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Author(s): Ning Zheng [1, 2]; Brenda A. Schulman [1, 3]; Langzhou Song [1, 2]; Julie J. Miller [1, 2]; Philip D. Jeffrey [1]; Ping Wang [1, 2]; Claire Chu [3]; [...]

Previous studies have found that cyclins and cyclin-dependent kinases (Cdks) perform a vital role in regulating cell cycle transitions. It was also observed that cyclins and Cdks collaborate with ubiquitin-mediated proteolysis to create a logical framework for cell cycle regulation. Furthermore, researches have found that two E3 complexes are responsible for regulating proteolysis. These include the cyclosome/anaphase=promoting complex and the SCF complex.

HIV-1 Vpr is similar to importin-B in the function in performs during HIV-1 nuclear import. This function is an essential part of the Vpr role in macrophage infection although not crucial for G2 arrest. The two functions of Vpr are genetically separable. When Vpr does not localize to the nuclear envelope HIV-1 cannot infect macrophages effectively.

Research has shown macromolecular movement across the nuclear envelope to be bidirectional, with proteins and RNAs being trafficked through nuclear pore complexes approximately 30 times the size of a ribosome. Of the various processes in nuclear transport, more is known about nuclear protein import than nuclear export. But in the light of several recent discoveries, it is now possible to propose a general mechanism for nuclear transport through the nuclear pore.

The bacteriophage λ cI protein (λcI), which is both a repressor and an activator of transcription (1), binds as a dimer to six 17-base pair (bp) operators on the phage [...], Two heterologous prokaryotic activators, the bacteriophage λ cI protein (λcl) and the Escherichia coli cyclic AMP receptor protein (CRP), were shown to activate transcription synergistically from an artificial promoter bearing binding sites for both proteins. The synergy depends on a functional activation (positive control) surface on each activator. These results imply that both proteins interact directly with RNA polymerase and thus suggest a precise mechanism for transcriptional synergy: the interaction of two activators with two distinct surfaces of RNA polymerase.

Abstract Maintenance of genomic integrity is important for cellular viability and proliferation. During DNA replication, cells respond to replication stress by activating checkpoint pathways that stabilize replication forks and prevent cell cycle progression. The Saccharomyces cerevisiae F-box protein Dia2 is a ubiquitin ligase component required for genomic stability and may help replication complexes negotiate damaged DNA or natural fragile sites. We recently implicated Dia2 in the replication stress response. We demonstrated that Dia2 is targeted for ubiquitin-mediated proteolysis and that activation of the S-phase checkpoint inhibits Dia2 protein turnover. S-phase checkpoint mutants fail to stabilize the Dia2 protein and checkpoint mutants that lack Dia2 exhibit increased sensitivity to replication stress. We also showed that Dia2 protein turnover is not the result of an autocatalytic mechanism. Instead, an N-terminal 20 amino acid motif that is also required for nuclear localization is necessary for Dia2 proteolysis. Dia2 mutants lacking this motif but modified with an exogenous strong nuclear localization signal are both nuclear and stable and disrupt cell cycle dynamics. In summary, our studies suggest that inhibition of Dia2 proteolysis is a novel target of the S-phase checkpoint. We think that this work will help to identify the mechanisms that function downstream of checkpoint activation and that intersect with cell cycle control pathways.

Control of cellular proliferation is critical to cell viability. The F-box protein Fbw7 (hAgo/hCdc4/FBXW7) functions as a specificity factor for the Skp1-Cul1-F-box protein (SCF) ubiquitin ligase complex and targets several proteins required for cellular proliferation for ubiquitin-mediated destruction. Fbw7 exists as three splice variants but the mechanistic role of each is not entirely clear. We examined the regulation of the Fbw7-ϒ isoform, which has been implicated in the degradation of c-Myc. We show here that Fbw7-ϒ is an unstable protein and that its turnover is proteasome-dependent in transformed cells. Using a two-hybrid screen, we identified a novel interaction partner, SLP-1, which binds the N-terminal domain of Fbw7-ϒ. Overexpression of SLP-1 inhibits the degradation of Fbw7-ϒ, suggesting that this interaction can happen in vivo. When Fbw7-ϒ is stabilized by overexpression of SLP-1, c-Myc protein abundance decreases, suggesting that the SCF Fbw7-ϒ complex maintains activity. We demonstrate that Cdk2 also binds the N-terminal domain of Fbw7-ϒ as well as SLP-1. Interestingly, co-expression of Cdk2 and SLP-1 does not inhibit Fbw7-ϒ degradation, suggesting that Cdk2 and SLP-1 may have opposing functions. [ABSTRACT FROM AUTHOR]

Studies of brain-specific kinase 2 (BRSK2), an AMP-activated protein kinase (AMPK)-related kinase, and its homologs suggest that they are multifunctional regulators of cell-cycle progression. BRSK2, which contains a ubiquitin-associated (UBA) domain, is polyubiquitinated in cells. However, the regulatory mechanisms and exact biological function of BRSK2 remain unclear. Herein, we show that BRSK2 co-localizes with the centrosomes during mitosis. We also demonstrate that BRSK2 protein levels fluctuate during the cell cycle, peaking during mitosis and declining in G1 phase. Furthermore, Cdh1, rather than Cdc20, promotes the degradation of BRSK2 in vivo. Consistent with this finding, knock-down of endogenous Cdh1 blocks BRSK2 degradation during the G1 phase. The conserved KEN box of BRSK2 is required for anaphase-promoting complex/cyclosome-Cdh1 (APC/CCdh1)-dependent degradation. Additionally, overexpression of either BRSK2(WT) or BRSK2(ΔKEN) increases the percentage of cells in G2/M. Thus, our results provide the first evidence that BRSK2 regulates cell-cycle progression controlled by APC/CCdh1 through the ubiquitin-proteasome pathway. [ABSTRACT FROM AUTHOR]

The COP9 signalosome (CSN) is a eukaryotic protein complex, which regulates a wide range of biological processes mainly through modulating the cullin ubiquitin E3 ligases in the ubiquitin-proteasome pathway. The CSN possesses a highly conserved deneddylase activity that centers at the JAMM motif of the Csn5 subunit but requires other subunits in a complex assembly. The classic CSN is composed of 8 subunits (Csn1-8), yet in several Ascomycota, the complex is smaller and lacks orthologs for a few CSN subunits, but nevertheless contains a conserved Csn5. This feature makes yeast a powerful model to determine the minimal assemblage required for deneddylation activity. Here we report, that Csi1, a diverged S. cerevisiae CSN subunit, displays significant homology with the carboxyl terminal domain of the canonical Csn6, but lacks the amino terminal MPN- domain. Through the comparative and experimental analyses of the budding yeast and the mammalian CSNs, we demonstrate that the MPN- domain of the canonical mouse Csn6 is not part of the CSN deneddylase core. We also show that the carboxyl domain of Csn6 has an indispensable role in maintaining the integrity of the CSN complex. The CSN complex assembled with the carboxyl fragment of Csn6, despite its lack of an MPN2 domain, is fully active in deneddylation of cullins. We propose that the budding yeast Csi1 is a functional equivalent of the canonical Csn6, and thus the composition of the CSN across phyla is more conserved than hitherto appreciated. [ABSTRACT FROM AUTHOR]

Swi1 and Swi3 form the replication fork protection complex and play critical roles in proper activation of the replication checkpoint and stabilization of replication forks in the fission yeast Schizosaccharomyces pombe. However, the mechanisms by which the Swi1-Swi3 complex regulates these processes are not well understood. Here, we report functional analyses of the Swi1-Swi3 complex in fission yeast. Swi1 possesses the DDT domain, a putative DNA binding domain found in a variety of chromatin remodeling factors. Consistently, the DDT domain-containing region of Swi1 interacts with DNA in vitro, and mutations in the DDT domain eliminate the association of Swi1 with chromatin in S. pombe cells. DDT domain mutations also render cells highly sensitive to S-phase stressing agents and induce strong accumulation of Rad22- DNA repair foci, indicating that the DDT domain is involved in the activity of the Swi1-Swi3 complex. Interestingly, DDT domain mutations also abolish Swi1's ability to interact with Swi3 in cells. Furthermore, we show that Swi1 is required for efficient chromatin association of Swi3 and that the Swi1 C- terminal domain directly interacts with Swi3. These results indicate that Swi1 associates with chromatin through its DDT domain and recruits Swi3 to function together as the replication fork protection complex. [ABSTRACT FROM AUTHOR]

SCF complexes are the largest family of E3 ubiquitin?protein ligases and mediate the ubiquitination of diverse regulatory and signalling proteins. Here we present the crystal structure of the Cul1?Rbx1?Skp1?F boxSkp2 SCF complex, which shows that Cul1 is an elongated protein that consists of a long stalk and a globular domain. The globular domain binds the RING finger protein Rbx1 through an intermolecular β-sheet, forming a two-subunit catalytic core that recruits the ubiquitin-conjugating enzyme. The long stalk, which consists of three repeats of a novel five-helix motif, binds the Skp1?F boxSkp2 protein substrate-recognition complex at its tip. Cul1 serves as a rigid scaffold that organizes the Skp1?F boxSkp2 and Rbx1 subunits, holding them over 100 Å apart. The structure suggests that Cul1 may contribute to catalysis through the positioning of the substrate and the ubiquitin-conjugating enzyme, and this model is supported by Cul1 mutations designed to eliminate the rigidity of the scaffold. [ABSTRACT FROM AUTHOR]

Presents findings of a study in which phosphorylated Cln1 was ubiquitinated by SCF complexes containing the F-box protein Grr1, Rbx1, and the E2Cdc34. How Rbx1 promotes association of Cdc34 with Cdc53 and stimulates Cdc34 auto-ubiquitination in the context of Cdc53 or SCF complexes.

MAP kinase signaling modules serve to transduce extracellular signals to the nucleus of eukaryotic cells, but little is known about how signals cross the nuclear envelope. Exposure of yeast cells to increases in extra-cellular osmolarity activates the HOG1 MAP kinase cascade, which is composed of three tiers of protein kinases, namely the SSK2, SSK22 and STE11 MAP-KKKs, the PBS2 MAPKK, and the HOG1 MAPK. Using green fluorescent protein (GFP) fusions of these kinases, we found that HOG1, PBS2 and STE11 localize to the cytoplasm of unstressed cells. Following osmotic stress, HOG1, but neither PBS2 nor STE11, translocates into the nucleus. HOG1 translocation occurs very rapidly, is transient, and correlates with the phosphorylation and activation of the MAP kinase by its MAPKK. HOG1 phosphorylation is necessary and sufficient for nuclear translocation, because a catalytically inactive kinase when phosphorylated is translocated to the nucleus as efficiently as the wild-type. Nuclear import of the MAPK under stress conditions requires the activity of the small GTP binding protein Ran­GSP1, but not the NLS-binding importin α/β heterodimer. Rather, HOG1 import requires the activity of a gene, NMD5, that encodes a novel importin β homolog. Similarly, export of dephosphorylated HOG1 from the nucleus requires the activity of the NES receptor XPO1/CRM1. Our findings define the requirements for the regulated nuclear transport of a stress-activated MAP kinase. [ABSTRACT FROM AUTHOR]

Cell division is controlled by a highly regulated program to accurately duplicate and segregate chromosomes. An important feature of the cell cycle regulatory program is that key cell cycle proteins are present and active during specific cell cycle stages but are later removed or inhibited to maintain appropriate timing. The ubiquitin-proteasome system has emerged as an important mechanism to target cell cycle proteins for degradation at critical junctures during cell division. Two key E3 ubiquitin ligase complexes that target key cell cycle proteins are the Skp1-Cul1-F-box protein complex and the anaphase-promoting complex/cyclosome. This chapter focuses on the role of these E3 ubiquitin ligases and how ubiquitin-dependent degradation of central cell cycle regulatory proteins advances the cell cycle.

Cell cycle progression is tightly regulated to prevent uncontrolled growth and division. Specific cell cycle factors are responsible for driving the cell from one cell cycle stage to the next. Many of these proteins are targeted for degradation by the ubiquitin proteasome system when their function is no longer required or may disrupt cell cycle progression. Here we describe a series of experiments used to study the ubiquitin-mediated degradation of cell cycle proteins. This collection of assays may be used to determine the requirement for individual proteins in the degradation and ubiquitination of cell cycle proteins in Saccharomyces cerevisiae.