Herein, we demonstrate that purified Stat1 binds to its tyrosine-phosphorylated docking site on the IFN gamma receptor alpha chain in a direct, specific, and reversible manner. Using surface plasmon resonance, we determine the affinity (KD = 137 nM) and specificity of the interaction and define the minimum affinity needed for receptor-mediated Stat1 activation. In addition, we quantitate the relative ability of purified Stat1 to interact with tyrosine-phosphorylated binding sites on other Stat proteins. Finally, we describe experiments that imply that the unidirectional release of activated Stat1 from the IFN gamma receptor reflects the preference of free tyrosine-phosphorylated Stat1 monomers to form high avidity reciprocal homodimers rather than reassociating with the receptor binding site. Our results demonstrate that IFN gamma-induced Stat1 activation is an ordered and affinity-driven process and we propose that this process may serve as a paradigm for Stat activation by other cytokine receptors.

Ubiquitination, endocytosis, and lysosomal degradation of the IFNAR1 (interferon ␣ receptor 1) subunit of the type I interferon (IFN) receptor is mediated by the SCF ␤-Trcp (Skp1-Cullin1-F-box protein ␤ transducin repeat-containing protein) E3 ubiquitin ligase in a phosphorylation-dependent manner. In addition, stability of IFNAR1 is regulated by its binding to Tyk2 kinase. Here we characterize the determinants of IFNAR1 ubiquitination and degradation. We found that the integrity of two Ser residues at positions 535 and 539 within the specific destruction motif present in the cytoplasmic tail of IFNAR1 is essential for the ability of IFNAR1 to recruit ␤-Trcp as well as to undergo efficient ubiquitination and degradation. Using an antibody that specifically recognizes IFNAR1 phosphorylated on Ser 535 we found that IFNAR1 is phosphorylated on this residue in cells. This phosphorylation is promoted by treatment of cells with IFN␣. Although the cytoplasmic tail of IFNAR1 contains seven Lys residues that could function as potential ubiquitin acceptor sites, we found that only three (Lys 501 , Lys 525 , and Lys 526 ), all located proximal to the destruction motif, are essential for ubiquitination and degradation of IFNAR1. Expression of Tyk2 stabilized IFNAR1 in a manner that was dependent neither on its binding to ␤-Trcp nor IFNAR1 ubiquitination. We discuss the complexities and specifics of the ubiquitination and degradation of IFNAR1, which is a ␤-Trcp substrate that undergoes degradation via a lysosomal pathway.Ubiquitin-mediated proteolysis plays an important regulatory role in many biological processes including cell cycle progression, transcription, and signal transduction (1). The process of protein ubiquitination is catalyzed by three distinct enzymes: ubiquitin-activating enzyme (E1), ubiquitin-conjugating enzyme (E2), and ubiquitin ligases (E3). The E1 enzyme activates ubiquitin in an ATP-dependent reaction and then transfers ubiquitin to an E2. The E2 enzyme then cooperates with an E3 ligase to catalyze the formation of an isopeptide bond between ubiquitin and substrate. E3 ligases, which bind both cognate E2 and the substrates, are responsible for conferring substrate specificity in the ubiquitination reaction. Proteins polyubiquitinated by these enzymes are degraded by the 26 S proteasome (1, 2).Alternately, oligo-or monoubiquitinated transmembrane proteins are endocytosed and degraded via the lysosomal pathway (3-6). E3 ligases that mediate the ubiquitination of cell surface receptors include c-Cbl/Hakai and Rsp5/NEDD4 family members (7,8). Recently we have shown that the SCF ␤-Trcp E3 ubiquitin ligase mediates ligand-induced ubiquitination of the IFNAR1 subunit of the type I interferon receptor complex. SCF ␤-Trcp -mediated ubiquitination of IFNAR1 1 leads to endocytosis of this receptor complex, followed by its degradation via lysosomal pathway, ultimately resulting in the down-regulation of the Type I IFN receptor. This mechanism plays a pivotal role in the negative regulation of IFN signaling (9).SC...

Interferon‐alpha (IFN alpha) induces rapid tyrosine phosphorylation of its receptors, two JAK kinases and three STAT transcription factors. One kinase, p135tyk2, is complexed with the IFNaR1 receptor, and may catalyze some of these phosphorylation events. We demonstrate that, in vitro, p135tyk2 phosphorylates two tyrosines on IFNaR1. A phosphopeptide corresponding to the major phosphorylation site (Tyr466) binds STAT2, but not STAT1, in an SH‐2‐dependent manner. Furthermore, only latent, non‐phosphorylated STAT2 interacts with this phosphopeptide. When this phosphopeptide is introduced into permeabilized cells, the IFN alpha‐dependent tyrosine phosphorylation of both STATs is blocked. Finally, mutant versions of IFNaR1, in which Tyr466 is changed to phenylalanine, can act in a dominant negative manner to inhibit phosphorylation of STAT2. These observations are consistent with a model in which IFNaR1 mediates the interaction between JAK kinases and the STAT transcription factors.

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MicroRNAs play important roles in regulating tumour development, progression and metastasis. Here we show that one of the miR-200 family members, miR-141, is under-expressed in several prostate cancer (PCa) stem/progenitor cell populations in both xenograft and primary patient tumours. Enforced expression of miR-141 in CD44+ and bulk PCa cells inhibits cancer stem cell properties including holoclone and sphere formation, as well as invasion, and suppresses tumour regeneration and metastasis. Moreover, miR-141 expression enforces a strong epithelial phenotype with a partial loss of mesenchymal phenotype. Whole-genome RNA sequencing uncovers novel miR-141-regulated molecular targets in PCa cells including the Rho GTPase family members (for example, CDC42, CDC42EP3, RAC1 and ARPC5) and stem cell molecules CD44 and EZH2, all of which are validated as direct and functionally relevant targets of miR-141. Our results suggest that miR-141 employs multiple mechanisms to obstruct tumour growth and metastasis.