Multiple molecular lesions in human cancers directly collaborate to deregulate proliferation and suppress apoptosis to promote tumorigenesis. The candidate tumor suppressor RASSF1A is commonly inactivated in a broad spectrum of human tumors and has been implicated as a pivotal gatekeeper of cell cycle progression. However, a mechanistic account of the role of RASSF1A gene inactivation in tumor initiation is lacking. Here we have employed loss-of-function analysis in human epithelial cells for a detailed investigation of the contribution of RASSF1 to cell cycle progression. We found that RASSF1A has dual opposing regulatory connections to G(1)/S phase cell cycle transit. RASSF1A associates with the Ewing sarcoma breakpoint protein, EWS, to limit accumulation of cyclin D1 and restrict exit from G(1). Surprisingly, we found that RASSF1A is also required to restrict SCF(betaTrCP) activity to allow G/S phase transition. This restriction is required for accumulation of the anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 and the concomitant block of APC/C-dependent cyclin A turnover. The consequence of this relationship is inhibition of cell cycle progression in normal epithelial cells upon RASSF1A depletion despite elevated cyclin D1 concentrations. Progression to tumorigenicity upon RASSF1A gene inactivation should therefore require collaborating genetic aberrations that bypass the consequences of impaired APC/C regulation at the G(1)/S phase cell cycle transition.

Protein arginine methylation is a eukaryotic posttranslational modification that plays a role in transcription, mRNA splicing and transport, in protein-protein interaction, and cell signaling. The type I protein arginine methyltransferase (PRMT) 8 is the only member of the human PRMT family that is localized at the cell membrane and its endogenous substrates have remained unknown as yet. Although PRMT8 was supposed to be expressed only in brain tissue, its presence in HEK 293 (T) cells could be demonstrated. We identified more than 20 PRMT8-binding partners in pull-down experiments using recombinant PRMT8 as bait followed by mass spectrometric identification of the bound proteins. Among the extracted proteins were several heterogeneous nuclear ribonucleoproteins (hnRNP), RNA-helicases (DEAD box proteins), the TET-family proteins TLS, Ewing's sarcoma (EWS), and TAF(II)68, and caprin, which all contain RGG methylation motifs and are potential substrates of PRMT8. Additionally, actin, tubulin, and heat shock proteins belong to the identified proteins. The interaction between PRMT8 and the EWS protein was characterized in more detail. Although binding of endogenous and recombinant EWS protein to PRMT8 as well as colocalization in HEK cells was observed, in vitro methylation assays revealed a rather poor methyltransferase activity of PRMT8 towards the EWS protein and a synthetic RGG-rich reference peptide (K(m), 1.3 microM; k(cat)/K(m), 2.8 x 10(-4) microM(-1) s(-1)) in comparison to PRMT1 (K(m), 0.8 microM; k(cat)/K(m), 8.1 x 10(-3) microM(-1) s(-1)). In contrast, substrate proteins within a cell extract could be methylated by PRMT8 as efficient as by PRMT1. The main interaction site of the EWS protein with PRMT8 was determined to be the C-terminal RGG box 3. Remarkably, complete methylation of the EWS protein did not abrogate the binding to PRMT8, pointing to an adapter role of PRMT8 for nuclear proteins at the cell membrane in addition to its methyltransferase activity.

Systematic mapping of protein-protein interactions, or 'interactome' mapping, was initiated in model organisms, starting with defined biological processes and then expanding to the scale of the proteome. Although far from complete, such maps have revealed global topological and dynamic features of interactome networks that relate to known biological properties, suggesting that a human interactome map will provide insight into development and disease mechanisms at a systems level. Here we describe an initial version of a proteome-scale map of human binary protein-protein interactions. Using a stringent, high-throughput yeast two-hybrid system, we tested pairwise interactions among the products of approximately 8,100 currently available Gateway-cloned open reading frames and detected approximately 2,800 interactions. This data set, called CCSB-HI1, has a verification rate of approximately 78% as revealed by an independent co-affinity purification assay, and correlates significantly with other biological attributes. The CCSB-HI1 data set increases by approximately 70% the set of available binary interactions within the tested space and reveals more than 300 new connections to over 100 disease-associated proteins. This work represents an important step towards a systematic and comprehensive human interactome project.