| dc.contributor.advisor | Schwappach-Pignataro, Blanche Prof. Dr. | |
| dc.contributor.author | Kilisch, Markus | |
| dc.date.accessioned | 2016-07-27T08:14:21Z | |
| dc.date.available | 2016-07-27T08:14:21Z | |
| dc.date.issued | 2016-07-27 | |
| dc.identifier.uri | http://hdl.handle.net/11858/00-1735-0000-0028-87D8-0 | |
| dc.identifier.uri | http://dx.doi.org/10.53846/goediss-5776 | |
| dc.language.iso | eng | de |
| dc.publisher | Niedersächsische Staats- und Universitätsbibliothek Göttingen | de |
| dc.rights.uri | http://creativecommons.org/licenses/by-nc-nd/4.0/ | |
| dc.subject.ddc | 540 | de |
| dc.title | Quantitative analysis of protein-protein interactions governing TASK-1/TASK-3 intracellular transport | de |
| dc.type | doctoralThesis | de |
| dc.contributor.referee | Diederichsen, Ulf Prof. Dr. | |
| dc.date.examination | 2016-06-01 | |
| dc.description.abstracteng | The transport of the K+-channels TASK-1 and TASK-3 to the cell surface is
regulated by protein-protein interactions with either the COPI vesicle coat, or
members of the phosphoadaptor protein family 14-3-3. Interactions are
mediated via a trafficking control region present at the distal C-terminus of
either K+-channel. This trafficking control region comprises a polybasic ER
retention and retrieval motif and an adjacent mode III 14-3-3 binding motif.
Phosphorylation of a conserved serine residue, as part of the mode III 14-3-3
binding motif, is followed by the recruitment of 14-3-3, thereby releasing the
channel from ER retention by sterically preventing the COPI vesicle coat from
binding to the overlapping ER retention and retrieval motif. Following
phosphorylation and 14-3-3 binding, the channel is transported forward to the
cell surface. In this thesis I determined the binding parameters of all seven
human 14-3-3 isoforms to the trafficking control regions of TASK-1 and TASK-
3. Furthermore, I investigated the direct effect of phosphorylation of the TASK-
1 and TASK-3 C-terminus on COPI binding. I observed distinctly different
binding parameters between individual 14-3-3 isoforms and different channel
C-termini demonstrating that 14-3-3 isoforms bind the same substrate in an
isoform specific manner. Surprisingly, the binding affinities determined for
TASK-1 were approximately two orders of magnitude lower than the binding
affinities determined for TASK-3. I explain these differences by small, but
physiologically relevant, amino acid sequence differences within the trafficking
control regions of TASK-1 and TASK-3. While TASK-3 presents a second
lysine residue that allows for high affinity binding of 14-3-3 proteins to this
trafficking control region, TASK-1 presents a second serine residue that upon
phosphorylation inhibits 14-3-3 binding. I further correlate my in vitro
observations with reporter protein assays performed in vivo (COS7),
assessing the relative cell surface expression of TASK-derived reporter
proteins. My findings indicate that the control of TASK-1 protein trafficking is
highly dynamic, modulated by COPI, 14-3-3, kinases and phosphatases.
Binding experiments performed with the yeast COPI vesicle coat and
phosphorylated or unphosphorylated constructs comprising the distal C-terminus of TASK-1 and TASK-3 (the last 15 amino acids) demonstrate that
the phosphorylation of these trafficking control regions is sufficient to interfere
with COPI binding, in absence of 14-3-3. In summary, my findings contribute
substantially to the quantitative understanding of events governing the
intracellular transport of TASK-1 and TASK-3. | de |
| dc.contributor.coReferee | Schwappach-Pignataro, Blanche Prof. Dr. | |
| dc.subject.eng | TASK-1 | de |
| dc.subject.eng | TASK-3 | de |
| dc.subject.eng | K2P | de |
| dc.subject.eng | COPI | de |
| dc.subject.eng | Secretory pathway | de |
| dc.subject.eng | 14-3-3 | de |
| dc.subject.eng | isoform specificity | de |
| dc.subject.eng | protein trafficking | de |
| dc.subject.eng | PKA | de |
| dc.subject.eng | phosphoadaptor protein | de |
| dc.identifier.urn | urn:nbn:de:gbv:7-11858/00-1735-0000-0028-87D8-0-2 | |
| dc.affiliation.institute | Fakultät für Chemie | de |
| dc.subject.gokfull | Chemie (PPN62138352X) | de |
| dc.identifier.ppn | 863978061 | |