Structural and functional analysis of a nuclear localization signal in SOCS1
Introduction
Suppressor of cytokine signaling 1 (SOCS1) is a member of the SOCS family consisting of eight proteins (SOCS1–7 and CIS). SOCS proteins are defined by key structural elements, namely the C-terminal SOCS-box, a cytokine-inducible Src-homology 2 (SH2) domain and an N-terminal region of variable length. SOCS1 down-regulates Janus kinases/signal transducers and activators of transcription (JAK/STAT) pathways in a mode of negative feedback inhibition (Endo et al., 1997, Naka et al., 1997, Starr et al., 1997). It has been shown that SOCS1 interacts with components of the cytokine receptor complex located at the plasma membrane. By now three different suppressive mechanisms of SOCS1 have been described: SOCS1 can bind to JAK2 and inhibit the catalytic activity; this function is mediated by an extended SH2 subdomain (eSS) and also depends on the kinase-inhibitory region (KIR) located N-terminally of the eSS (Yasukawa et al., 1999). Moreover, SOCS1 protein can bind membrane proximal cytokine receptor domains and occupy binding sites for STATs, thereby acting as a competitive inhibitor (Fenner et al., 2006, Qing et al., 2005). Finally, SOCS1 targets JAK cytokine receptor complexes for proteasomal degradation by means of the SOCS-box domain, which acts as E3 ubiquitin ligase (Vuong et al., 2004, Zhang et al., 1999). SOCS1 mediated proteasomal degradation has also been suggested for inhibition of TIRAP/Mal in Toll-like receptor (TLR) signaling (Mansell et al., 2006).
Unexpectedly, SOCS1 was recently detected in the nucleus by different groups (Baetz et al., 2008, Kamio et al., 2004, Lee et al., 2008). We were able to identify a bipartite functional nuclear localization domain (NLS) in SOCS1 that resides between the SH2 domain and the SOCS-box and which was proven to mediate nuclear transport (Baetz et al., 2008). Mutations of critical basic amino acids within the predicted NLS resulted in the inversion of predominant nuclear to cytoplasmic localization. However, the exact length and sequence requirements of the NLS in SOCS1 remained ill-defined and the functional role of SOCS1 in the cell nucleus is only poorly understood. It was hypothesized that a functional role of SOCS1 in the nucleus could be exerted by its ability to act as E3 ubiquitin ligase and it was shown that SOCS1 induces proteasomal degradation of NFκB in the cell nucleus (Maine et al., 2007, Ryo et al., 2003).
In the present study we aimed to analyze the exact composition of the NLS in SOCS1 in more detail and to examine the function of non-nuclear mutants of SOCS1. Our results reveal the particular importance of the second cluster of basic amino acids for the function of the NLS in SOCS1. Furthermore we identify a role of nuclear SOCS1 localization in association with a SOCS-box mediated function in regulating IFN-signaling, possibly in a proteasome dependent manner.
Section snippets
Cell culture and transfection
HEK293 cells were maintained in DMEM (Dulbecco's modified Eagle's medium, Biochrom AG, Berlin, Germany). BEAS-2B cells were cultured in RPMI 1640 (Biochrom AG). All media were further supplemented with 10% (v/v) fetal calf-serum (Biowest, Nuaillé, France), 100 U/ml penicillin and 100 μg/ml streptomycin (PAA Laboratories, Pasching, Austria). Transient transfection of cells was carried out using Lipofectamine 2000 (Invitrogen, Karlsruhe, Germany).
Mutations
Point mutations and deletions were generated using
Nuclear localization of SOCS1 is mainly dependent on the second cluster of basic amino acids in the NLS
Previously, we reported that SOCS1 is localized predominantly in the nucleus. We were able to identify a bipartite nuclear localization signal within the SOCS1 sequence (Fig. 1A). This NLS consists of two separated clusters of basic residues, CL1 and CL2 (two and four arginine amino acids, respectively). These clusters had not been mutated individually in previous work. To define the regions required for the nuclear localization more exactly we decided to mutate the first and second NLS cluster
Discussion
The balance of stimulation and termination of STAT activity is essential for proper control of cytokine activity to prevent damage during inflammation. One key regulator of STAT1/2 activity is SOCS1, acting as a classical feedback inhibitor in JAK/STAT signaling. The current understanding of the inhibitory capacity of SOCS1 in JAK/STAT signaling is based upon the function to suppress tyrosine-phosphorylation of STAT1/2 by regulating activation of the cytokine receptor complex (Yoshimura et al.,
Acknowledgement
This work was supported by grants of the German Research Foundation to A.D. (Da592/3).
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