Signalling through JAKs and STATs

Cytokines normally activate the JAK/STAT and additional signalling pathways in a modular fashion through different receptors. There appear to be additional JAK pathways and cross talk between pathways. A mammalian expression and purification system was established to identify phosphorylation sites in JAK1 (Janus Kinase 1) which may modulate activity and/or mediate additional protein-protein interactions and to find new proteins, which are recruited by human Jak1. Highly purified hJAK1 and different analytical mass spectrometry approaches were used to identify potential serine and threonine sites. The functional significance of any sites identified was analysed by mutagenesis and following assaying of different aspects in JAK/STAT signalling. The system was modified to co-purify constitutively and/or inducibly associated proteins. The binding of interferons (IFNs) to their receptors leads to the phosphorylation and activation of signal transducers and activators of transcription (STATs) and their translocation from the cytoplasm to the nucleus. We could show that IFN-α and -γ signalling and STAT1 translocation are independent of the actin cytoskeleton or microtubules. Using fluorescence loss in photobleaching (FLIP) and fluorescence recovery after photobleaching (FRAP) experiments the mobility of a fusion protein of STAT1 with green fluorescent protein (STAT1-GFP) was compared with that of GFP and protein kinase C (PKC)-GFP. In IFN-γ treated and control cells cytoplasmic STAT1-GFP shows high, energy independent, mobility comparable to that of freely diffusible GFP. A random walk model for movement of STAT1 from the plasma membrane to the nuclear pore is, therefore, indicated. Nuclear STAT1-GFP showed similar high mobility, with exclusion from nucleoli, consistent with high rates of association and dissociation of STAT1-DNA and/or -protein complexes in the nucleoplasm of the cell