The Small Ubiquitin-related Modifier SUMO-1 belongs to the ubiquitin (Ub) and ubiquitin-like (Ubl) protein family. SUMO conjugation occurs on specific lysine residues within protein targets, regulating pathways involved in differentiation, apoptosis, the cell cycle, and responses to stress by altering protein function through changes in activity, cellular localization, or by protecting substrates from ubiquitination 1,2 . Ub/Ubl conjugation occurs in sequential steps and requires the concerted action of E2 conjugating proteins and E3 ligases 1,2 . In addition to being a SUMO E3, the nucleoporin Nup358/RanBP2 localizes SUMO conjugated RanGAP1 to the cytoplasmic face of the nuclear pore complex (NPC) via interactions in a complex that also includes Ubc9, the SUMO E2 conjugating protein 3-6 . We present the 3.0 Å crystal structure of a four protein complex between Ubc9, a Nup358/ RanBP2 E3 ligase domain (IR1-M), and SUMO-1 conjugated to the C-terminal domain of RanGAP1. Structural insights, combined with biochemical and kinetic data using additional substrates, support a model whereby Nup358/RanBP2 acts as an E3 by binding both SUMO and Ubc9 to position the SUMO-E2-thioester in an optimal orientation to enhance conjugation.Ub/Ubls are activated by E1 and transferred to E2 to form E2-Ub/Ubl-thioesters. While competent for Ub/Ubl ligation to lysine ɛ-amino groups, E2s generally require E3 ligases to specifically recognize substrate lysine residues. Most E3s belong to either RING or HECT families and while RING E3s recruit substrate and bind the E2-Ub/Ubl via a zinc domain to promote conjugation to lysine residues 7 , HECT E3s recruit E2-Ub/Ubl to generate E3-Ub/Ublthioesters for conjugation 8 . The SUMO E2 can directly bind the consensus sequence Ψ-K-x-D/E where Ψ is hydrophobic, K is the substrate lysine, x is any amino acid, and D or E is acidic 9 , although several SUMO E3s facilitate conjugation in vivo and in vitro and include RING-type E3s 10,11 , Nup358/RanBP2 12 , and Pc2 13 . Nup358/RanBP2 and Pc2 appear unrelated to either RING or HECT E3 ligases.One of the first discovered functions for SUMO-1 was its role in nucleocytoplasmic trafficking 3-5 . SUMO conjugation localizes RanGAP1 to the NPC in a complex that includes the SUMO E2 Ubc9 and Nup358/RanBP2, a multi-domain 3,224 amino acid (aa) nucleoporin that also interacts with Ran and other nuclear transport factors 3-6,14,15 . The SUMORanGAP1-Ubc9-Nup358/RanBP2 complex does not dissociate upon entry into mitosis and NPC disassembly, but redistributes to kinetochores and contributes to the stability of kinetochore-microtubule interactions 16 . A ~30kDa Nup358/RanBP2 fragment named IR1-M-IR2 binds Ubc9 and promotes SUMO E3 activity in vitro and in vivo 12,17-19 , although domains flanking IR1-M-IR2 also contribute to SUMO-RanGAP1 interactions 6 . IR1-M-IR2 was parsed into three elements: IR1 (aa To determine the molecular details of this system, SUMO-1 was conjugated to RanGAP1, combined with Ubc9 and Nup358/RanBP2, purified by gel filtration, and cry...

SUMMARY RASSF1A is a tumor suppressor gene that is epigenetically silenced in a wide variety of sporadic human malignancies. Expression of alternative RASSF1 isoforms cannot substitute for RASSF1A-promoted cell-cycle arrest and apoptosis. Apoptosis can be driven by either activating Bax or by activation of MST kinases. The Raf1 proto-oncogene binds to MST2, preventing its activation and proapoptotic signaling. Here we show that key steps in RASSF1A-induced apoptosis are the disruption of the inhibitory Raf1-MST2 complex by RASSF1A and the concomitant enhancement of MST2 interaction with its substrate, LATS1. Subsequently, RASSF1A-activated LATS1 phosphorylates and releases the transcriptional regulator YAP1, allowing YAP1 to translocate to the nucleus and associate with p73, resulting in transcription of the proapoptotic target gene puma. Our results describe an MST2-dependent effector pathway for RASSF1A proapoptotic signaling and indicate that silencing of RASSF1A in tumors removes a proapoptotic signal emanating from p73.

First identified in the early 1980s as retroviral oncogenes, the Raf proteins have been the objects of intense research. The discoveries 10 years later that the Raf family members (Raf-1, B-Raf, and A-Raf) are bona fide Ras effectors and upstream activators of the ubiquitous ERK pathway increased the interest in these proteins primarily because of the central role that this cascade plays in cancer development. The important role of Raf in cancer was corroborated in 2002 with the discovery of B-Raf genetic mutations in a large number of tumors. This led to intensified drug development efforts to target Raf signaling in cancer. This work yielded not only recent clinical successes but also surprising insights into the regulation of Raf proteins by homodimerization and heterodimerization. Surprising insights also came from the hunt for new Raf targets. Although MEK remains the only widely accepted Raf substrate, new kinase-independent roles for Raf proteins have emerged. These include the regulation of apoptosis by suppressing the activity of the proapoptotic kinases, ASK1 and MST2, and the regulation of cell motility and differentiation by controlling the activity of Rok-α. In this review, we discuss the regulation of Raf proteins and their role in cancer, with special focus on the interacting proteins that modulate Raf signaling. We also describe the new pathways controlled by Raf proteins and summarize the successes and failures in the development of efficient anticancer therapies targeting Raf. Finally, we also argue for the necessity of more systemic approaches to obtain a better understanding of how the Ras-Raf signaling network generates biological specificity.

Modification of cellular proteins by the ubiquitin-like protein SUMO is essential for nuclear metabolism and cell cycle progression in yeast. X-ray structures of the human Senp2 catalytic protease domain and of a covalent thiohemiacetal transition-state complex obtained between the Senp2 catalytic domain and SUMO-1 revealed details of the respective protease and substrate surfaces utilized in interactions between these two proteins. Comparative biochemical and structural analysis between Senp2 and the yeast SUMO protease Ulp1 revealed differential abilities to process SUMO-1, SUMO-2, and SUMO-3 in maturation and deconjugation reactions. Further biochemical characterization of the three SUMO isoforms into which an additional Gly-Gly di-peptide was inserted, or whereby the respective SUMO tails from the three isoforms were swapped, suggests a strict dependence for SUMO isopeptidase activity on residues C-terminal to the conserved Gly-Gly motif and preferred cleavage site for SUMO proteases.

Signal transduction requires the coordination of activities between different pathways. In mammalian cells, Raf-1 regulates the MST-LATS and MEK-ERK pathways. We found that a complex circuitry of competing protein interactions coordinates the crosstalk between the ERK and MST pathways. Combining mathematical modelling and experimental validation we show that competing protein interactions can cause steep signalling switches through phosphorylation-induced changes in binding affinities. These include Akt phosphorylation of MST2 and a feedback phosphorylation of Raf-1 Ser 259 by LATS1, which enables Raf-1 to suppress both MST2 and MEK signalling. Mutation of Raf-1 Ser 259 stimulates both pathways, simultaneously driving apoptosis and proliferation, whereas concomitant MST2 downregulation switches signalling to cell proliferation, transformation and survival. Thus, competing protein interactions provide a versatile regulatory mechanism for signal distribution through the dynamic integration of graded signals into switch-like responses.