Quantitative analysis of protein-protein interactions governing TASK-1/TASK-3 intracellular transport

by Markus Kilisch
Doctoral thesis
Date of Examination:2016-06-01
Date of issue:2016-07-27
Advisor:Prof. Dr. Blanche Schwappach-Pignataro
Referee:Prof. Dr. Ulf Diederichsen
Referee:Prof. Dr. Blanche Schwappach-Pignataro
Persistent Address: http://dx.doi.org/10.53846/goediss-5776

 

 

Files in this item

Name:20160422_PhD_Thesis_Markus_Kilisch.pdf
Size:4.23Mb
Format:PDF
Description:2016_Kilisch_PhD_Thesis_AK_Schwappach

The following license files are associated with this item:

Abstract

English

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.
Keywords: TASK-1; TASK-3; K2P; COPI; Secretory pathway; 14-3-3; isoform specificity; protein trafficking; PKA; phosphoadaptor protein
 
Statistik