Target recognition by the ubiquitin system is mediated by E3 ubiquitin ligases. Nedd4 family members are E3 ligases comprised of a C2 domain, 2-4 WW domains that bind PY motifs (L/PPxY) and a ubiquitin ligase HECT domain. The nine Nedd4 family proteins in mammals include two close relatives: Nedd4 (Nedd4-1) and Nedd4L (Nedd4-2), but their global substrate recognition or differences in substrate specificity are unknown. We performed in vitro ubiquitylation and binding assays of human Nedd4-1 and Nedd4-2, and rat-Nedd4-1, using protein microarrays spotted with B8200 human proteins. Top hits (substrates) for the ubiquitylation and binding assays mostly contain PY motifs. Although several substrates were recognized by both Nedd4-1 and Nedd4-2, others were specific to only one, with several Tyr kinases preferred by Nedd4-1 and some ion channels by Nedd4-2; this was subsequently validated in vivo. Accordingly, Nedd4-1 knockdown or knockout in cells led to sustained signalling via some of its substrate Tyr kinases (e.g. FGFR), suggesting Nedd4-1 suppresses their signalling. These results demonstrate the feasibility of identifying substrates and deciphering substrate specificity of mammalian E3 ligases.
cAMP-dependent Ras activation has been demonstrated in numerous cell types, particularly of neuronal (including melanoma cells) and endocrine origin, but the Ras activator involved has not been identified. In B16 melanoma cells, cAMP activates the Ras/Erk pathway, leading initially to stimulation but subsequently to long term (>24-h) inhibition of melanogenesis (dendrite extension and melanin production). Here we identify CNrasGEF as the Ras guanine nucleotide exchange factor (GEF) involved. We demonstrate that CNrasGEF is expressed endogenously in B16 melanoma cells and that cAMP-mediated activation of Ras and Erk1/2 in these cells can be augmented by CNrasGEF overexpression and reduced by its knockdown by RNA interference. Moreover, we show that CNras-GEF participates in the regulation of melanogenesis. Knockdown of CNrasGEF leads to increased dendrite extension and melanin production observed ϳ50 h after forskolin/isobutylmethylxanthine treatment, suggesting that CNrasGEF inhibits melanogenesis in the long term. Independently, we find that overexpression of CNras-GEF leads to apoptosis, whereas its knockdown by RNAi enhances cell proliferation, independent of cAMP. Collectively, these results suggest that CNrasGEF regulates melanogenesis but that it also has a distinct role in regulating cell proliferation/apoptosis. cAMP exhibits differential mitogenic effects in different cell types (1). In several endocrine, neuronal, or Schwann cells and in some 3T3 cells, cAMP promotes cell proliferation, often by activating Ras. For example, in thyroid cells, cAMP-stimulated mitogenesis following thyroid stimulating hormone binding to its receptor is mediated via Ras activation, independent of protein kinase A (2). Moreover, cAMP-dependent activation of Erk in NIH-3T3 cells was recently demonstrated to be carried out independently of Rap1-Epac and instead was proposed to be mediated via Ras activation (3). The cAMP-dependent Ras activator involved in these cases has not been identified. Recently, a pathway for cAMPdependent Ras/Erk activation (independent of protein kinase A or Rap1) has been proposed in melanoma cells (4).Melanocytes are specialized epidermal cells of neurocrest origin that synthesize melanin and are responsible for skin pigmentation and protection from UV radiation (5, 6). Melanoma cells are transformed melanocytes that give rise to a very aggressive skin cancer, melanoma. B16 mouse melanoma cells, particularly B16-F10, have been characterized extensively with regard to their tumorigenic and metastatic potential (e.g. Refs. 7-9) and the signaling pathways responsible for melanin production (5). Melanin synthesis takes place in intracellular organelles called melanosomes. Upon stimulation by UV radiation, several factors are released, including ␣-melanocyte-stimulating hormone (10), a strong melanogenic factor that acts by binding to the melanocortin receptor, MC1R. MC1R is a G protein-coupled receptor coupled to G␣ s , causing elevation of intracellular cAMP upon ligand binding. cAMP is a critical com...
Several scientists and science communicators have turned to the Internet to discuss science through weblogs (blogs). An interview with five science bloggers suggests that scientists and non-scientists alike benefit in various ways from reading and writing science blogs. What is a blog? According to blog-monitoring website Technorati, there are currently about 48 million blogs on the Internet (1). Blogs, short for weblogs, are websites that are frequently updated with short articles and often allow readers to respond by leaving a comment. Of the almost 50 million blogs out there, quite a lot serve as personal journals, and the ease with which they are updated is an invitation for some people to put their entire private life or work details online. As a result, there are cases in which people have been fired for blogging at work or about work (2,3). This is unfortunate, because when used right, blogs have the capacity to be a valuable part of a person's work. They are a perfect tool for informal interactive discussions by allowing the authors to post an article about any topic they want and having visitors interact through comments.
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