Charakterisierung von Punktmutanten in der Linker-Domäne des humanen STAT1-Proteins

Characterization of point mutants in the linker - domain of the human STAT1 - protein

by Jessica Grebe
Doctoral thesis
Date of Examination:2016-07-19
Date of issue:2016-07-14
Advisor:Prof. Dr. Thomas Meyer
Referee:Prof. Dr. Thomas Meyer
Referee:Prof. Dr. Dieter Kube
Persistent Address: http://dx.doi.org/10.53846/goediss-5750

 

 

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Abstract

English

Signal transducers and activators of transcription (STATs) are components of an old phylogenetic and highly conserved signal transduction pathway in metazoans. As key elements of the JAK-STAT signal pathway, members of this protein family are involved in immune processes, cell growth and homeostasis. Upon external stimuli, cytoplasmatic STAT proteins are phosphorylated on a single tyrosine residue in their carboxy-termini and are then transported in the nucleus as dimeric transcription factors to drive STAT-dependent gene expression. In the present study, four point mutants were generated in the linker domain of the STAT1 molecules (W504, F506, W539 and F554). The exchange of a highly conserved phenylalanine to alanine in position 554 resulted in reduced levels of tyrosine phosphorylation. The STAT1-F554A mutant also showed decreased transcriptional activation of STAT1-driven reporter genes and endogenous target genes. The phenotype of this mutant is explained by decreased stability of the parallel conformer of STAT1 dimers in favor of the antiparallel conformation. The altered conformational equilibrium results in enhanced dephosphorylation by the inactivating TC-45 tyrosine phosphatase. In contrast, STAT1-W504A was slightly hyperphosphorylated on the critical tyrosine residue 701 and consequently showed a higher level of transcriptional activation in reporter gene assays and reverse-transcriptase PCR assays. In summary, these data confirm that the linker domain of the STAT1 molecule plays an important role in tyrosine phosphorylation and gene expression. Further research should address the physiological functions of the linker domain in an organismic context.
Keywords: STAT1; linker domain; JAK-STAT signaling
Schlagwörter: STAT1; Linker Domäne; JAK-STAT Signalweg
 
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