3 DISTINCT DOMAINS OF SSI-1 SOCS-1/JAB PROTEIN ARE REQUIRED FOR ITS SUPPRESSION OF INTERLEUKIN-6 SIGNALING/

Cytokine inducible protein SSI-1 [signal transducers and activators of transcription (STAT)-induced STAT inhibitor 1, also referred to as SO CS-1 (suppressor of cytokine signaling 1) or JAB (Janus kinase-binding protein)] negatively regulates cytokine receptor signaling by inhibit ion of JAK kinases. The SSI family of proteins includes eight members that are structurally characterized by an SH2 domain and a C-terminal conserved region that we have called the SC-motif, In this study, we i nvestigated the roles of these domains in the function of SSI-1, Resul ts of reporter assays demonstrated that the pre-SH2 domain (24 aa in f ront of the SH2 domain) and the SH2 domain of SSI-1 were required for the suppression by SSI-1 of interleukin 6 signaling. Coexpression stud ies of COS7 cells revealed that these domains also were required for i nhibition of three JAKs (JAK1, JAK2, and TYK2), Furthermore, deletion of the SH2 domain, but not the pre-SH2 domain, resulted in loss of ass ociation of SSI-I with TIW, Thus, SSI-I associates with JAK family kin ase via its SH2 domain, and the pre-SH2 domain is required for the fun ction of SSI-I, Deletion of the SC-motif markedly reduced expression o f SSI-I protein in M1 cells, and this reduction was reversed by treatm ent with proteasome inhibitors, suggesting that this motif is required to protect the SSI-I molecule from proteolytic degradation. Based on these findings, we concluded that three distinct domains of SSI-1 (the pre-SH2 domain, the SH2 domain, and the SC-motif) cooperate in the su ppression of interleukin 6 signaling.