignant phenotypes of genetically diverse glioblastoma cells. We reasoned that ubiquitin-dependent regulation of pluripotency-related transcription factor SOX2, which is indispensable for the maintenance of tumorigenic glioblastoma stem-like cells (GSC), may represent one such mechanism. TRIM26, an E3-ubiquitin ligase with immune-related functions, is highly expressed in glioblastoma tumors compared to normal brain. Immunoprecipitation followed by mass spectrometry suggested TRIM26 interacts with SOX2. We hypothesized that TRIM26 plays an essential role in GSCs by regulating SOX2 function. METHODS Direct protein-protein interactions were assessed by in vitro binding assays. In Vitro ubiquitination assays were performed. Lentiviral TRIM26 overexpression and knockdown were used to test the role of TRIM26 in regulating SOX2 stability, activity, and ubiquitination. The functional relevance of TRIM26 in GSCs was assessed by in Vitro self-renewal and in Vivo tumorigenicity assays. RESULTS We found that TRIM26 directly interacts with SOX2 via the C-terminal PRY-SPRY domain. Unexpectedly, TRIM26 overexpression resulted in decreased SOX2 polyubiquitination in cells. In line with this observation, TRIM26 knockdown in GSCs decreased SOX2 protein stability without changing SOX2 mRNA levels. Functionally, TRIM26 knockdown reduced SOX2 transcriptional activity, self-renewal, and in Vivo tumorigenicity in multiple genetically divergent GSC lines. Mechanistically, we discovered TRIM26 stabilizes SOX2 protein by competitively reducing the interaction of SOX2 with WWP2, a bonafide SOX2 E3 ligase in GSCs. Accordingly; WWP2 depletion in the setting of TRIM26 knockdown in GSCs rescued SOX2 protein levels, self-renewal, and tumorigenicity. CONCLUSION Together, these results suggest that TRIM26 maintains GSCs by protecting SOX2 from WWP2-mediated ubiquitination and subsequent proteasomal degradation. These findings raise the intriguing possibility that modulating ubiquitin-dependent regulation of SOX2 in genetically heterogeneous GSCs may represent a unifying therapeutic strategy.

scholarly journals LPTO-06. A NOVEL BRAIN-PERMEANT CHEMOTHERAPEUTIC AGENT FOR THE TREATMENT OF BREAST CANCER LEPTOMENINGEAL METASTASIS

2019 ◽  
Vol 1 (Supplement_1) ◽  
pp. i7-i7
Author(s):  
Jiaojiao Deng ◽  
Sophia Chernikova ◽  
Wolf-Nicolas Fischer ◽  
Kerry Koller ◽  
Bernd Jandeleit ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Brain Tumors ◽  
Cell Lines ◽  
Chemotherapeutic Agent ◽  
Leptomeningeal Metastasis ◽  
Breast Cancer Cell Lines ◽  
Normal Brain ◽  
Breast Cancers

Abstract Leptomeningeal metastasis (LM), a spread of cancer to the cerebrospinal fluid and meninges, is universally and rapidly fatal due to poor detection and no effective treatment. Breast cancers account for a majority of LMs from solid tumors, with triple-negative breast cancers (TNBCs) having the highest propensity to metastasize to LM. The treatment of LM is challenged by poor drug penetration into CNS and high neurotoxicity. Therefore, there is an urgent need for new modalities and targeted therapies able to overcome the limitations of current treatment options. Quadriga has discovered a novel, brain-permeant chemotherapeutic agent that is currently in development as a potential treatment for glioblastoma (GBM). The compound is active in suppressing the growth of GBM tumor cell lines implanted into the brain. Radiolabel distribution studies have shown significant tumor accumulation in intracranial brain tumors while sparing the adjacent normal brain tissue. Recently, we have demonstrated dose-dependent in vitro and in vivo anti-tumor activity with various breast cancer cell lines including the human TNBC cell line MDA-MB-231. To evaluate the in vivo antitumor activity of the compound on LM, we used the mouse model of LM based on the internal carotid injection of luciferase-expressing MDA-MB-231-BR3 cells. Once the bioluminescence signal intensity from the metastatic spread reached (0.2 - 0.5) x 106 photons/sec, mice were dosed i.p. twice a week with either 4 or 8 mg/kg for nine weeks. Tumor growth was monitored by bioluminescence. The compound was well tolerated and caused a significant delay in metastatic growth resulting in significant extension of survival. Tumors regressed completely in ~ 28 % of treated animals. Given that current treatments for LM are palliative with only few studies reporting a survival benefit, Quadriga’s new agent could be effective as a therapeutic for both primary and metastatic brain tumors such as LM. REF: https://onlinelibrary.wiley.com/doi/full/10.1002/pro6.43

scholarly journals All-Trans Retinoic Acid Increases DRP1 Levels and Promotes Mitochondrial Fission

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1202
Author(s):  
Bojjibabu Chidipi ◽  
Syed Islamuddin Shah ◽  
Michelle Reiser ◽  
Manasa Kanithi ◽  
Amanda Garces ◽  
...  
Keyword(s):  
Retinoic Acid ◽  
Mitochondrial Fission ◽  
Normal Function ◽  
Mitochondrial Network ◽  
Protein Levels ◽  
Trans Retinoic Acid ◽  
All Trans Retinoic Acid ◽  
Area And Perimeter

In the heart, mitochondrial homeostasis is critical for sustaining normal function and optimal responses to metabolic and environmental stressors. Mitochondrial fusion and fission are thought to be necessary for maintaining a robust population of mitochondria, and disruptions in mitochondrial fission and/or fusion can lead to cellular dysfunction. The dynamin-related protein (DRP1) is an important mediator of mitochondrial fission. In this study, we investigated the direct effects of the micronutrient retinoid all-trans retinoic acid (ATRA) on the mitochondrial structure in vivo and in vitro using Western blot, confocal, and transmission electron microscopy, as well as mitochondrial network quantification using stochastic modeling. Our results showed that ATRA increases DRP1 protein levels, increases the localization of DRP1 to mitochondria in isolated mitochondrial preparations. Our results also suggested that ATRA remodels the mitochondrial ultrastructure where the mitochondrial area and perimeter were decreased and the circularity was increased. Microscopically, mitochondrial network remodeling is driven by an increased rate of fission over fusion events in ATRA, as suggested by our numerical modeling. In conclusion, ATRA results in a pharmacologically mediated increase in the DRP1 protein. It also results in the modulation of cardiac mitochondria by promoting fission events, altering the mitochondrial network, and modifying the ultrastructure of mitochondria in the heart.

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