SUMMARY Lysine63-linked ubiquitin (K63-Ub) chains represent a particular ubiquitin topology that mediates proteasome-independent signaling events. The deubiquitinating enzyme (DUB) BRCC36 segregates into distinct nuclear and cytoplasmic complexes that are specific for K63-Ub hydrolysis. RAP80 targets the five-member nuclear BRCC36 complex to K63-Ub chains at DNA double-strand breaks. The alternative four-member BRCC36 containing complex (BRISC) lacks a known targeting moiety. Here we identify Serine Hydroxymethyltransferase (SHMT) as a heretofore-unappreciated component that fulfills this function. SHMT directs BRISC activity at K63-Ub chains conjugated to the type 1 interferon (IFN) receptor chain 1 (IFNAR1). BRISC-SHMT2 complexes localize to and deubiquitinate actively engaged IFNAR1, thus limiting its K63-Ub mediated internalization and lysosomal degradation. BRISC deficient cells and mice exhibit attenuated responses to IFN and are protected from IFN-associated immunopathology. These studies reveal a novel mechanism of DUB regulation, and suggest a therapeutic use of BRISC inhibitors for treating pathophysiologic processes driven by elevated IFN responses.

Cells respond to a milieu of extracellular regulators by timely activation of diverse intracellular signaling cascades. Cells also restrict the magnitude and duration of these signaling events by ligand-inducible degradation of the cognate receptors (reviewed in references 8 and 19). Delineating the mechanisms that underlie specific proteolytic elimination of receptors is important for understanding the pathogenesis of numerous human disorders elicited by an unabated signaling. Conversely, an intimate knowledge of targets that can be used to interfere with such "eliminative signaling" should expand our abilities to augment the therapeutic efficacy of the ligands of medical importance.Among such ligands are type I interferons (IFNs), including alpha and beta interferons (IFN-␣ and IFN-␤), which exhibit potent antitumor, antiviral, and immunomodulatory activities and which are widely used in therapy of human tumors (25), chronic viral infections (5), and multiple sclerosis (24). These cytokines trigger their signaling via activating the cognate cell surface receptor assembled by the IFN-␣/␤ receptor chain 1 (IFNAR1) and IFNAR2. This event is followed by activation of Janus tyrosine kinases (JAK) JAK1 and Tyk2, tyrosine phosphorylation of receptors, and recruitment of the signal transducers and activators of transcription (STAT1 and STAT2) that induce gene expression through binding to the IFN-stimulated response element (ISRE) within the promoters of IFNstimulated genes (reviewed in references 1, 29, 39, and 46). This pathway is under the control of several mechanisms of negative regulation (including the effects of tyrosine dephosphorylation, JAK inhibition and degradation, and STAT sumoylation) that are common to numerous JAK-STAT-activating cytokines and polypeptide hormones (reviewed in reference 17). Conversely, the ligand-specific rapid termination of IFN-␣/␤ signaling is mediated by elimination of its receptor that depends on endocytosis and subsequent lysosomal degradation of the IFNAR1 chain (4).Degradation of IFNAR1 is stimulated by its ubiquitination, which is facilitated by the SCF ␤Trcp E3 ubiquitin ligase. This ligase is recruited to IFNAR1 in a manner that depends upon phosphorylation of specific serine residues within a well-defined degron (26-28). Serine phosphorylation of the IFNAR1 degron on Ser535 is essential for IFNAR1 ubiquitination and degradation. The physiologic IFN-␣-or IFN-␤-inducible pathway requires catalytic activity of Tyk2 to stimulate phosphorylation of the IFNAR1 degron, recruitment of ␤Trcp, and IFNAR1 ubiquitination and degradation (27,32,34). Alternatively, the basal ligand-and JAKindependent phosphorylation (32) is mediated by casein kinase 1␣ (CK1␣) (30) in a manner that is regulated by the priming phosphorylation of IFNAR1 (2). This priming phosphorylation can be further stimulated by inducers of unfolded protein response such as thapsigargin (TG) (31).Potent catalytic activity and abundance of CK1␣ hindered the efforts to identify the long-sought serine kinase(s) that medi...

The ubiquitination of the receptor that mediates signaling induced by the polypeptide pituitary hormone prolactin (PRL) has been shown to lead to the degradation of this receptor and to the ensuing negative regulation of cellular responses to PRL. However, the mechanisms of PRL receptor (PRLr) proteolysis remain largely to be determined. Here we provide evidence that PRLr is internalized and primarily degraded via the lysosomal pathway. Ubiquitination of PRLr is essential for the rapid internalization of PRLr, which proceeds through a pathway dependent on clathrin and the assembly polypeptide 2 (AP2) adaptor complexes. Recruitment of AP2 to PRLr is stimulated by PRLr ubiquitination, which also is required for the targeting of already internalized PRLr to the lysosomal compartment. While mass spectrometry analysis revealed that both monoubiquitination and polyubiquitination (via both K48-and K63-linked chains) occur on PRLr, the results of experiments using forced expression of ubiquitin mutants indicate that PRLr polyubiquitination via K63-linked chains is important for efficient interaction of PRLr with AP2 as well as for efficient internalization, postinternalization sorting, and proteolytic turnover of PRLr. We discuss how specific ubiquitination may regulate early and late stages of endocytosis of PRLr and of related receptors to contribute to the negative regulation of the magnitude and duration of downstream signaling.Endocytosis of signaling receptors is a major mechanism used by cells to restrict the magnitude and duration of signal transduction induced by extracellular ligands. Ligand-induced endocytosis of cell surface receptors may occur through clathrin-dependent or -independent pathways. The clathrin-dependent pathway links receptors with clathrin-coated vesicles, which are specialized invaginations of the plasma membrane that concentrate receptors that become internalized. This pathway relies on the interaction of the assembly polypeptide 2 (AP2) clathrin adaptor complexes with specific endocytic signals located within the cytoplasmic domain of the receptors (5).AP2 complexes, which are involved in the assembly of clathrin triskelions at the plasma membrane, are composed of four components, including two adaptin subunits (␣ and ␤2) and two smaller subunits ( 2 and 2); among these subunits, each has different biological functions (34). There are specific endocytic motifs that are essential for receptor clustering on the membrane and clathrin-dependent internalization of receptors. For example, both tyrosine-and leucine-based motifs can be recognized by the AP2 complex through the interaction with its 2 subunit and with the ␤2 or ␣/ 2 hemicomplexes, respectively (5,8,13).The process of receptor endocytosis is not exclusively regulated by the linear motifs located on the cytoplasmic tail of receptors. Posttranslational modification by ubiquitination has also emerged as an important factor in the endocytosis and sorting of surface receptors. Ubiquitin is a 76-amino-acid protein that forms an isopeptide...

Linear endocytic motifs of signaling receptors as well as their ubiquitination determine the rate of ligand-induced endocytosis that mediates down-regulation of these receptors and restricts the magnitude and duration of their respective signal transduction pathways. We previously hypothesized that, in the absence of its cognate ligand, type I interferon (IFN), the IFN␣ receptor chain 1 (IFNAR1) receptor chain is protected from basal endocytosis by a hypothetical masking complex that prevents the Tyr-based endocytic motif within IFNAR1 from interacting with components of the adaptin protein complex 2 (AP2). Here we identify a member of the Janus kinase (Jak) family, Tyk2, as a component of such a masking complex. In the absence of ligand or of receptor chain ubiquitination, binding of Janus kinase Tyk2 within the proximity of the Tyr-based linear motif of IFNAR1 is required to prevent IFNAR1 internalization and to maintain its cell surface expression. Furthermore, interaction experiments revealed that Tyk2 physically shields this Tyrbased motif from the recognition by the AP50 subunit of AP2. These data delineate a long-sought ligand-and ubiquitin-independent mechanism by which Tyk2 contributes to both the regulation of total IFNAR1 levels as well as the regulation of the cell surface density of this receptor chain.Cells react to diverse environmental stimuli by expressing specific receptors that recognize these stimuli and initiate specific signaling pathways that enable a cell or a tissue to cope with an altered environment. Down-regulation of these signaling receptors represents the most specific mode of limiting the magnitude and duration of given signal transduction pathways. For transmembrane receptors displayed at the cell surface, ligand-stimulated endocytosis is a major mechanism by which the ability of a cell to react to a ligand is restricted. In addition, basal ligand-independent internalization may determine how responsive a naive cell could be to a subsequent encounter with a particular ligand.Mechanisms mediating internalization of signaling receptors involve a dynamic plasma membrane exchange resulting in bulk endocytosis and a cargo-specific clathrin-dependent endocytosis. Interaction of clathrin lattices formed on the plasma membrane with cargo receptors relies on adaptor complexes such as the adaptin protein-2 complex (AP2), 4 which recognizes specific linear endocytic motifs present within the cytoplasmic domains of the target receptor. For example, the 2 subunit of AP2 (also termed AP50) is known to recognize Tyr-based linear endocytic motifs and to enable the AP2-dependent tethering of cargo to clathrin molecules (reviewed in Refs. 1-4).Receptor ubiquitination has also emerged as a key endocytic signal for numerous eukaryotic cell surface receptors (reviewed in Refs. 5-8). For several receptors (such as epidermal growth factor receptor, growth hormone receptor, interferon ␣ receptor chain 1 (IFNAR1)), ubiquitination is stimulated by the ligand. This stimulation is mediated by the ligand-ind...

From its traditional identity as a hormone involved in growth and differentiation of mammary epithelium and in lactation, to having a pertinent role in the development of mammary carcinoma, the peptide hormone/cytokine prolactin (PRL) has emerged as a versatile signaling molecule. There has been significant progress in our understanding of the fine working of PRL in the past several years. Notably, much effort has been concentrated on the mediator of PRL action, namely, the prolactin receptor (PRLr). The causal link between increased PRLr expression and breast cancer is being increasingly appreciated. Considering that the level of the receptor on the surface is a critical determinant of signaling output in response to PRL, the uncovering of regulatory elements that control receptor expression becomes important. The principle focus of this review is on the regulation of PRLr expression and activity in breast cancer with a brief overview of different isoforms of PRLr, their expression, signaling capabilities and the biological outcomes of PRL/PRLr signaling.