The regulation of the transcription factor TFEB in B cells via pathways of the B cell antigen receptor
by Jakob Christoph Willenbrink
Date of Examination:2025-04-08
Date of issue:2025-03-25
Advisor:Prof. Dr. Jürgen Wienands
Referee:Prof. Dr. Jürgen Wienands
Referee:Prof. Dr. Björn Chapuy
Persistent Address:
http://dx.doi.org/10.53846/goediss-11172
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Abstract
English
The signalling pathways downstream of the BCR are vital for B cell function and previous to this work, it was shown that TFEB is a transcription factor implicated in the cellular re sponse of B cells to the stimulation of their BCR. Recent findings suggest that TFEB is placed at an essential point of B cell biology, at the one hand inducing apoptosis to prevent autoreactive B cells from causing damage, while on the other hand equipping the B cell with receptors for costimulatory signals and increasing MHC II-mediated antigen presentation to provide an escape from the induced cell death. It was also shown that different subsets of B cells vary in their subcellular localisation of TFEB. For instance, unswitched and switched memory B cells hold increased amounts of TFEB in their nuclei even in a resting state. This work was able to provide information on the possible signalling basis of these differ ences in between different subsets of B cells. By Single Cell Dilution of WEHI-231 B cells as well as Ramos B cells, subpopulations with differing translocation rates of TFEB could be generated. Moreover, it was shown that these different subpopulations have a conspicu ous divergence within one of the identified pathways for TFEB downstream of the BCR. Whereas the investigated parts of the BCR-PLC-PKC axis displayed no differences in terms of expression, enzymatic activity or signalling amplitude, this work was able to link the dif fering translocation rates to a stronger signalling amplitude within the CD19-PI3K-Akt axis. This was demonstrated by significant disparities of Akt phosphorylation. Subpopulations with strong nuclear translocation of TFEB also showed strong Akt phosphorylation and in contrast, subpopulations with low translocation rates also were found to have less phosphor ylated Akt. Furthermore, this work was able to implicate multiple phosphorylation sites of TFEB in the regulation of its subcellular localisation. Constitutive dephosphorylation of these phosphor ylation sites was mimicked by introducing single amino acid substitutions via site-directed mutagenesis. The simulation of constitutive dephosphorylation at these sites led to a very significant increase in nuclear localisation of TFEB, thus indicating the involvement of these sites in the regulation of TFEB. The phosphorylation sites which were found to be involved are not exclusively sites that previously have been reported to be phosphorylated by PKC and GSK3, the kinases responsible for TFEB phosphorylation in B cells according to our findings. These sites also include phosphorylation sites that are described as being phosphor ylated by other kinases such as mTOR and Akt. While these findings prove that multiple phosphorylation sites are involved in the regulation of the subcellular localisation of TFEB, it was also shown that the regulation does not simply involve all phosphorylation sites of TFEB. Substituting S3 with alanine and thus mimicking dephosphorylation at this site did not have any notable influence on the subcellular localisation of TFEB. Making the targeted phosphorylation sites of PKC and GSK3 unavailable to phosphorylation was thought to diminish the effect these kinases have on the subcellular localisation of TFEB. Hence, inhibitors of these enzymes were used to investigate, if their influence on TFEB’s localisation was eliminated. The findings showed that inhibition of PKC and GSK3 still led to a significant increase of nuclear translocation of TFEB, suggesting that these two kinases also use other phosphorylation sites which have not been implicated in this process, yet. In summary, this work was able to shed light on many different aspects of the regulation of TFEB downstream of the BCR in B cells. The findings of this work verified numerous pre vious results from our institute regarding the pathways for TFEB regulation and moreover provided many new information on the possible foundation of different translocation rates of TFEB in subsets of B cells as well as the role of multiple phosphorylation sites in its regulation of the subcellular localisation. Since the nuclear translocation of TFEB appears as a hallmark event of BCR signalling, addressing the new questions this work created as well as the unresolved ones might prove to be a promising field to future research.
Keywords: B-cells; B-cell; B cell receptor; TFEB; Immunology; Signalling
