| 1. |
- Ottmann, Christian, et al.
(author)
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Phosphorylation-independent interaction between 14-3-3 and exoenzyme S : from structure to pathogenesis.
- 2007
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In: EMBO Journal. - : Wiley. - 0261-4189 .- 1460-2075. ; 26:3, s. 902-913
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Journal article (peer-reviewed)abstract
- 14-3-3 proteins are phosphoserine/phosphothreonine-recognizing adapter proteins that regulate the activity of a vast array of targets. There are also examples of 14-3-3 proteins binding their targets via unphosphorylated motifs. Here we present a structural and biological investigation of the phosphorylation-independent interaction between 14-3-3 and exoenzyme S (ExoS), an ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. ExoS binds to 14-3-3 in a novel binding mode mostly relying on hydrophobic contacts. The 1.5 A crystal structure is supported by cytotoxicity analysis, which reveals that substitution of the corresponding hydrophobic residues significantly weakens the ability of ExoS to modify the endogenous targets RAS/RAP1 and to induce cell death. Furthermore, mutation of key residues within the ExoS binding site for 14-3-3 impairs virulence in a mouse pneumonia model. In conclusion, we show that ExoS binds 14-3-3 in a novel reversed orientation that is primarily dependent on hydrophobic residues. This interaction is phosphorylation independent and is required for the function of ExoS.
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| 2. |
- Saline, Maria, et al.
(author)
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AMPK and AKT protein kinases hierarchically phosphorylate the N-terminus of the FOXO1 transcription factor, modulating interactions with 14-3-3 proteins
- 2019
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In: Journal of Biological Chemistry. - 0021-9258 .- 1083-351X. ; 294:35, s. 13106-13116
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Journal article (peer-reviewed)abstract
- Forkhead box protein O1 (FOXO1) is a transcription factor involved in various cellular processes such as glucose metabolism, development, stress resistance, and tumor suppression. FOXO1's transcriptional activity is controlled by different environmental cues through a myriad of posttranslational modifications. In response to growth factors, the serine/threonine kinase AKT phosphorylates Thr24 and Ser256 in FOXO1 to stimulate binding of 14-3-3 proteins, causingFOXO1inactivation. In contrast, low nutrient and energy levels induce FOXO1 activity. AMP-activated protein kinase (AMPK), a master regulator of cellular energy homeostasis, partly mediates this effect through phosphorylation of Ser383 and Thr649 in FOXO1. In this study, we identified Ser22 as an additional AMPK phosphorylation site in FOXO1's N terminus, with Ser22 phosphorylation preventing binding of 14-3-3 proteins. The crystal structure of a FOXO1 peptide in complex with 14-3-3 σ at 2.3 Å resolution revealed that this is a consequence of both steric hindrance and electrostatic repulsion. Furthermore, we found that AMPK-mediated Ser22 phosphorylation impairs Thr24 phosphorylation by AKT in a hierarchical manner. Thus, numerous mechanisms maintain FOXO1 activity via AMPK signaling. AMPK-mediated Ser22 phosphorylation directly and indirectly averts binding of 14-3-3 proteins, whereas phosphorylation of Ser383 and Thr649 complementarily stimulates FOXO1 activity. Our results shed light on a mechanism that integrates inputs from both AMPK and AKT signaling pathways in a small motif to fine-tune FOXO1 transcriptional activity.
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| 3. |
- Scheepstra, Marcel, et al.
(author)
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Ligand Dependent Switch from RXR Homo- to RXR-NURR1 Heterodimerization
- 2017
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In: ACS Chemical Neuroscience. - : American Chemical Society (ACS). - 1948-7193. ; 8:9, s. 2065-2077
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Journal article (peer-reviewed)abstract
- Retinoid X receptors (RXRs) play key roles in many physiological processes in both the periphery and central nervous system. In addition, RXRs form heterodimers with other nuclear receptors to exert their physiological effects. The nuclear receptor related 1 protein (NURR1) is particularly interesting because of its role in promoting differentiation and survival of dopamine neurons. However, only a small number of RXR-heterodimer selective modulators are available, with limited chemical diversity. This work describes the synthesis, biochemical evaluation, and structural elucidation of a novel series of RXR ligands with strongly biased interactions with RXRα-NURR1 heterodimers. Targeted modifications to the small molecule biaryl scaffold caused local RXRα side-chain disturbances and displacement of secondary structural elements upon ligand binding. This resulted in the repositioning of protein helices in the heterodimer interface of RXRα, alterations in homo- versus heterodimer formation, and modulation of activation function 2 (AF2). The data provide a rationale for the design of RXR ligands consisting of a highly conserved hydrophilic region, strongly contributing to the ligand affinity, and a variable hydrophobic region, which efficiently probes the effects of structural changes at the level of the ligand on co-regulator recruitment or the RXRα-NURR1 dimerization interface.
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| 4. |
- Schönherr, Christina, et al.
(author)
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The neuroblastoma ALK(I1250T) mutation is a kinase-dead RTK in vitro and in vivo
- 2011
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In: Translational Oncology. - : Elsevier BV. - 1944-7124 .- 1936-5233. ; 4:4, s. 258-265
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Journal article (other academic/artistic)abstract
- Activating mutations in the kinase domain of anaplastic lymphoma kinase (ALK) have recently been shown to be an important determinant in the genetics of the childhood tumor neuroblastoma. Here we discuss an in-depth analysis of one of the reported gain-of-function ALK mutations—ALKI1250T—identified in the germ line DNA of one patient. Our analyses were performed in cell culture-based systems and subsequently confirmed in a Drosophila model. The results presented here indicate that the germ line ALKI1250T mutation is most probably not a determinant for tumor initiation or progression and, in contrast, seems to generate a kinase-dead mutation in the ALK receptor tyrosine kinase (RTK). Consistent with this, stimulation with agonist ALK antibodies fails to lead to stimulation of ALKI1250T and we were unable to detect tyrosine phosphorylation under any circumstances. In agreement, ALKI1250T is unable to activate downstream signaling pathways or to mediate neurite outgrowth, in contrast to the activated wild-type ALK receptor or the activating ALKF1174S mutant. Identical results were obtained when the ALKI1250T mutant was expressed in a Drosophila model, confirming the lack of activity of this mutant ALK RTK. We suggest that the ALKI1250T mutation leads to a kinase-dead ALK RTK, in stark contrast to assumed gain-of-function status, with significant implications for patients reported to carry this particular ALK mutation.
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| 5. |
- Sundén, Henrik, 1978, et al.
(author)
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Chiral Dihydrobenzofuran Acids Show Potent Retinoid X Receptor-Nuclear Receptor Related 1 Protein Dimer Activation
- 2016
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In: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 59:3, s. 1232-1238
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Journal article (peer-reviewed)abstract
- The nuclear receptor Nurr1 can be activated by RXR via heterodimerization (RXR-Nurr1) and is a promising target for treating neurodegenerative diseases. We herein report the enantioselective synthesis and SAR of sterically constricted benzofurans at RXR. The established SAR, using whole cell functional assays, lead to the full agonist 9a at RXR (pEC50 of 8.2) and RXR-Nurr1. The X-ray structure shows enantiomeric discrimination where 9a optimally addresses the ligand binding pocket of RXR.
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| 6. |
- Yasmin, Lubna, et al.
(author)
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Electrostatic interactions play a minor role in the binding of ExoS to 14-3-3 proteins
- 2010
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In: Biochemical Journal. - : Portland Press. - 0264-6021 .- 1470-8728. ; 427:2, s. 217-224
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Journal article (peer-reviewed)abstract
- 14-3-3 proteins belong to a family of conserved molecules expressed in all eukaryotic cells that play an important role in a multitude of signalling pathways. 14-3-3 proteins bind either to phosphoserine/phosphothreonine residues or to sequence-specific non-phosphorylated motifs in more than 200 interaction partners [Pozuelo Rubio, Geraghty, Wong, Wood, Campbell, Morrice and Mackintosh (2004) Biochem. J. 379, 395-408]. These interactions result in cell-cycle regulation, apoptosis, stress responses, cell metabolism and malignant transformation. One example of a phosphorylation-independent interaction is the binding of 14-3-3 to ExoS (exoenzyme S), a bacterial ADP-ribosyltransferase toxin of Pseudomonas aeruginosa. In the present study, we have utilized additional biochemical and infection analyses to define further the structural basis of the interaction between ExoS and 14-3-3. An ExoS leucine-substitution mutant dramatically reduced the interaction potential with 14-3-3 suggesting that Leu422, Leu423, Leu426 and Leu428 of ExoS are important for its interaction with 14-3-3, its enzymatic activity and cytotoxicity. However, ExoS substitution mutants of residues that interact with 14-3-3 through an electrostatic interaction, such as Ser416, His418, Asp424 and Asp427, showed no reduction in their interaction potential with 14-3-3. These ExoS substitution mutants were also as aggressive as wild-type ExoS at inducing cell death and to modify endogenous ExoS target within the cell. In conclusion, electrostatic interaction between ExoS and 14-3-3 via polar residues (Ser416, His418, Asp424 and Asp427) appears to be of secondary importance. Thus the interaction between the 'roof' of the groove of 14-3-3 and ExoS relies more on hydrophobic interaction forces, which probably contributes to induce cell death after ExoS infection and activation.
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