| 1. |
- Masson, Kristina, et al.
(författare)
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Direct binding of Cbl to Tyr(568) and Tyr(936) of the stem cell factor receptor/c-Kit is required for ligand-induced ubiquitination, internalization and degradation
- 2006
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Ingår i: Biochemical Journal. - 0264-6021. ; 399, s. 59-67
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Tidskriftsartikel (refereegranskat)abstract
- The ubiquitin E3 ligase Cbl has been shown to negatively regulate tyrosine kinase receptors, including the stem cell factor receptor/c-Kit. Impaired recruitment of Cbl to c-Kit results in a deregulated positive signalling that eventually can contribute to carcinogenesis. Here, we present results showing that Cbl is activated by the SFKs (Src family kinases) and recruited to c-Kit in order to trigger receptor ubiquitination. We demonstrate that phosphorylated Tyr(568) and Tyr(936) in c-Kit are involved in direct binding and activation of Cbl and that binding of the TKB domain (tyrosine kinase binding domain) of Cbl to c-Kit is specified by the presence of an isoleucine or leucine residue in position +3 to the phosphorylated tyrosine residue on c-Kit. Apart from the direct association between Cbl and c-Kit, we show that phosphorylation of Cbl by SFK members is required for activation of Cbl to occur. Moreover, we demonstrate that Cbl mediates mono-ubiquitination of c-Kit and that the receptor is subsequently targeted for lysosomal degradation. Taken together, our findings reveal novel insights into the mechanisms by which Cbl negatively regulates c-Kit-mediated signalling.
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| 2. |
- 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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| 3. |
- Neuschäfer-Rube, Frank, et al.
(författare)
-
Identification of a Ser/Thr cluster in the C-terminal domain of the human prostaglandin receptor EP4 that is essential for agonist-induced beta-arrestin1 recruitment but differs from the apparent principal phosphorylation site
- 2004
-
Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 379:Pt 3, s. 573-585
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Tidskriftsartikel (refereegranskat)abstract
- hEP4-R (human prostaglandin E2 receptor, subtype EP4) is a G(s)-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 beta-arrestin complex formation, which is stabilized by phosphorylation. Subsequently beta-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 beta-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 beta-arrestin1. However, phosphorylation greatly enhanced the stability of the beta-arrestin1-receptor complexes. For maximal agonist-induced internalization, phosphorylation of the principal phosphorylation site was not required, but both beta-arrestin1 recruitment and the presence of Ser/Thr residues in the distal half of the C-terminal domain were necessary.
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| 4. |
- Wollberg, Patrik, et al.
(författare)
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The adapter protein APS associates with the multifunctional docking sites Tyr-568 and Tyr-936 in c-Kit
- 2003
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Ingår i: Biochemical Journal. - 0264-6021 .- 1470-8728. ; 370:Pt 3, s. 1033-1038
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Tidskriftsartikel (refereegranskat)abstract
- The adapter protein APS has previously been shown to be involved in recruiting the ubiquitin E3 ligase c-Cbl to the insulin receptor, the platelet-derived growth factor beta-receptor and the erythropoietin receptor, leading to increased degradation of the receptors and inhibition of mitogenesis. Here we demonstrate, by use of immobilized synthetic phosphopeptides corresponding to various autophosphorylated tyrosine residues in the receptor for stem-cell factor (c-Kit), that APS preferentially associates with phosphorylated Tyr-568 and Tyr-936. Tyr-568 has previously been identified as the binding site of the Src family of tyrosine kinases, the Csk-homologous kinase CHK, and the protein tyrosine phosphatase SHP-2. We have recently demonstrated that Tyr-936 is an autophosphorylation site involved in binding the adapter proteins Grb2 and Grb7. We could further demonstrate that the critical determinant for binding of APS is the presence of either a leucine or an isoleucine residue in the position +3 to the phosphorylated tyrosine. This allowed us to design mutants that selectively failed to associate with APS, while still associating with Src family members, SHP-2 and Grb2, respectively.
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