Tyk2-interacting partners (M. Algarté, C. Steindler, T. Yeh, M. Oloomi)
The large amino-terminal moiety of the JAK proteins carries the molecular determinant for binding to cytokine receptors. Sequence analysis of the amino-terminal region of JAKs has revealed the presence of a band 4.1-related domain, named FERM. This domain was first identified in cytoskeletal proteins of the ERM family (ezrin, radixin, moesin) where it mediates intramolecular interactions and membrane targeting. Based on the conviction that they are still missing elements that physically interact with Tyk2, a large effort has been put on identifying novel partners of Tyk2. A yeast two-hybrid screen was performed using as bait the FERM domain of Tyk2 and eleven positive clones were isolated, six of them containing overlapping sequences. Experiments are in progress to characterize two novel proteins and to analyze the physiological role of their interaction with Tyk2.
Nuclear localization of Tyk2 (J. Ragimbeau, A. Vasserot)
We have studied the subcellular distribution of the wild-type Tyk2 protein and of several mutants expressed in Tyk2-deficient human cells. Direct GFP-associated fluorescence and immunostaining showed a diffuse localization of Tyk2 throughout the cell, including the nuclear compartment. The nuclear localization of Tyk2 requires a NLS-like motif, rich in arginine residues, that maps within the region mediating interaction with cytokine receptors. Our studies demonstrate that Tyk2 can reside in the nucleus independently of receptor binding and that the nuclear pool is dispensable for the transcriptional and anti-vesicular stomatitis virus responses induced by IFN-a/b. Current work is aimed at understanding the molecular mechanism of nuclear import and the function of Tyk2 in the nucleus.
Down modulation of IFN-a/b responses by receptor cross-interference (E. Dondi)
In contrast to the large number of cytokine receptors, only four JAK proteins exists in mammalian cells, implying the shared use of these kinases by many different receptor complexes and the possibility of interference if receptor numbers exceed the amount of available JAK. In a collaborative effort, we have investigated whether cross-competition among Tyk2-interacting receptors can occur. Our results suggest that exogenously expressed Tyk2-interacting receptors can sequester and titer out the kinase away from the endogenous IFNAR1 receptor chain, leading to a reduced IFN-a/b response. This represents a novel mechanism of trans-modulation between cytokine responses based on competition for a shared receptor-associated tyrosine kinase.
Study of the IFN-a/b response in differentiating human T cells (E. Dondi)
A critical aspect of the immune response to pathogens is mediated by the helper function of CD4+ T cells which are activated and differentiate into two functionally distinct subsets, Th1 and Th2. IFN-a/b produced in response to a viral or bacterial infection or by activated dendritic cells can direct human CD4+ T cells toward Th1 development, thus linking innate and adaptive immunity. We have recently found that human differentiated Th1 and Th2 cell lines display a strongly reduced sensitivity to IFN-a/b as compared to their naïve precursors. This down-modulation of IFN sensitivity appears intrinsic to the differentiation process itself. Considering the impact of IFN-a/b in determining T cell fate, the reduction of sensitivity that we have found may represent an important regulatory mechanism. Thus, we want to investigate the molecular mechanism(s) underlying the reduced IFN-a/b sensitivity of differentiated cells and the physiological relevance of this phenomenon.