Comparison of substrate specificity of the ubiquitin ligases Nedd4 and Nedd4-2 using proteome arrays.

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 approximately 8200 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.

The guanine nucleotide exchange factor CNrasGEF regulates melanogenesis and cell survival in melanoma cells.

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 CNrasGEF participates in the regulation of melanogenesis. Knockdown of CNrasGEF leads to increased dendrite extension and melanin production observed approximately 50 h after forskolin/isobutylmethylxanthine treatment, suggesting that CNrasGEF inhibits melanogenesis in the long term. Independently, we find that overexpression of CNrasGEF 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.