The production of pigment by melanocytic cells of the skin involves a series of enzymatic reactions that take place in specialized organelles called melanosomes. Melan-A/MART-1 is a melanocytic transmembrane protein with no enzymatic activity that accumulates in vesicles at the trans side of the Golgi and in melanosomes. We show here that, in melanoma cells, Melan-A associates with two homologous to E6-AP C-terminus (HECT)-E3 ubiquitin ligases, NEDD4 and Itch, and is ubiquitylated. Both NEDD4 and Itch participate in the degradation of Melan-A. A mutant Melan-A lacking ubiquitinacceptor residues displays increased half-life and, in pigmented cells, accumulates in melanosomes. These results suggest that ubiquitylation regulates the lysosomal sorting and degradation of Melan-A/MART-1 from melanosomes in melanocytic cells.

Recent experiments have unravelled novel signal transduction pathways that involve the SRC homology 2 (SH2) domain adapter protein SHB. SHB is ubiquitously expressed and contains proline rich motifs, a phosphotyrosine binding (PTB) domain, tyrosine phosphorylation sites and an SH2 domain and serves a role in generating signaling complexes in response to tyrosine kinase activation. SHB mediates certain responses in platelet-derived growth factor (PDGF) receptor-, fibroblast growth factor (FGF) receptor-, neural growth factor (NGF) receptor TRKA-, T cell receptor-, interleukin-2 (IL-2) receptor- and focal adhesion kinase- (FAK) signaling. Upstream of SHB in some cells lies the SRC-like FYN-Related Kinase FRK/RAK (also named BSK/IYK or GTK). FRK/RAK and SHB exert similar effects when overexpressed in rat phaeochromocytoma (PC12) and beta-cells, where they both induce PC12 cell differentiation and beta-cell proliferation. Furthermore, beta-cell apoptosis is augmented by these proteins under conditions that cause beta-cell degeneration. The FRK/RAK-SHB responses involve FAK and insulin receptor substrates (IRS) -1 and -2. Besides regulating apoptosis, proliferation and differentiation, SHB is also a component of the T cell receptor (TCR) signaling response. In Jurkat T cells, SHB links several signaling components with the TCR and is thus required for IL-2 production. In endothelial cells, SHB both promotes apoptosis under conditions that are anti-angiogenic, but is also required for proper mitogenicity, spreading and tubular morphogenesis. In embryonic stem cells, dominant-negative SHB (R522K) prevents early cavitation of embryoid bodies and reduces differentiation to cells expressing albumin, amylase, insulin and glucagon, suggesting a role of SHB in development. In summary, SHB is a versatile signal transduction molecule that produces diverse biological responses in different cell types under various conditions. SHB operates downstream of GTK in cells that express this kinase.

Stimulation of the T cell antigen receptor (TCR) induces tyrosine phosphorylation of numerous intracellular proteins. We have recently investigated the role of the adaptor protein Shb in the early events of T cell signaling and observed that Shb associates with Grb2, linker for activation of T cells (LAT) and the TCR -chain in Jurkat cells. We now report that Shb also associates with phospholipase C-␥1 (PLC-␥1) in these cells. Overexpression of Src homology 2 domain defective Shb caused diminished phosphorylation of LAT and consequently the activation of mitogen-activated protein kinases was decreased upon TCR stimulation. In addition, the Shb mutant also blocked phosphorylation of PLC-␥1 and the increase in cytoplasmic Ca 2؉ following TCR stimulation. Nuclear factor for activation of T cells is a major target for Ras and calcium signaling pathways in T cells following TCR stimulation, and the overexpression of the mutant Shb prevented TCR-dependent activation of the nuclear factor for activation of T cells. Consequently, endogenous interleukin-2 production was decreased under these conditions. The results indicate a role for Shb as a link between the TCR and downstream signaling events involving LAT and PLC-␥1 and resulting in the activation of transcription of the interleukin-2 gene.The T cell response is initiated by the presentation of an antigen to the T cell receptor (TCR).1 This is followed by rapid phosphorylation of tyrosines on both the receptor itself and cytoplasmic proteins, the initiation of several signaling cascades, and subsequently the activation of interleukin-2 (IL-2) gene transcription and other T cell immune functions. The early events in T cell signaling involve the activation of several protein tyrosine kinases. After TCR -chain phosphorylation (by the protein tyrosine kinases Lck or Fyn), a Syk family protein tyrosine kinase, ZAP70, binds the chain immunoreceptor tyrosine-based activation motifs (ITAMs) of the CD3 complex (1) and consequently becomes phosphorylated and activated. Active ZAP70 is then responsible for phosphorylation and activation of many additional substrates: for example, phospholipase C-␥1 (PLC-␥1), p36/38 linker for activation of T cells (LAT) (2), the guanine nucleotide exchange factor Vav, and the adaptor protein SLP-76 (3). In the course of these events, PLC-␥1, Grb2, and phosphoinositide 3-kinase all associate with tyrosine-phosphorylated p36/38 LAT (4 -7). Phosphorylation of PLC-␥1 activates this enzyme, which then hydrolyzes phosphatidylinositol phosphate to yield diacylglycerol and inositol 1,4,5-tris-phosphate, the latter messenger mobilizing intracellular Ca 2ϩ . The resulting increase in cytoplasmic Ca 2ϩ activates calcineurin, which mediates nuclear translocation and activation of the T cell-specific transcription factor nuclear factor for activation of T cells (NFAT) (8).Activation of the Ras signaling pathway in response to TCR stimulation involves p36/38 LAT, Grb2 and Sos (5). As a consequence, mitogen-activated protein (MAP) kinases are stimulated, caus...

The level of intracellular proteins is mainly regulated through modifications by ubiquitin ligases that target them for degradation. Members of the NEDD4 family of E3 ubiquitin ligases, such as Itch (atrophin‐1 interacting protein 4), possess up to four WW domains for specific association with PY motif‐containing substrates. We have identified sorting nexin 9 (SNX9), a protein involved in endocytic processes, as a new substrate of Itch. Itch ubiquitylates SNX9 and regulates intracellular SNX9 levels. Using truncated proteins, we found that the interaction with SNX9 is mediated by the proline‐rich domain (PRD) of Itch, a domain distinct from the conventional WW recognition domain, and the SH3 domain of SNX9. Interaction with the PRD of Itch is essential for SNX9 ubiquitylation and degradation. Furthermore, this effect is specific for Itch, as NEDD4, a related PRD‐containing E3 ligase, does not bind SNX9. SNX18, a second member of the SNX family containing an SH3 domain, was also found to bind to Itch. Our results indicate that the pool of substrates of NEDD4 family E3 ubiquitin ligases extends beyond proteins containing PY motifs. Structured digital abstract  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7889719: SNX18 (uniprotkb:http://www.uniprot.org/uniprot/Q96RF0) physically interacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) with ITCH (uniprotkb:http://www.uniprot.org/uniprot/Q96J02) by anti tag coimmunoprecipitation (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0007)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7889571, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7889619: ITCH (uniprotkb:http://www.uniprot.org/uniprot/Q96J02) physically interacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) with SNX9 (uniprotkb:http://www.uniprot.org/uniprot/Q9Y5X1) by pull down (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0096)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7889653: Melan‐A (uniprotkb:http://www.uniprot.org/uniprot/Q16655) physically interacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0914) with NEDD4 (uniprotkb:http://www.uniprot.org/uniprot/P46934) and ITCH (uniprotkb:http://www.uniprot.org/uniprot/Q96J02) by pull down (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0096)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7889591, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7889673, http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7890033: SNX9 (uniprotkb:http://www.uniprot.org/uniprot/Q9Y5X1) physically interacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) with ITCH (uniprotkb:http://www.uniprot.org/uniprot/Q96J02) by anti tag coimmunoprecipitation (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0007)  http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7889689: SNX9 (uniprotkb:http://www.uniprot.org/uniprot/Q9Y5X1) physically interacts (htt...