ESCRT complexes form the main machinery driving protein sorting from endosomes to lysosomes. Currently, the picture regarding assembly of ESCRTs on endosomes is incomplete. The structure of the conserved heterotrimeric ESCRT-I core presented here shows a fan-like arrangement of three helical hairpins, each corresponding to a different subunit. Vps23/Tsg101 is the central hairpin sandwiched between the other subunits, explaining the critical role of its "steadiness box" in the stability of ESCRT-I. We show that yeast ESCRT-I links directly to ESCRT-II, through a tight interaction of Vps28 (ESCRT-I) with the yeast-specific zinc-finger insertion within the GLUE domain of Vps36 (ESCRT-II). The crystal structure of the GLUE domain missing this insertion reveals it is a split PH domain, with a noncanonical lipid binding pocket that binds PtdIns3P. The simultaneous and reinforcing interactions of ESCRT-II GLUE domain with membranes, ESCRT-I, and ubiquitin are critical for ubiquitinated cargo progression from early to late endosomes.

We describe a genome-wide gain-of-function screen for regulators of NF-kappaB, and identify Rap1 (Trf2IP), as an essential modulator of NF-kappaB-mediated pathways. NF-kappaB is induced by ectopic expression of Rap1, whereas its activity is inhibited by Rap1 depletion. In addition to localizing on telomeres, mammalian Rap1 forms a complex with IKKs (IkappaB kinases), and is crucial for the ability of IKKs to be recruited to, and phosphorylate, the p65 subunit of NF-kappaB to make it transcriptionally competent. Rap1-mutant mice display defective NF-kappaB activation and are resistant to endotoxic shock. Furthermore, levels of Rap1 are positively regulated by NF-kappaB, and human breast cancers with NF-kappaB hyperactivity show elevated levels of cytoplasmic Rap1. Similar to inhibiting NF-kappaB, knockdown of Rap1 sensitizes breast cancer cells to apoptosis. These results identify the first cytoplasmic role of Rap1 and provide a mechanism through which it regulates an important signalling cascade in mammals, independent of its ability to regulate telomere function.

The structure confirms the importance of the HLH and leucine zipper motifs in dimerization as well as the mode of E box recognition which was previously analyzed by X-ray crystallography of shortened constructs. The disorder observed in the C-terminal domain suggests that contacts with additional protein components of the transcription machinery are necessary for ordering the secondary structure. The tetramers seen in the crystal are consistent with the tendency of Max and other bHLHZ and HLH proteins to form higher order oligomers in solution and may play a role in DNA looping. The location of the two phosphorylation sites at Ser1 and Ser10 (the latter is the N-cap of the basic helix) suggests how phosphorylation could disrupt DNA binding.

Post-translational modifications of NF-kappaB through phosphorylations enhance its transactivation potential. Much is known about the kinases that phosphorylate NF-kappaB, but little is known about the phosphatases that dephosphorylate it. By using a genome-scale siRNA screen, we identified the WIP1 phosphatase as a negative regulator of NF-kappaB signalling. WIP1-mediated regulation of NF-kappaB occurs in both a p38-dependent and independent manner. Overexpression of WIP1 resulted in decreased NF-kappaB activation in a dose-dependent manner, whereas WIP1 knockdown resulted in increased NF-kappaB function. We show that WIP1 is a direct phosphatase of Ser 536 of the p65 subunit of NF-kappaB. Phosphorylation of Ser 536 is known to be essential for the transactivation function of p65, as it is required for recruitment of the transcriptional co-activator p300. WIP1-mediated regulation of p65 regulated binding of NF-kappaB to p300 and hence chromatin remodelling. Consistent with our results, mice lacking WIP1 showed enhanced inflammation. These results provide the first genetic proof that a phosphatase directly regulates NF-kappaB signalling in vivo.