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The tumor suppressor gene 101 (tsg101) regulates vesicular trafficking processes in yeast and mammals. We report a novel protein, Tal (Tsg101-associated ligase), whose RING finger is necessary for multiple monoubiquitylation of Tsg101. Bivalent binding of Tsg101 to a tandem tetrapeptide motif (PTAP) and to a central region of Tal is essential for Tal-mediated ubiquitylation of Tsg101. By studying endocytosis of the epidermal growth factor receptor and egress of the human immunodeficiency virus, we conclude that Tal regulates a Tsg101-associated complex responsible for the sorting of cargo into cytoplasm-containing vesicles that bud at the multivesicular body and at the plasma membrane.[Keywords: Endocytosis; growth factor; HIV; ubiquitin; signal transduction] Supplemental material is available at http://www.genesdev.org.

Suppressors of cytokine signaling (SOCS) are Src homology-2-containing proteins originally identified as negative regulators of cytokine signaling. Accumulating evidence indicates a role for SOCS proteins in the regulation of additional signaling pathways including receptor tyrosine kinases. Notably, SOCS36E, the Drosophila ortholog of mammalian SOCS5, was recently implicated as a negative regulator of the Drosophila ortholog of EGFR. In this study, we aimed at characterizing the role of SOCS5 in the negative regulation of EGFR. Here we show that the expression of SOCS5 and its closest homolog SOCS4 is elevated in cells following treatment with EGF, similar to several negative feedback regulators of EGFR whose expression is up-regulated upon receptor activation. The expression of SOCS5 led to a marked reduction in EGFR expression levels by promoting EGFR degradation. The reduction in EGFR levels and EGF-induced signaling in SOCS5-expressing cells requires both the Src homology-2 and SOCS box domains of SOCS5. Interestingly, EGFR is degraded by SOCS5 prior to EGF treatment in a ligand-and c-Cbl-independent manner. SOCS5 can associate with EGFR and can also bind the ElonginBC protein complex via its SOCS box, which may recruit an E3 ubiquitin ligase to promote EGFR degradation. Thus, we have characterized a novel function for SOCS5 in regulating EGFR and discuss its potential role in controlling EGFR homeostasis.

When appended to the epidermal growth factor receptor (EGFR), ubiquitin serves as a sorting signal for lysosomal degradation. Here we demonstrate that the ubiquitin ligase of EGFR, namely c-Cbl, also mediates receptor modification with the ubiquitin-like molecule Nedd8. EGF stimulates receptor neddylation, which enhances subsequent ubiquitylation, as well as sorting of EGFR for degradation. Multiple lysine residues, located within the tyrosine kinase domain of EGFR, serve as attachment sites for Nedd8. A set of clathrin coat-associated binders of ubiquitin also bind Nedd8, but they undergo ubiquitylation, not neddylation. We discuss the emerging versatility of the concerted action of ubiquitylation and neddylation in the process that desensitizes growth factor-activated receptor tyrosine kinases.Growth factors and their transmembrane receptors, harboring intrinsic tyrosine kinase activity, play essential roles in cell fate determination. Whereas the process leading to growth factor-induced activation of signaling pathways is relatively well understood, mechanisms that initiate signal desensitization are only beginning to be unraveled (reviewed in Ref. 1). In the case of the epidermal growth factor receptor (EGFR) 2 and related receptor tyrosine kinases, the major signal attenuation process involves ligand-induced internalization of activated receptors and their sorting to degradation in lysosomes (reviewed in Ref.2). In analogy to the pivotal role played by phosphotyrosine and respective binding domains (e.g. Src homology domain 2) in positive signaling pathways, ubiquitin and ubiquitin-binding domains (e.g. ubiquitin-interacting motifs (UIMs)) regulate receptor endocytosis and sorting for lysosomal degradation (3). For example, recruitment of an E3 ubiquitin ligase, called c-Cbl (4 -7), enables subsequent conjugation of ubiquitin to multiple lysines of EGFR (8 -10). The appended ubiquitins are thought to recruit a set of endocytic proteins (e.g. Eps15) through their UIMs (reviewed in Ref. 11).In contrast to the well understood cellular functions of protein ubiquitylation, the roles played by ubiquitin-like proteins such as Nedd8 and SUMO are less characterized (12). Nedd8 is the closest kin of ubiquitin and it is linked to other proteins by an amide bond linking the carboxyl-terminal carboxylate to lysine residues. Neddylation is initiated by a heterodimeric complex comprising Uba3 and the amyloid precursor proteinbinding protein (APP-BP1). Ubc12, an E2-like component, then mediates conjugation of Nedd8 (13), but the identity and function of Nedd8-specific E3 ligases are less understood. Modification of Cullins, the first discovered neddylation substrate, is promoted by Roc1/Rbx, a RING finger protein (14, 15), which recruits Ubc12 (16, 17). Cullins are shared subunits of SCF (Skp1-Cdc53/CUL-F-box) complexes, assembled E3 ubiquitin ligases that regulate ubiquitylation of proteins involved primarily in cell cycle control. Neddylation of Cullin1 and Cullin2 activates their E3 ubiquitin ligase activity toward subs...

IntroductionLipid rafts are plasma membrane microdomains postulated to function in signaling and membrane trafficking. [1][2][3] Lipid rafts are enriched in gangliosides (glycosphingolipids) and cholesterol, which form liquid-ordered domains of decreased membrane fluidity. The long, saturated acyl chains of gangliosides impart a high degree of order further stabilized by intercalating cholesterol molecules, leading to the insolubility of lipid rafts in nonionic detergents. Lipid rafts can be isolated based on their detergent insolubility and low-buoyancy density using discontinuous sucrose gradient ultracentrifugation of nonionic detergent lysates. Lipid rafts are not artifacts of detergent extraction. They have been detected in living cells using chemical cross-linking and fluorescence resonance energy transfer. 4,5 The modification of proteins with saturated acyl groups can result in their localization within lipid rafts. Thus, these microdomains are enriched in glycosyl-phosphatidyl-inositol anchored proteins (GPI-AP) and in many signaling molecules such as Src family protein tyrosine kinases (PTKs), the adaptor protein LAT, heterotrimeric and small G-proteins, and phosphoinositides. 3 In addition, several transmembrane receptors are inducibly recruited to or stabilized within lipid rafts, including T-cell receptor (TCR), B-cell receptor (BCR), and Fc⑀RI. 2 Subsequent activation of signaling molecules in lipid rafts may facilitate signaling through these immunoreceptors. Lipid rafts may act to segregate molecules in the plasma membrane and to regulate signaling through the spatial coordination of intermolecular associations.Stimulation of T cells through TCR results in the activation of multiple signaling pathways, leading to interleukin-2 (IL-2) responsiveness and the secretion of IL-2 and resulting in autocrine cell growth. 6 The high-affinity receptor for IL-2 is composed of the IL-2R␣ chain, which functions solely in IL-2 binding, and IL-2R␤ and IL-2R␥ c , which contribute to IL-2 binding and mediate signal transduction. 7 IL-2-induced proliferation requires activation of the Janus family kinases JAK1 and JAK3, which are constitutively associated with IL-2R␤ and IL-2R␥, respectively. 8,9 Ligandinduced IL-2R aggregation leads to the juxtaposition of JAK1 and JAK3, resulting in their phosphorylation and activation. Subsequent phosphorylation of tyrosine residues in receptor chains leads to the SH2-domain-mediated recruitment of signal transducer and activator of transcription (STAT) proteins STAT5a and STAT5b. 7 JAK-mediated phosphorylation of STAT proteins leads to their dimerization by SH2 domain-phosphotyrosine interactions and their translocation to the nucleus, where STAT proteins regulate gene transcription. Signaling through the IL-2R also induces the recruitment and activation of phosphatidylinositol 3 kinase (PI3K), which is implicated in IL-2-mediated proliferation and survival through its downstream effector protein kinase B/Akt. 10,11 In addition, the tyrosine phosphorylation of IL-2R␤ results i...

Accumulating evidence suggests that proteins tethered to the plasma membrane through glycosylphosphatidylinositol (GPI) anchors share common biological properties. In the present study we demonstrate that GPI-anchored proteins regulate T cell growth. Specifically, anti-TCR-induced proliferation was profoundly inhibited by co-immobilized mAb specific for Thy-1, CD48 and Ly6A/E. However, neither IL-2 production nor the effector function of cytotoxic T lymphocytes was impaired in these circumstances. Analysis of the IL-2 receptor (IL-2R) signaling pathway revealed that the association of IL-2R beta and gamma chains with the Janus kinases, JAK1 and JAK3, was not perturbed in the presence of mAb specific for GPI-linked proteins. However, in these conditions, IL-2-mediated recruitment of IL-2Ralpha, beta and gamma chains, resulting in the formation of the high-affinity hetero-trimeric IL-2R, was inhibited. The resulting phosphorylation of JAK1 and JAK3, indicative of their activation states, was correspondingly reduced. These results characterize a novel state of T cell physiology in which effector function is maintained, in the absence of clonal expansion. A physiological role for GPI-anchored proteins in the maintenance of cellular homeostasis and function is discussed.

Exposure of normal juvenile chicken bone marrow cells to the replication defective avian reticuloendotheliosis virus strain T (REV-T) (chicken syncytial virus [CSV]) in vitro resulted in the generation of transformed cell lines containing T cells. The transformed T cells derived from bone marrow included cells expressing either alpha/beta or gamma/delta T cell receptors (TCRs) in proportions roughly equivalent to the proportions of TCR-alpha/beta and TCR-gamma/delta T cells found in the normal bone marrow in vivo. Essentially all TCR-alpha/beta-expressing transformed bone marrow-derived T cells expressed CD8, whereas few, if any, expressed CD4. In contrast, among TCR-gamma/delta T cells, both CD8+ and CD8- cells were derived, all of which were CD4-. Exposure of ex vivo spleen cells to REV-T(CSV) yielded transformed polyclonal cell lines containing > 99% B cells. However, REV-T(CSV) infection of mitogen-activated spleen cells in vitro resulted in transformed populations containing predominantly T cells. This may be explained at least in part by in vitro activation resulting in dramatically increased levels of T cell REV-T(CSV) receptor expression. In contrast to REV-T(CSV)-transformed lines derived from normal bone marrow, transformed lines derived from activated spleen cells contained substantial numbers of CD4+ cells, all of which expressed TCR-alpha/beta. While transformed T cells derived from bone marrow were stable for extended periods of in vitro culture and were cloned from single cells, transformed T cells from activated spleen were not stable and could not be cloned. We have therefore dissociated the initial transformation of T cells with REV-T(CSV) from the requirements for long-term growth. These results provide the first demonstration of efficient in vitro transformation of chicken T lineage cells by REV-T(CSV). Since productive infection with REV-T(CSV) is not sufficient to promote long-term growth of transformed cells, these results further suggest that immortalization depends not only upon expression of the v-rel oncogene but also on intracellular factor(s) whose expression varies according to the state of T cell physiology and/or activation.