The importance of homotypic interactions of unphosphorylated STAT proteins in cytokine-induced signal transduction
by Priyanka Rajeev Menon
Date of Examination:2021-02-25
Date of issue:2021-03-12
Advisor:Prof. Dr. Thomas Meyer
Referee:Prof. Dr. Thomas Meyer
Referee:Prof. Dr. Oliver Wirths
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EnglishSeveral cytokines mediate cellular responses via the family of transcription factors called signal transducers and activators of transcription (STAT). Though phosphorylated STATs (P-STAT) form the active and extensively studied component of STAT-mediated signal transduction, recent studies report novel physiological roles for unphosphorylated STATs (U-STAT). However, the interactions within U-STATs and between P-STATs and U-STATs are not very well understood. This project investigates how U-STATs interact with themselves and other P-STATs and the impact of this interaction on cytokine-induced signal transduction. While the U-STAT anti-parallel dimer interface has been reported and studied in STAT1, despite of high sequence homology, a similar interface has not been examined in STAT3. Through site-directed mutagenesis, two STAT3 point mutations V77A in the amino-terminal domain and F174A in the coiled-coil domain have been studied to determine the relevance of the anti-parallel dimer in STAT3-mediated signal transduction. Both V77A and F174A displayed a predominant nuclear accumulation in resting cells and cytokine stimulation led to slight increase in nuclear accumulation. The STAT3 mutants showed elevated levels of cytokine-stimulated tyrosine phosphorylation, while the affinity of mutant P-STAT3 bound to in vitro GAS sites was unaltered. The high concentration of mutant P-STAT3 in V77A- and F174A-expressing cells resulted in an elevated transcription of reporter constructs and endogenous target genes containing one-and-a-half GAS sites in their promoter region, but did not affect other genes with a single GAS promoter. Both V77A and F174A behaved similar to the wild-type (WT) in in vitro phosphorylation and dephosphorylation assays, but showed a resistance to in vivo dephosphorylation induced by the kinase inhibitor staurosporine. By an addition of GAS sequences to in vitro dephosphorylation assays, it was confirmed that increased nuclear presence and binding to DNA drove the hyper-phosphorylated status of these mutants. These results provide the evidence of an anti-parallel dimer interface in STAT3, the disruption of which leads to STAT3 hyperactivity. Constitutively active STAT3 has been implicated to drive several cancers, and these results present the anti-parallel dimer of U-STATs as an important mechanism to regulate STAT3 signalling. Though U-STATs have been recently identified to have their distinct gene activation pattern, whether U-STATs have an impact on P-STAT-mediated signal transduction is unknown. Therefore, the unphosphorylated and dimerization-deficient double mutant R602L/Y701F was generated in STAT1, to identify any possible interactions between U-STAT1 and P-STAT1. This double mutant, when co-expressed in cells with WT STAT1, lead to an inhibition of the IFNα- and IFNγ-induced nuclear import of STAT1. Additionally, mutating residues involved in the anti-parallel dimer interface in mutant U-STAT1 did not abolish the import block of P-STAT1. Experiments revealed that dimerization-deficient U-STAT1 inhibited the nuclear import of co-expressed P-STAT1 in a concentration-dependent manner with no major changes in the DNA binding and transcriptional activities of P-STAT1. The co-expressed WT molecule phosphorylated and dephosphorylated similarly in the presence and absence of mutant U-STAT1, but its nuclear import was blocked. Through mutagenesis and deletion experiments, the interaction between mutant U-STAT1 and P-STAT1 was identified to the amino-terminus of the mutant protein. Therefore, a transient import block mediated by high cellular concentration of U-STAT1 is proposed, wherein the assembly of the import structure of P-STAT1 is disrupted by interaction via the amino-terminus of U-STAT1. This STAT1 nuclear import block structure, that does not completely inhibit STAT1-mediated signal transduction in the nucleus, can be used to promote intracellular STAT1-mediated processes other than cytokine signalling, in cells primed with IFNγ.
Keywords: cytokine signalling; STAT1; STAT3; dimerization; unphosphorylated STAT; interferon priming; gain-of-function mutation; JAK-STAT pathway