1.
2.
Agarwal, Shruti, et al.
(författare)
Phosphorylation of the activation loop tyrosine 823 in c-Kit is crucial for cell survival and proliferation.
2013
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 288:31, s. 22460-22468
Tidskriftsartikel (refereegranskat) 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.
3.
Alvarado-Kristensson, Maria, et al.
(författare)
p38-MAPK signals survival by phosphorylation of caspase-8 and caspase-3 in human neutrophils
2004
Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 199:4, s. 449-458
Tidskriftsartikel (refereegranskat) abstract
Neutrophil apoptosis occurs both in the bloodstream and in the tissue and is considered essential for the resolution of an inflammatory process. Here, we show that p38-mitogen-activated protein kinase (MAPK) associates to caspase-8 and caspase-3 during neutrophil apoptosis and that p38-MAPK activity, previously shown to be a survival signal in these primary cells, correlates with the levels of caspase-8 and caspase-3 phosphorylation. In in vitro experiments, immunoprecipitated active p38-MAPK phosphorylated and inhibited the activity of the active p20 subunits of caspase-8 and caspase-3. Phosphopeptide mapping revealed that these phosphorylations occurred on serine-364 and serine-150, respectively. Introduction of mutated (S150A), but not wild-type, TAT-tagged caspase-3 into primary neutrophils made the Fas-induced apoptotic response insensitive to p38-MAPK inhibition. Consequently, p38-MAPK can directly phosphorylate and inhibit the activities of caspase-8 and caspase-3 and thereby hinder neutrophil apoptosis, and, in so doing, regulate the inflammatory response.
4.
Arora, D, et al.
(författare)
Protein tyrosine phosphatase DEP-1 controls receptor tyrosine kinase FLT3 signaling
2011
Ingår i: Journal of Biological Chemistry. - 1083-351X. ; 286:13, s. 10918-10929
Tidskriftsartikel (refereegranskat) abstract
Fms-like tyrosine kinase 3 (FLT3) plays an important role in hematopoietic differentiation and constitutively active FLT3 mutant proteins contribute to the development of acute myeloid leukemia (AML). Little is known about the protein tyrosine phosphatases (PTP) affecting the signaling activity of FLT3. To identify such PTP, myeloid cells expressing wild type FLT3 were infected with a panel of lentiviral pseudotypes carrying shRNA expression cassettes targeting different PTP. Out of 20 PTP tested, expressed in hematopoietic cells or presumed to be involved in oncogenesis or tumor suppression, DEP-1 (PTPRJ) was identified as a PTP negatively regulating FLT3 phosphorylation and signaling. Stable myeloid cell lines with strongly reduced DEP-1 levels showed site-selective hyperphosphorylation of FLT3. Similarly, acute depletion of DEP-1 in the human AML cell line THP-1 caused elevated FLT3 phosphorylation. Particularly, the sites pY589, pY591, and pY842 involved in the FLT3 ligand (FL) -mediated activation of FLT3 were hyperphosphorylated most. Enhanced FLT3 phosphorylation in DEP-1 depleted cells was accompanied by enhanced FLT3-dependent activation of ERK and cell proliferation. Overexpression of DEP-1 resulted in opposite effects on FL-mediated receptor phosphorylation and signaling activity. Furthermore, FL-mediated colony formation in methylcellulose of 32D cells expressing FLT3 was induced in response to shRNA-mediated DEP-1 knockdown. This transforming effect of DEP-1 knockdown was consistent with a moderately increased activation of STAT5 upon FL-stimulation, but did not translate into myeloproliferative disease formation in the 32D-C3H/HeJ mouse model. The data indicate that DEP-1 is negatively regulating FLT3 signaling activity and its loss may contribute to, but is not sufficient for leukemogenic cell transformation.
5.
Hassel, Sylke, et al.
(författare)
Interaction and functional cooperation between the serine/threonine kinase bone morphogenetic protein type II receptor with the tyrosine kinase stem cell factor receptor
2006
Ingår i: Journal of Cellular Physiology. - : Wiley. - 0021-9541 .- 1097-4652. ; 206:2, s. 457-467
Tidskriftsartikel (refereegranskat) abstract
Transmembrane receptors with intrinsic serine/threonine or tyrosine kinase domains regulate vital functions of cells in multicellular eukaryotes, e.g., differentiation, apoptosis, and proliferation. Here, we show that bone morphogenetic protein type II receptor (BMPR-II) which has a serine/threonine kinase domain, and stem cell factor receptor (c-kit) which contains a tyrosine kinase domain form a complex in vitro and in vivo; the interaction is induced upon treatment of cells with BMP2 and SCF. Stem cell factor (SCF) modulated BMP2-dependent activation of Smad1/5/8 and phosphorylation of Erk kinase. SCF also enhanced BMP2-dependent differentiation of C2C12 cells. We found that BMPR-II was phosphorylated at Ser757 upon co-expression with and activation of c-kit. BMPR-II phosphorylation required intact kinase activity of BMPR-II. Abrogation of the c-kit/SCF-dependent phosphorylation of BMPR-II at the Ser757 interfered with the cooperative effect of BMP2 and SCF. Our data suggest that the complex formation between c-kit and BMPR-II leads to phosphorylation of BMPR-II at Ser757, which modulates BMPR-II-dependent signaling.
6.
Hägg, Maria, et al.
(författare)
EGF and dextran-conjugated EGF induces differential phosphorylation of the EGF receptor
2002
Ingår i: International Journal of Molecular Medicine. - 1107-3756 .- 1791-244X. ; 10:5, s. 655-659
Tidskriftsartikel (refereegranskat) abstract
Dextran-conjugated EGF (EGF-dextran) has a potential use for targeted radionuclide therapy of tumors that overexpress the epidermal growth factor receptor (EGFR). There are plans to treat both bladder carcinomas and malignant gliomas with local injections of radiolabeled EGF-dextran since these tumors often express high levels of EGFR. In this report we show that EGF and EGF-dextran differentially activate the EGFR. In the human glioma cell line U-343, activation of the serine/threonine kinases Erk and Akt is identical upon stimulation with EGF or EGF-dextran. However, the effect on phospholipase Cgamma1 (PLCgamma1) phosphorylation differs. In cells stimulated with EGF-dextran, the PLCgamma1 phosphorylation is lower than in cells stimulated with EGF. This observation could be explained by the fact that the PLCgamma1 association sites in the EGFR, tyrosine residues 992 and 1173, were phosphorylated to a lower degree when the receptor was stimulated with EGF-dextran as compared to with EGF.
7.
Kazi, Julhash U., et al.
(författare)
Src-Like Adaptor Protein (SLAP) Binds to the Receptor Tyrosine Kinase Flt3 and Modulates Receptor Stability and Downstream Signaling.
2012
Ingår i: PLoS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 7:12
Tidskriftsartikel (refereegranskat) abstract
Fms-like tyrosine kinase 3 (Flt3) is an important growth factor receptor in hematopoiesis. Gain-of-function mutations of the receptor contribute to the transformation of acute myeloid leukemia (AML). Src-like adaptor protein (SLAP) is an interaction partner of the E3 ubiquitin ligase Cbl that can regulate receptor tyrosine kinases-mediated signal transduction. In this study, we analyzed the role of SLAP in signal transduction downstream of the type III receptor tyrosine kinase Flt3. The results show that upon ligand stimulation SLAP stably associates with Flt3 through multiple phosphotyrosine residues in Flt3. SLAP constitutively interacts with oncogenic Flt3-ITD and co-localizes with Flt3 near the cell membrane. This association initiates Cbl-dependent receptor ubiquitination and degradation. Depletion of SLAP expression by shRNA in Flt3-transfected Ba/F3 cells resulted in a weaker activation of FL-induced PI3K-Akt and MAPK signaling. Meta-analysis of microarray data from patient samples suggests that SLAP mRNA is differentially expressed in different cancers and its expression was significantly increased in patients carrying the Flt3-ITD mutation. Thus, our data suggest a novel role of SLAP in different cancers and in modulation of receptor tyrosine kinase signaling apart from its conventional role in regulation of receptor stability.
8.
Kazi, Julhash U., et al.
(författare)
Src-Like Adaptor Protein (SLAP) differentially regulates normal and oncogenic c-Kit signaling
2014
Ingår i: Journal of Cell Science. - : The Company of Biologists. - 0021-9533 .- 1477-9137. ; 127:3, s. 653-662
Tidskriftsartikel (refereegranskat) 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.
9.
Lennartsson, Johan, et al.
(författare)
C-Kit signal transduction and involvement in cancer
2005
Ingår i: Cancer Therapy. ; 3, s. 5-28
Forskningsöversikt (refereegranskat) abstract
Receptor tyrosine kinases, such as c-Kit, are proteins whose function it is to transduce signals from the environment into the cell leading to complex behaviors such as proliferation, migration, survival and differentiation. Many of these behaviors are deregulated in cancer, which is characterized by uncontrolled proliferation, insensitivity towards death stimuli, migration of tumor cells away from the primary tumor site and in some cases also block of cellular differentiation leaving the cell in an immature proliferative state. To be able to target these processes it is vital to have a detailed understanding of the receptor function and the downstream pathways activated. In this article we will review the mechanisms by which c-Kit induces signal transduction as well as describing tumors in which c-Kit function is perturbed.
10.
Lennartsson, Johan, et al.
(författare)
The stem cell factor receptor/c-Kit as a drug target in cancer
2006
Ingår i: Current Cancer Drug Targets. - 1568-0096 .- 1873-5576. ; 6:1, s. 65-75
Tidskriftsartikel (refereegranskat) abstract
Tyrosine phosphorylation has a key role in intracellular signaling. Inappropriate proliferation and survival cues in tumor cells often occur as a consequence of unregulated tyrosine kinase activity. Much of the current development of anti-cancer therapies tries to target causative proteins in a specific manner to minimize side-effects. One attractive group of target proteins is the kinases. c-Kit is a receptor tyrosine kinase that normally controls the function of primitive hematopoietic cells, melanocytes and germ cells. It has become clear that uncontrolled activity of c-Kit contributes to formation of an array of human tumors. The unregulated activity of c-Kit may be due to overexpression, autocrine loops or mutational activation. This makes c-Kit an excellent target for cancer therapies in these tumors. In this review we will highlight the current knowledge on the signal transduction molecules and pathways activated by c-Kit under normal conditions and in cancer cells, and the role of aberrant c-Kit signaling in cancer progression. Recent advances in the development of specific inhibitors interfering with these signal transduction pathways will be discussed.
