| 1. |
- Agarwal, Shruti, et al.
(author)
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Phosphorylation of the activation loop tyrosine 823 in c-Kit is crucial for cell survival and proliferation.
- 2013
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In: Journal of Biological Chemistry. - 1083-351X. ; 288:31, s. 22460-22468
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Journal article (peer-reviewed)abstract
- The receptor tyrosine kinase c-Kit, also known as the stem cell factor receptor, plays a key role in several developmental processes. Activating mutations in c-Kit lead to alteration of these cellular processes and have been implicated in many human cancers such as gastrointestinal stromal tumors (GISTs), acute myeloid leukemia (AML), testicular seminomas and mastocytosis. Regulation of the catalytic activity of several kinases is known to be governed by phosphorylation of tyrosine residues in the activation loop of the kinase domain. However, in the case of c-Kit phosphorylation of Y823 has been demonstrated to be a late event that is not required for kinase activation. However, since phosphorylation of Y823 is a ligand-activated event, we sought to investigate the functional consequences of Y823 phosphorylation. By using a tyrosine to phenylalanine mutant of tyrosine 823 we investigated the impact of Y823 on c-Kit signaling. We here demonstrate that Y823 is crucial for cell survival and proliferation and mutation of Y823 to phenylalanine leads to decreased sustained phosphorylation and ubiquitination of c-Kit as compared to the wild-type receptor. Furthermore, the mutated receptor was upon ligand-stimulation quickly internalized and degraded. Phosphorylation of the E3 ubiquitin ligase, Cbl was transient followed by a substantial reduction in phosphorylation of downstream signaling molecules such as Akt, Erk, Shc and Gab2. Thus, we propose that activation loop tyrosine 823 is crucial for activation of both the MAPK and PI3K pathways and that its disruption leads to a destabilization of the c-Kit receptor and decreased survival of cells.
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| 2. |
- Agarwal, Shruti, et al.
(author)
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The activation loop tyrosine 823 is essential for the transforming capacity of the c-Kit oncogenic mutant D816V.
- 2015
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In: Oncogene. - : Springer Science and Business Media LLC. - 1476-5594 .- 0950-9232. ; 34:35, s. 4581-4590
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Journal article (peer-reviewed)abstract
- Oncogenic c-Kit mutations have been shown to display ligand-independent receptor activation and cell proliferation. A substitution of aspartate to valine at amino acid 816 (D816V) is one of the most commonly found oncogenic c-Kit mutations and is found in >90% of cases of mastocytosis and less commonly in germ-cell tumors, core-binding factor acute myeloid leukemia and mucosal melanomas. The mechanisms by which this mutation leads to constitutive activation and transformation are not fully understood. Previous studies have shown that the D816V mutation causes a structural change in the activation loop (A-loop), resulting in weaker binding of the A-loop to the juxtamembrane domain. In this paper, we have investigated the role of Y823, the only tyrosine residue in the A-loop, and its role in oncogenic transformation by c-Kit/D816V by introducing the Y823F mutation. Although dispensable for the kinase activity of c-Kit/D816V, the presence of Y823 was crucial for cell proliferation and survival. Furthermore, mutation of Y823 selectively downregulates the Ras/Erk and Akt pathways as well as the phosphorylation of STAT5 and reduces the transforming capacity of the D816V/c-Kit in vitro. We further show that mice injected with cells expressing c-Kit/D816V/Y823F display significantly reduced tumor size as well as tumor weight compared with controls. Finally, microarray analysis, comparing Y823F/D816V cells with cells expressing c-Kit/D816V, demonstrate that mutation of Y823 causes upregulation of proapoptotic genes, whereas genes of survival pathways are downregulated. Thus, phosphorylation of Y823 is not necessary for kinase activation, but essential for the transforming ability of the c-Kit/D816V mutant.Oncogene advance online publication, 1 December 2014; doi:10.1038/onc.2014.383.
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| 3. |
- Heiberg, Einar, et al.
(author)
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Longitudinal strain from velocity encoded cardiovascular magnetic resonance: a validation study
- 2013
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In: Journal of Cardiovascular Magnetic Resonance. - 1097-6647. ; 15
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Journal article (peer-reviewed)abstract
- Background: Regional myocardial function is typically evaluated by visual assessment by experienced users, or by methods requiring substantial post processing time. Visual assessment is subjective and not quantitative. Therefore, the purpose of this study is to develop and validate a simple method to derive quantitative measures of regional wall function from velocity encoded Cardiovascular Magnetic Resonance (CMR), and provide associated normal values for longitudinal strain. Method: Both fast field echo (FFE) and turbo field echo (TFE) velocity encoded CMR images were acquired in three long axis planes in 36 healthy volunteers (13 women, 23 men), age 35 +/- 12 years. Strain was also quantified in 10 patients within one week after myocardial infarction. The user manually delineated myocardium in one time frame and strain was calculated as the myocardium was tracked throughout the cardiac cycle using an optimization formulation and mechanical a priori assumptions. A phantom experiment was performed to validate the method with optical tracking of deformation as an independent gold standard. Results: There was an excellent agreement between longitudinal strain measured by optical tracking and longitudinal strain measured with TFE velocity encoding. Difference between the two methods was 0.0025 +/- 0.085 (ns). Mean global longitudinal strain in the 36 healthy volunteers was -0.18 +/- 0.10 (TFE imaging). Intra-observer variability for all segments was 0.00 +/- 0.06. Inter-observer variability was -0.02 +/- 0.07 (TFE imaging). The intra-observer variability for radial strain was high limiting the applicability of radial strain. Mean longitudinal strain in patients was significantly lower (-0.15 +/- 0.12) compared to healthy volunteers (p<0.05). Strain (expressed as percentage of normal strain) in infarcted regions was lower compared to remote areas (p<0.01). Conclusion: In conclusion, we have developed and validated a robust and clinically applicable technique that can quantify longitudinal strain and regional myocardial wall function and present the associated normal values for longitudinal strain.
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| 4. |
- Kazi, Julhash U., et al.
(author)
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Src-Like Adaptor Protein (SLAP) differentially regulates normal and oncogenic c-Kit signaling
- 2014
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In: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 127:3, s. 653-662
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Journal article (peer-reviewed)abstract
- The Src-Like Adaptor Protein (SLAP) is an adaptor protein sharing considerable structural homology with Src. SLAP is expressed in variety of cells regulating receptor tyrosine kinase signaling by direct association. In this report, we show that SLAP associates with both wild-type and oncogenic c-Kit (c-Kit-D816V). The association involves SLAP SH2 domain and receptor phosphotyrosine residues different from those mediating Src interaction. Association of SLAP triggers c-Kit ubiquitination which, in turn, is followed by receptor degradation. Although SLAP depletion potentiates c-Kit downstream signaling by stabilizing the receptor, it remains non-functional in c-Kit-D816V signaling. Ligand-stimulated c-Kit or c-Kit-D816V did not alter membrane localization of SLAP. Interestingly oncogenic c-Kit-D816V, but not wild-type c-Kit, phosphorylates SLAP on Y120, Y258 and Y273 residues. Physical interaction between c-Kit-D816V and SLAP is mandatory for the phosphorylation to take place. Although tyrosine phosphorylated SLAP does not affect c-Kit-D816V signaling, mutation of these tyrosine sites to phenylalanine can restore SLAP activity. Taken together the data demonstrate that SLAP negatively regulates wild-type c-Kit signaling, but not its oncogenic counterpart, indicating a possible mechanism by which the oncogenic c-Kit bypasses the normal cellular negative feedback control.
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| 5. |
- Kazi, Julhash U., et al.
(author)
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The tyrosine kinase CSK associates with FLT3 and c-Kit receptors and regulates downstream signaling.
- 2013
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In: Cellular Signalling. - : Elsevier BV. - 1873-3913 .- 0898-6568. ; 25:9, s. 1852-1860
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Journal article (peer-reviewed)abstract
- Type III receptor tyrosine kinases (RTKs), FLT3 and c-Kit play important roles in a variety of cellular processes. A number of SH2-domain containing proteins interact with FLT3 and c-Kit and regulate downstream signaling. The SH2-domain containing non-receptor protein tyrosine kinase CSK is mainly studied in context of regulating Src family kinases. Here we present an addition role of this kinase in RTK signaling. We show that CSK interacts with FLT3 and c-Kit in a phosphorylation dependent manner. This interaction is facilitated through the SH2-domain of CSK. Under basal conditions CSK is mainly localized throughout the cytosolic compartment but upon ligand stimulation it is recruited to the inner side of cell membrane. CSK association did not alter receptor ubiquitination or phosphorylation but disrupted downstream signaling. Selective depletion of CSK using siRNA, or inhibition with CSK inhibitor, led to increased phosphorylation of Akt and Erk, but not p38, upon FLT3 ligand (FL) stimulation. Stem cell factor (SCF)-mediated Akt and Erk activation was also elevated by CSK inhibition. However, siRNA mediated CSK knockdown increased SCF stimulated Akt phosphorylation but decreased Erk phosphorylation. CSK depletion also significantly increased both FL- and SCF-induced SHC, Gab2 and SHP2 phosphorylation. Furthermore, CSK depletion contributed to oncogenic FLT3- and c-Kit-mediated cell proliferation, but not to cell survival. Thus, the results indicate that CSK association with type III RTKs, FLT3 and c-Kit can have differential impact on receptor downstream signaling.
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