4 results on '"Kastein, Mirja"'
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2. Dephosphorylation of human dopamine transporter at threonine 48 by protein phosphatase PP1/2A up-regulates transport velocity
- Author
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Yang, Jae-Won, primary, Larson, Garret, additional, Konrad, Lisa, additional, Shetty, Madhur, additional, Holy, Marion, additional, Jäntsch, Kathrin, additional, Kastein, Mirja, additional, Heo, Seok, additional, Erdem, Fatma Asli, additional, Lubec, Gert, additional, Vaughan, Roxanne A., additional, Sitte, Harald H., additional, and Foster, James D., additional
- Published
- 2019
- Full Text
- View/download PDF
3. Phosphorylation–mediated regulation of the Serotonin Transporter
- Author
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Kastein, Mirja
- Abstract
Posttranslationale Modifikationen von Proteinen spielen eine wichtige Rolle in verschiedenen molekularen Prozessen. Aufgrund der Existenz vieler Kinasen und Phosphatasen in Eukaryonten scheint die Phosphorylierung eine besondere Bedeutung für die Koordinierung des zellulären Netzwerkes zu haben. Obwohl die genauen molekularen Abläufe noch unerforscht sind, wird die Aktivität, die Interaktion mit anderen Proteinen sowie die zelluläre Verteilung des Serotonintransporters (SERT) durch Phosphorylierung reguliert. Der Serotonintransporter enthält Konsensusmotive für Kinasen wie die Proteinkinase C (PKC) und die p38 MAPK. Beide Kinasen sind bereits in Verbindung gebracht worden, die Aktivität und Oberflächenexpression des Transporters zu beeinflussen. Dennoch sind spezifische Phosphorylierungsstellen, die mit der Regulierung der Aktivität und des Transports verbunden sind, noch nicht genau beschrieben worden. Aufgrund dessen war das Ziel, den Einfluss der Kinasenaktivierung auf die zelluläre Umverteilung von SERT zu untersuchen sowie spezifische Phosphorylierungsstellen zu iden-tifizieren, die für die SERT-Funktion und -Oberflächenexpression bzw. ER-Retention wichtig sind. Immunoblot-Analysen zeigten, dass die unglykosylierte Form von SERT als Folge der Behandlung mit dem p38 MAPK-Aktivator Anisomcyin in YFP-SERT stabil exprimierenden HEK293-Zellen reduziert wurde. Im Gegensatz dazu führte der Einsatz von β-PMA (PKC-Aktivator) zu einer erhöhten Expression der Retentions-Form. Da Anisomycin keinen Effekt in Dopamintransporter exprimierenden Zellen zeigte, wird angekommen, dass die Anisomycin-vermittelte zelluläre Umverteilung spezifisch für SERT ist. Desweiteren wurden p38 MAPK und PKC spezifische Phosphorylierungsstellen im C-Terminus von SERT via In vitro Phosphorylierung und Massenspektrometrie identifiziert. Obwohl durchgeführte In vivo Phosphorylierungsexperimente S52 und S62 als Anisomycin oder Okadasäure (Phosphatase 1/2 A Inhibitor) induzierte Phosphorylierungsstellen identifizierten, zeigten generierte phosphorylierungsresistente und phosphorylierte Mutan-ten der beiden Stellen das gleiche Anisomycin-vermittelte Expressionsmuster wie im SERT-Wildtyp. Dagegen zeigte die phosphorylierungsresistente Mutante der identifizierten p38 MAPK-spezifischen Phosphorylierungsstelle Thr616 eine signifikante Expressionsreduzierung der unglykosylierten SERT Form durch Anisomycin. Zusammenfassend lässt sich sagen, dass nur die Phosphorylierung des C-Terminus des Serotonintransporters Einfluss auf die zelluläre Umverteilung von SERT hat., Posttranslational modifications of proteins play an important role in several molecular processes. Especially, phosphorylation seems to play an important role in the coordination of the cellular network due to the presence of many different kinases and phosphatases in eukaryotes. Among other proteins, this rule also applies to the serotonin transporter (SERT). In particular, the regulation of SERT trafficking, activity and interaction with other proteins is regulated by phosphorylation, although it still contains many unexplored mo-lecular processes. SERT contains consensus sites for kinases, such as protein kinase C (PKC) and p38 MAPK, which are already known to influence SERT activity and surface expression. However, specific phosphorylation sites involved in the regulation of transporter activity and trafficking have not been well identified. Therefore, it was aimed to determine the cellular compartment distribution of SERT by kinase activation and identify specific phosphorylation sites implicated in the SERT function and surface expression/ER retention. Immunoblot analysis showed that the ER retained form of SERT was decreased in HEK293 cells stably expressing YFP-SERT due to treatment of a p38 MAPK activator anisomcyin. By contrast, treatment with β-PMA, a PKC activator, caused an increase of the ER-resident SERT expression. Anisomycin-mediated effect on ER retention form was detected in SERT but not in DAT expressing cells, leading to the assumption of specificity of anisomycin-mediated redistribution for SERT. In addition, in vitro phosphorylation assay followed by mass spectrometry only identified only phosphorylation sites in the C-terminal of SERT specific for p38 MAPK and PKC. Even though further in vivo phosphorylation experiments determined S52 and S62 as phosphorylation sites induced by anisomycin or okadaic acid, a phosphatase 1/2 A inhibitor, generated phosphomimetic and dephosphomimetic mutants of both sites still showed the same redistribution of SERT by anisomycin treatment as observed in the wild type SERT. However, dephosphomimetic mutant at identified p38 MAPK-specific Thr616 phosphorylation site showed a significant reduction of the anisomycin effect on ER-resident SERT. Taken together, phosphorylation at the C-terminus of SERT but not at the N-terminus regulates the ER retention of SERT.
- Published
- 2014
- Full Text
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4. Dephosphorylation of human dopamine transporter at threonine 48 by protein phosphatase PP1/2A up-regulates transport velocity.
- Author
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Jae-Won Yang, Larson, Garret, Konrad, Lisa, Shetty, Madhur, Holy, Marion, Jäntsch, Kathrin, Kastein, Mirja, Seok Heo, Erdem, Fatma Asli, Lubec, Gert, Vaughan, Roxanne A., Sitte, Harald H., and Foster, James D.
- Subjects
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CALMODULIN , *PHOSPHOPROTEIN phosphatases - Abstract
Several protein kinases, including protein kinase C, Ca2+/calmodulin-dependent protein kinase II, and extracellular signal-regulated kinase, play key roles in the regulation of dopamine transporter (DAT) functions. These functions include surface expression, internalization, and forward and reverse transport, with phosphorylation sites for these kinases being linked to distinct regions of the DAT N terminus. Protein phosphatases (PPs) also regulate DAT activity, but the specific residues associated with their activities have not yet been elucidated. In this study, using co-immunoprecipitation followed by MS and immunoblotting analyses, we demonstrate the association of DAT with PP1 and PP2A in the mouse brain and heterologous cell systems. By applying MS in conjunction with a metabolic labeling method, wedefined a PP1/2A-sensitive phosphorylation site at Thr-48 in human DAT, a residue that has not been previously reported to be involved in DAT phosphorylation. Site-directed mutagenesis of Thr-48 to Ala (T48A) to prevent phosphorylation enhanced dopamine transport kinetics, supporting a role for this residue in regulating DAT activity. Moreover, T48A-DAT displayed increased palmitoylation, suggesting that phosphorylation/dephosphorylation at this site has an additional regulatory role and reinforcing a previously reported reciprocal relationship between C-terminal palmitoylation and N-terminal phosphorylation. [ABSTRACT FROM AUTHOR]
- Published
- 2019
- Full Text
- View/download PDF
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