Impairment of ribosomal biogenesis can activate the p53 protein independently of DNA damage. The ability of ribosomal proteins L5, L11, L23, L26, or S7 to bind Mdm2 and inhibit its ubiquitin ligase activity has been suggested as a critical step in p53 activation under these conditions. Here, we report that L5 and L11 are particularly important for this response. Whereas several other newly synthesized ribosomal proteins are degraded by proteasomes upon inhibition of Pol I activity by actinomycin D, L5 and L11 accumulate in the ribosome-free fraction where they bind to Mdm2. This selective accumulation of free L5 and L11 is due to their mutual protection from proteasomal degradation. Furthermore, the endogenous, newly synthesized L5 and L11 continue to be imported into nucleoli even after nucleolar disruption and colocalize with Mdm2, p53, and promyelocytic leukemia protein. This suggests that the disrupted nucleoli may provide a platform for L5-and L11-dependent p53 activation, implying a role for the nucleolus in p53 activation by ribosomal biogenesis stress. These findings may have important implications with respect to understanding the pathogenesis of diseases caused by impaired ribosome biogenesis.proteasome | ribosomal stress

SignificanceMacropinocytosis and phagocytosis are two Ras-regulated, highly related processes of great physiological relevance collectively termed large-scale endocytosis. Both are actin-driven and entail engulfment of extracellular material by crown-like protrusions. Aside from the Arp2/3 complex, which serves as the main nucleator of branched actin filaments at the cup rim, the underlying mechanisms of actin assembly still remain elusive. Here, we analyzed the role of Diaphanous-related formin G (ForG) from Dictyostelium by biochemical, genetic, and imaging techniques. Our data demonstrate that this formin exhibits a rather weak nucleation activity and imply that ForG-mediated filament elongation synergizes with the Arp2/3 complex in actin assembly. Finally, we identify ForG as a Ras-regulated formin and show its significance for actin assembly in endocytic structures.

SummaryRac proteins are the only canonical Rho family GTPases in Dictyostelium, where they act as key regulators of the actin cytoskeleton. To monitor the dynamics of activated Rac1 in Dictyostelium cells, a fluorescent probe was developed that specifically binds to the GTPbound form of Rac1. The probe is based on the GTPase-binding domain (GBD) from PAK1 kinase, and was selected on the basis of yeast two-hybrid, GST pull-down and fluorescence resonance energy transfer assays. The PAK1 GBD localizes to leading edges of migrating cells and to endocytotic cups. Similarly to its role in vertebrates, activated Rac1 therefore appears to control de novo actin polymerization at protruding regions of the Dictyostelium cell. Additionally, we found that the IQGAP-related protein DGAP1, which sequesters active Rac1 into a quaternary complex with actin-binding proteins cortexillin I and cortexillin II, localizes to the trailing regions of migrating cells. Notably, PAK1 GBD and DGAP1, which both bind to Rac1-GTP, display mutually exclusive localizations in cell migration, phagocytosis and cytokinesis, and opposite dynamics of recruitment to the cell cortex upon stimulation with chemoattractants. Moreover, cortical localization of the PAK1 GBD depends on the integrity of the actin cytoskeleton, whereas cortical localization of DGAP1 does not. Taken together, these results imply that Rac1 GTPases play a dual role in regulation of cell motility and polarity in Dictyostelium.

Macropinocytosis and phagocytosis are evolutionarily conserved forms of bulk endocytosis used by cells to ingest large volumes of fluid and solid particles, respectively. Both processes are regulated by Ras signaling, which is precisely controlled by mechanisms involving Ras GTPase activating proteins (RasGAPs) responsible for terminating Ras activity on early endosomes. While regulation of Ras signaling during large-scale endocytosis in WT Dictyostelium has been, for the most part, attributed to the Dictyostelium ortholog of human RasGAP NF1, in commonly used axenic laboratory strains, this gene is mutated and inactive. Moreover, none of the RasGAPs characterized so far have been implicated in the regulation of Ras signaling in large-scale endocytosis in axenic strains. In this study, we establish, using biochemical approaches and complementation assays in live cells, that Dictyostelium IQGAP-related protein IqgC interacts with active RasG and exhibits RasGAP activity toward this GTPase. Analyses of iqgC− and IqgC-overexpressing cells further revealed participation of this GAP in the regulation of both types of large-scale endocytosis and in cytokinesis. Moreover, given the localization of IqgC to phagosomes and, most prominently, to macropinosomes, we propose IqgC acting as a RasG-specific GAP in large-scale endocytosis. The data presented here functionally distinguish IqgC from other members of the Dictyostelium IQGAP family and call for repositioning of this genuine RasGAP outside of the IQGAP group.

Cancer-causing human papillomavirus (HPV) E6 oncoproteins have a class I PDZ-binding motif (PBM) on their C termini, which play critical roles that are related to the HPV life cycle and HPV-induced malignancies. E6 oncoproteins use these PBMs to interact with, to target for proteasome-mediated degradation, a plethora of cellular substrates that contain PDZ domains and that are involved in the regulation of various cellular pathways. In this study, we show that both HPV-16 and HPV-18 E6 oncoproteins can interact with Na+/H+ exchange regulatory factor 2 (NHERF-2), a PDZ domain-containing protein, which among other cellular functions also behaves as a tumor suppressor regulating endothelial proliferation. The interaction between the E6 oncoproteins and NHERF-2 is PBM dependent and results in proteasome-mediated degradation of NHERF-2. We further confirmed this effect in cells derived from HPV-16- and HPV-18-positive cervical tumors, where we show that NHERF-2 protein turnover is increased in the presence of E6. Finally, our data indicate that E6-mediated NHERF-2 degradation results in p27 downregulation and cyclin D1 upregulation, leading to accelerated cellular proliferation. To our knowledge, this is the first report to demonstrate that E6 oncoproteins can stimulate cell proliferation by indirectly regulating p27 through targeting a PDZ domain-containing protein. IMPORTANCE This study links HPV-16 and HPV-18 E6 oncoproteins to the modulation of cellular proliferation. The PDZ domain-containing protein NHERF-2 is a tumor suppressor that has been shown to regulate endothelial proliferation; here, we demonstrate that NHERF-2 is targeted by HPV E6 for proteasome-mediated degradation. Interestingly, this indirectly affects p27, cyclin D1, and CDK4 protein levels and, consequently, affects cell proliferation. Hence, this study provides information that will improve our understanding of the molecular basis for HPV E6 function, and it also highlights the importance of the PDZ domain-containing protein NHERF-2 and its tumor-suppressive role in regulating cell proliferation.