| 1. |
- Alvarado-Kristensson, Maria, et al.
(författare)
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p38-MAPK signals survival by phosphorylation of caspase-8 and caspase-3 in human neutrophils
- 2004
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Ingår i: Journal of Experimental Medicine. - : Rockefeller University Press. - 0022-1007 .- 1540-9538. ; 199:4, s. 449-458
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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.
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| 2. |
- Blaukat, Andree, et al.
(författare)
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Determination of bradykinin B2 receptor in vivo phosphorylation sites and their role in receptor function
- 2001
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 276:44, s. 40431-40440
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Tidskriftsartikel (refereegranskat)abstract
- Reversible phosphorylation plays important roles in G protein-coupled receptor signaling, desensitization, and endocytosis, yet the precise location and role of in vivo phosphorylation sites is unknown for most receptors. Using metabolic 32P labeling and phosphopeptide sequencing we provide a complete phosphorylation map of the human bradykinin B2 receptor in its native cellular environment. We identified three serine residues, Ser(339), Ser(346), and Ser(348), at the C-terminal tail as principal phosphorylation sites. Constitutive phosphorylation occurs at Ser(348), while ligand-induced phosphorylation is found at Ser(339) and Ser(346)/Ser(348) that could be executed by several G protein-coupled receptor kinases. In addition, we found a protein kinase C-dependent phosphorylation of Ser(346) that was mutually exclusive with the basal phosphorylation at Ser(348) and therefore may be implicated in differential regulation of B2 receptor activation. Functional analysis of receptor mutants revealed that a low phosphorylation stoichiometry is sufficient to initiate receptor sequestration while a clustered phosphorylation around Ser(346) is necessary for desensitization of the B2 receptor-induced phospholipase C activation. This was further supported by the specifically reduced Ser(346)/Ser(348) phosphorylation observed upon stimulation with a nondesensitizing B2 receptor agonist. The differential usage of clustered phosphoacceptor sites points to distinct roles of multiple kinases in controlling G protein-coupled receptor function.
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| 3. |
- Chiara, Federica, et al.
(författare)
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A gain of function mutation in the activation loop of platelet-derived growth factor beta-receptor deregulates its kinase activity
- 2004
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 279:41, s. 42516-42527
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Tidskriftsartikel (refereegranskat)abstract
- The platelet-derived growth factor receptors (PDGFRs) are receptor tyrosine kinases implicated in multiple aspects of cell growth, differentiation, and survival. Recently, a gain of function mutation in the activation loop of the human PDGFRalpha has been found in patients with gastrointestinal stromal tumors. Here we show that a mutation in the corresponding codon in the activation loop of the murine PDGFRbeta, namely an exchange of asparagine for aspartic acid at amino acid position 849 (D849N), confers transforming characteristics to embryonic fibroblasts from mutant mice, generated by a knock-in strategy. By comparing the enzymatic properties of the wild-type versus the mutant receptor protein, we demonstrate that the D849N mutation lowers the threshold for kinase activation, causes a dramatic alteration in the pattern of tyrosine phosphorylation kinetics following ligand stimulation, and induces a ligand-independent phosphorylation of several tyrosine residues. These changes result in deregulated recruitment of specific signal transducers. The GTPase-activating protein for Ras (RasGAP), a negative regulator of the Ras mitogenic pathway, displayed a delayed binding to the mutant receptor. Moreover, we have observed enhanced ligand-independent ERK1/2 activation and an increased proliferation of mutant cells. The p85 regulatory subunit of the phosphatidylinositol 3 '-kinase was constitutively associated with the mutant receptor, and this ligand-independent activation of the phosphatidylinositol 3'-kinase pathway may explain the observed strong protection against apoptosis and increased motility in cellular wounding assays. Our findings support a model whereby an activating point mutation results in a deregulated PDGFRbeta with oncogenic predisposition.
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| 4. |
- Dixelius, Johan, et al.
(författare)
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Ligand-induced vascular endothelial growth factor receptor-3 (VEGFR-3) heterodimerization with VEGFR-2 in primary lymphatic endothelial cells regulates tyrosine phosphorylation sites
- 2003
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Ingår i: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 278, s. 40973-
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Tidskriftsartikel (refereegranskat)abstract
- Vascular endothelial growth factors (VEGFs) regulate the development and growth of the blood and lymphatic vascular systems. Of the three VEGF receptors (VEGFR), VEGFR-1 and -2 are expressed on blood vessels; VEGFR-2 is found also on lymphatic vessels. VEGFR-3 is expressed mainly on lymphatic vessels but it is also up-regulated in tumor angiogenesis. Although VEGFR-3 is essential for proper lymphatic development, its signal transduction mechanisms are still incompletely understood. Trans-phosphorylation of activated, dimerized receptor tyrosine kinases is known to be critical for the regulation of kinase activity and for receptor interaction with signal transduction molecules. In this study, we have identified five tyrosyl phosphorylation sites in the VEGFR-3 carboxyl-terminal tail. These sites were used both in VEGFR-3 overexpressed in 293 cells and when the endogenous VEGFR-3 was activated in lymphatic endothelial cells. Interestingly, VEGF-C stimulation of lymphatic endothelial cells also induced the formation of VEGFR-3/VEGFR-2 heterodimers, in which VEGFR-3 was phosphorylated only at three of the five sites while the two most carboxyl-terminal tyrosine residues appeared not to be accessible for the VEGFR-2 kinase. Our data suggest that the carboxyl-terminal tail of VEGFR-3 provides important regulatory tyrosine phosphorylation sites with potential signal transduction capacity and that these sites are differentially used in ligand-induced homo- and heterodimeric receptor complexes.
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| 5. |
- Galindo, Mario, et al.
(författare)
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Chromatin from two classes of platyhelminthes display both protist H1 and higher eukaryote core histones
- 2004
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Ingår i: FEBS Letters. - : Wiley. - 0014-5793 .- 1873-3468. ; 567:2-3, s. 225-229
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Tidskriftsartikel (refereegranskat)abstract
- Histones from the parasitic platyhelminthes, Echinococcus granulosus and Fasciola hepatica, were systematically characterized. Core histones H2A, H2B, H3 and H4, which were identified on the basis of amino acid sequencing and mass spectrometry data, showed conserved electrophoretic patterns. Histones H1, identified on the basis of physicochemical properties, amino acid composition and amino acid sequencing, showed divergence, both in their number and electrophoretic mobilities, between the two species and among other organisms. According to these data, core histones but not H1 histones, would be stabilized during evolution at the level of platyhelminthes.
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| 6. |
- Groth, Anja, et al.
(författare)
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Human Tousled like kinases are targeted by an ATM- and Chk1-dependent DNA damage checkpoint
- 2003
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Ingår i: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 22:7, s. 1676-1687
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Tidskriftsartikel (refereegranskat)abstract
- All eukaryotes respond to DNA damage by modulation of diverse cellular processes to preserve genomic integrity and ensure survival. Here we identify mammalian Tousled like kinases (Tlks) as a novel target of the DNA damage checkpoint. During S-phase progression, when Tlks are maximally active, generation of DNA double-strand breaks (DSBs) leads to rapid and transient inhibition of Tlk activity. Experiments with chemical inhibitors, genetic models and gene targeting through RNA interference demonstrate that this response to DSBs requires ATM and Chk1 function. Chk1 phosphorylates Tlk1 on serine 695 (S695) in vitro, and this UCN-01- and caffeine-sensitive site is phosphorylated in vivo in response to DNA damage. Substitution of S695 to alanine impaired efficient downregulation of Tlk1 after DNA damage. These findings identify an unprecedented functional co- operation between ATM and Chk1 in propagation of a checkpoint response during S phase and suggest that, through transient inhibition of Tlk kinases, the ATM-Chk1-Tlk pathway may regulate processes involved in chromatin assembly.
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| 7. |
- Klint, Peter, et al.
(författare)
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Translocated in liposarcoma (TLS) is a substrate for fibroblast growth factor receptor-1.
- 2004
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Ingår i: Cellular signalling. - : Elsevier BV. - 0898-6568. ; 16:4, s. 515-20
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Tidskriftsartikel (refereegranskat)abstract
- Binding of fibroblast growth factor (FGF) to the high affinity receptor-1 (FGFR-1) leads to activation of its endogenous tyrosine kinase activity. A number of substrates for the FGFR-1 kinase have been identified. Among those, FGF receptor-substrate-2 (FRS-2) was identified by virtue of its interaction with p13suc, a yeast protein involved in cell cycle regulation. We have used immobilized p13suc to identify a new substrate for FGRF-1, which is identical to "translocated in liposarcoma" (TLS). TLS is a RNA/DNA-binding protein which occurs in fusion products with different transcription factors in a variety of solid tumours. We show that TLS is tyrosine phosphorylated in intact cells by a number of different growth factors, indicating a role in growth regulation.
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| 8. |
- Lennartsson, Johan, et al.
(författare)
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Identification of Tyr900 in the kinase domain of c-Kit as a Src-dependent phosphorylation site mediating interaction with c-Crk
- 2003
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Ingår i: Experimental Cell Research. - 0014-4827 .- 1090-2422. ; 288:1, s. 110-118
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Tidskriftsartikel (refereegranskat)abstract
- We have previously demonstrated that ligand-stimulation of c-Kit induces phosphorylation of Tyr568 and Tyr570 in the juxtamembrane region of the receptor, leading to recruitment, phosphorylation and activation of members of the Src family of tyrosine kinases. In this paper, we demonstrate that members of the Src family of tyrosine kinases are able to phosphorylate c-Kit selectively on one particular tyrosine residue, Tyr900, located in the second part of the tyrosine kinase domain. In order to identify potential docking partners of Tyr900, a synthetic phosphopeptide corresponding to the amino acid sequence surrounding Tyr900 was used as an affinity matrix. By use of MALDI-TOF mass spectrometry, CrkII was identified as a protein that specifically bound to Tyr900 in a phosphorylation dependent manner, possibly via the p85 subunit of PI3-kinase. Expression of a mutant receptor where Tyr900 had been replaced with a phenylalanine residue (Y900F) resulted in a receptor with reduced ability to phosphorylate CrkII. Together these data support a model where c-Src phosphorylates the receptor, thereby creating docking sites for SH2 domain containing proteins, leading to recruitment of Crk to the receptor.
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| 9. |
- Lorenzo, Carmen, et al.
(författare)
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Echinococcus granulosus antigen 5 is closely related to proteases of the trypsin family
- 2003
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 369:Pt 1, s. 191-198
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Tidskriftsartikel (refereegranskat)abstract
- Antigen 5 (Ag5) is a dominant secreted component of the larval stage of Echinococcus granulosus, and is highly immunogenic in human infections. Although the diagnostic value of Ag5 has been thoroughly evaluated, there has been little progress in its molecular characterization and the understanding of its biological role. In the present study, the Ag5 gene was cloned by reverse transcription-PCR on the basis of the amino acid sequences of tryptic fragments. The nucleotide sequence indicates that Ag5 is synthesized as a single polypeptide chain that is afterwards processed into single disulphide-bridged 22 and 38 kDa subunits. Whereas the 22 kDa component contains a highly conserved glycosaminoglycan-binding motif that may help to confine Ag5 in the host tissue surrounding the parasite, the 38 kDa subunit is closely related to serine proteases of the trypsin family. The sequences in the vicinity of the active-site histidine, aspartic acid and serine residues, and critical cysteine residues involved in disulphide formation, are well conserved, but the catalytic serine residue is replaced by threonine. Since there are no significant chemical differences between the O gamma atoms of these residues, we performed a series of enzymic assays to find out whether Ag5 is a catalytic molecule. Neither proteolytic activity nor binding to protease inhibitors could be detected using the native purified antigen. Thus it may be possible that Ag5 possesses a highly specific physiological substrate or, more likely, that trypsin-like folding has been recruited to fulfil novel functions.
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| 10. |
- Neuschäfer-Rube, Frank, et al.
(författare)
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Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin EP4-R essential for agonist-induced beta-arrestin1 recruitment that differs from the apparent principal phosphorylation site.
- 2004
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Ingår i: Biochemical Journal. - 0264-6021. ; 379:3, s. 573-585
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Tidskriftsartikel (refereegranskat)abstract
- hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a Gs-linked heterotrimeric GPCR (G-protein-coupled receptor). It undergoes agonist-induced desensitization and internalization that depend on the presence of its C-terminal domain. Desensitization and internalization of GPCRs are often linked to agonist-induced b-arrestin complex formation, which is stabilized by phosphorylation. Subsequently b-arrestin uncouples the receptor from its G-protein and links it to the endocytotic machinery. The C-terminal domain of hEP4-R contains 38 Ser/Thr residues that represent potential phosphorylation sites. The present study aimed to analyse the relevance of these Ser/Thr residues for agonist-induced phosphorylation, interaction with b-arrestin and internalization. In response to agonist treatment, hEP4-R was phosphorylated. By analysis of proteolytic phosphopeptides of the wild-type receptor and mutants in which groups of Ser/Thr residues had been replaced by Ala, the principal phosphorylation site was mapped to a Ser/Thr-containing region comprising residues 370–382, the presence of which was necessary and sufficient to obtain full agonist-induced phosphorylation. A cluster of Ser/Thr residues (Ser-389–Ser-390–Thr-391–Ser-392) distal to this site, but not the principal phosphorylation site, was essential to allow agonist-induced recruitment of b-arrestin1. However, phosphorylation greatly enhanced the stability of the b-arrestin1–receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both b-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary.
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