Deanna M. Koepp

Structure of the Cul1-Rbx1-Skp1-F boxSkp2 SCF ubiquitin ligase complex.

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.

How the Cyclin Became a Cyclin: Regulated Proteolysis in the Cell Cycle

As is readily apparent, proteolysis plays a tremendously important and varied role in cell cycle regulation. While the SCF and APC are key regulators, other proteolytic systems may also contribute to cell cycle control. A well-documented case is the stabilization of p53 in response to DNA damage. In addition, a novel proteolyic system cabable of cleaving cyclin A was recently found to be activated by the presence of the CKI p27 in cell extracts (Bastians et al. 1998xBastians, H, Townsley, F.M, and Ruderman, J.V. Proc. Natl. Acad. Sci. USA. 1998; 95: 15374–15381Crossref | PubMed | Scopus (26)See all ReferencesBastians et al. 1998). Furthermore, the regulation of protein stability at the proteolytic step rather than the ubiquitination step is an interesting and unexplored avenue for cell cycle control. A possible example of this may come from the recent observation that the Cdk subunit Cks1 (Suc1 in S. pombe) associates with the proteasome and may regulate it (Kaiser et al. 1999xKaiser, P, Moncollin, V, Clarke, D.J, Watson, M.H, Bertolaet, B.L, Reed, S.I, and Bailly, E. Genes Dev. 1999; in pressSee all ReferencesKaiser et al. 1999). The significance of these new discoveries is not yet clear but they are certain to keep cell cycle research on the cutting edge.§To whom correspondence should be addressed (e-mail: selledge@bcm.tmc.edu).

REGULATED NUCLEO/CYTOPLASMIC EXCHANGE OF HOG1 MAPK REQUIRES THE IMPORTIN BETA HOMOLOGS NMD5 AND XPO1

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 extracellular osmolarity activates the HOG1 MAP kinase cascade, which is composed of three tiers of protein kinases, namely the SSK2, SSK22 and STE11 MAPKKKs, 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 Ran–GSP1, 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.

A family of mammalian F-box proteins

Ubiquitin-mediated destruction of regulatory proteins is a frequent means of controlling progression through signaling pathways [1]. F-box proteins [2] are components of modular E3 ubiquitin protein ligases called SCFs, which function in phosphorylation-dependent ubiquitination ([3] [4] [5], reviewed in [6] [7]). F-box proteins contain a carboxy-terminal domain that interacts with substrates and a 42-48 amino-acid F-box motif which binds to the protein Skp1 [2] [3] [4]. Skp1 binding links the F-box protein with a core ubiquitin ligase composed of the proteins Cdc53/Cul1, Rbx1 (also called Hrt1 and Roc1) and the E2 ubiquitin-conjugating enzyme Cdc34 [8] [9] [10] [11]. The genomes of the budding yeast Saccharomyces cerevisiae and the nematode worm Caenorhabditis elegans contain, respectively, 16 and more than 60 F-box proteins [2] [7]; in S. cerevisiae, the F-box proteins Cdc4, Grr1 and Met30 target cyclin-dependent kinase inhibitors, G1 cyclins and transcriptional regulators for ubiquitination ([3] [4] [5] [8] [10], reviewed in [6] [7]). Only four mammalian F-box proteins (Cyclin F, Skp1, beta-TRCP and NFB42) have been identified so far [2] [12]. Here, we report the identification of a family of 33 novel mammalian F-box proteins. The large number of these proteins in mammals suggests that the SCF system controls a correspondingly large number of regulatory pathways in vertebrates. Four of these proteins contain a novel conserved motif, the F-box-associated (FBA) domain, which may represent a new protein-protein interaction motif. The identification of these genes will help uncover pathways controlled by ubiquitin-mediated proteolysis in mammals.

A GTPase controlling nuclear trafficking: running the right way or walking RANdomly?

The entry of transport factors into the nucleus during nuclear protein import provokes two questions: how do the transport factors exit the nucleus, and do import factors play a role in RNA export as well? Gorlich et al. (1996 [this issue of Cell]) now present data implicating importin α directly in export of snRNAs. They find that a significant fraction of yeast importin α associates with the cap binding complex (CBC), a multicomponent structure which shuttles between the nucleus and the cytoplasm while directing the export of capped U snRNAs. While it is not surprising that importin α should bind to a protein targeted to the nucleus, it is unusual that importin α does not dissociate from CBC upon nuclear entry. Even more intriguing is the observation that importin β can dissociate RNA from the importin α–CBC complex. This result leads Gorlich et al. 1996xGorlich, D, Kraft, R, Kostka, S, Vogel, F, Hartmann, E, Laskey, R.A, Mattaj, I.W, and Izaurralde, E. Cell. 1996; 87Abstract | Full Text | Full Text PDF | PubMed | Scopus (150)See all ReferencesGorlich et al. 1996 to suggest a model (shown in Figure 8 of their paper) in which a cytoplasmic complex of importin α and β function in the import of CBC and a nuclear complex of importin α and CBC function in the export of snRNA. An alternative interpretation is that importin α “hitches a ride” with CBC to exit the nucleus and plays no direct role in snRNA export. Furthermore, export of importin α in complex with CBC cannot be the only means of exiting the nucleus for importin α, as the absence of CBC is not lethal, at least for the yeast cell. In addition, an untested prediction of this model is that mutant alleles of importin α should exhibit defects in the export of capped U snRNAs.The export of mRNA from the nucleus may proceed by a similar pathway that is, in principle, the reciprocal of the protein import pathway. Numerous RNA binding proteins, such as some hnRNPs, have been shown to shuttle between the nucleus and the cytoplasm (seeIzaurralde and Mattaj 1995xIzaurralde, E and Mattaj, I.W. Cell. 1995; 81: 153–159Abstract | Full Text PDF | PubMed | Scopus (201)See all ReferencesIzaurralde and Mattaj 1995, for review). As such, these shuttling RNA-binding proteins have been proposed to act as carriers of mRNA out of the nucleus. Once in the cytoplasm, the mRNA dissociates from its carrier, perhaps by replacement with cytoplasmic RNA-binding proteins or recruitment into ribosomes. The carrier proteins would then reenter the nucleus for additional rounds of export. Support for such a mechanism comes from experiments with the yeast shuttling protein Npl3p; mutations in Npl3p block both mRNA export and export of Npl3p from the nucleus. Similarly, export of Npl3p depends on ongoing RNA synthesis (Lee et al. 1996xLee, M.S, Henry, M, and Silver, P.A. Genes Dev. 1996; 10: 1233–1246Crossref | PubMedSee all ReferencesLee et al. 1996). Taken together, Npl3p and similar proteins, such as hnRNPA1 and the HIV Rev protein, exhibit characteristics expected for RNA transporters. It remains to be shown whether or not these proteins, like CBC, are coupled to importins for export. However, the observation that the nucleotide bound state of Ran causes defects in both mRNA and importin α export provides an important link.Implicit in these proposed models is the notion that some or all of the “import” factors must cycle between the nucleus and the cytoplasm. The molecular details of the export of transport factors themselves are not clear. Nevertheless, we can speculate about the export process using the principles already discussed (Figure 3Figure 3). Once inside the nucleus, importin α must dissociate from the NLS-bearing substrate, which may be accomplished by competition with RNA-binding proteins. Ran may move out of the nucleus as a complex of Ran-GTP–importin β. Dissociation of these two proteins could be a result of the GAP activity of Rna1p, either inside the NPC or on the cytoplasmic face of the NPC. There is evidence that Rna1p can interact with importin β (Koepp et al. 1996xKoepp, D.M, Wong, D.H, Corbett, A.H, and Silver, P.A. J. Cell Biol. 1996; 133: 1163–1176Crossref | PubMed | Scopus (108)See all ReferencesKoepp et al. 1996). The precise signal for an irreversible step of export is unclear, but it is possible that free importin β could dissociate importin α from RNA-binding proteins. Thus, the key players in nuclear protein import would be regenerated for another round of transport.Figure 3Model for Transport Factor CyclingNuclear transport factors cycle between the nucleus and the cytoplasm in order to promote multiple rounds of transport. See text for details.View Large Image | View Hi-Res Image | Download PowerPoint Slide

Mutants in a yeast Ran binding protein are defective in nuclear transport

Ran, a Ras‐like GTPase, has been implicated in controlling the movement of proteins and RNAs in and out of the nucleus. We have constructed strains of Saccharomyces cerevisiae which produce fusion proteins containing glutathione‐S‐transferase (GST) fused to Gsp1p, which encodes the essential yeast Ran homolog, and a mutant form of Gsp1p that mimics the GTP‐bound state. A major protein with the apparent size of 34 kDa co‐purifies with the GTP‐bound form of Gsp1p. This protein was identified as Yrb1p (Yeast Ran Binding Protein) and stimulates GTP hydrolysis by Gsp1p in the presence of Rna1p, the Gsp1 GTPase activating protein. Yrb1p is located in the cytoplasm with some concentration at the nuclear periphery. Temperature‐sensitive yrb1 mutants are defective in nuclear protein import and RNA export. A mutation in the highly conserved Ran binding region of Yrb1p reduces its ability to interact with Gsp1p. These data indicate that Yrb1p functions with Gsp1p and suggest that together they can control transport of macromolecules across the nuclear envelope.

Fbw7 Isoform Interaction Contributes to Cyclin E Proteolysis

The ubiquitin proteasome system plays important roles in regulating cell growth and proliferation. Many proteins that function in ubiquitin-mediated destruction have been linked to tumorigenesis. The putative tumor-suppressor protein Fbw7 (hAgo/hCdc4) is a specificity factor for the Skp1-Cul1-F-box protein ubiquitin ligase complex and targets a number of proto-oncogene products for ubiquitin-mediated destruction, including the cell cycle regulator cyclin E. In mammals, there are three splice variants of Fbw7 that use distinct first exons, resulting in proteins that have unique NH2 termini but are otherwise identical. Here, we show that the Fbw7 splice variants interact with each other through an NH2-terminal region common to all of the Fbw7 isoforms. Other F-box proteins have been shown to regulate substrate binding or turnover by forming homodimeric or heterodimeric complexes, which are dependent on a sequence motif called the D domain. Fbw7 and its orthologues exhibit significant sequence similarity to such F-box proteins, including the D domain. Fbw7 mutants that lack the region encompassing the D domain fail to bind other Fbw7 isoforms, despite being properly localized and binding both cyclin E and Skp1. Finally, we show the functional significance of this region as mutants lacking the NH2-terminal region involved in Fbw7 binding exhibit reduced rates of cyclin E protein turnover, indicating that Fbw7 isoform interaction is important for the efficiency of cyclin E turnover. Overall, this study contributes to the current understanding of the regulation of the Fbw7 tumor-suppressor protein. (Mol Cancer Res 2006;4(12):935–43)

The C. elegans L1CAM homologue LAD-2 functions as a coreceptor in MAB-20/Sema2–mediated axon guidance

The L1 cell adhesion molecule (L1CAM) participates in neuronal development. Mutations in the human L1 gene can cause the neurological disorder CRASH (corpus callosum hypoplasia, retardation, adducted thumbs, spastic paraplegia, and hydrocephalus). This study presents genetic data that shows that L1-like adhesion gene 2 (LAD-2), a Caenorhabditis elegans L1CAM, functions in axon pathfinding. In the SDQL neuron, LAD-2 mediates dorsal axon guidance via the secreted MAB-20/Sema2 and PLX-2 plexin receptor, the functions of which have largely been characterized in epidermal morphogenesis. We use targeted misexpression experiments to provide in vivo evidence that MAB-20/Sema2 acts as a repellent to SDQL. Coimmunoprecipitation assays reveal that MAB-20 weakly interacts with PLX-2; this interaction is increased in the presence of LAD-2, which can interact independently with MAB-20 and PLX-2. These results suggest that LAD-2 functions as a MAB-20 coreceptor to secure MAB-20 coupling to PLX-2. In vertebrates, L1 binds neuropilin1, the obligate receptor to the secreted Sema3A. However, invertebrates lack neuropilins. LAD-2 may thus function in the semaphorin complex by combining the roles of neuropilins and L1CAMs.

Yra1 Is Required for S Phase Entry and Affects Dia2 Binding to Replication Origins

ABSTRACT The Saccharomyces cerevisiae F-box protein Dia2 is important for DNA replication and genomic stability. Using an affinity approach, we identified Yra1, a transcription-coupled mRNA export protein, as a Dia2 interaction partner. We find that yra1 mutants are sensitive to DIA2 expression levels. Like Dia2, Yra1 associates with chromatin and binds replication origins, suggesting that they may function together in DNA replication. Consistent with this idea, Yra1 and Dia2 coimmunoprecipitate with Hys2, a subunit of DNA polymerase δ. The C terminus of Yra1 is required to interact with Dia2. A yra1 mutant that lacks this domain is temperature sensitive yet has no apparent defect in RNA export. Remarkably, this mutant also fails to enter S phase at the nonpermissive temperature. Significantly, other mutants in transcription-coupled export do not exhibit S phase entry defects or sensitivity to DIA2 expression levels. Together, these results indicate that Yra1 has a role in DNA replication distinct from its role in mRNA export. Furthermore, Dia2 binding to replication origins is significantly reduced when association with Yra1 is compromised, suggesting that one aspect of the role of Yra1 in DNA replication may involve recruiting Dia2 to chromatin.

Activation of the S-Phase Checkpoint Inhibits Degradation of the F-Box Protein Dia2

ABSTRACT A stable genome is critical to cell viability and proliferation. During DNA replication, the S-phase checkpoint pathway responds to replication stress. In budding yeast, the chromatin-bound F-box protein Dia2 is required to maintain genomic stability and may help replication complexes overcome sites of damaged DNA and natural fragile regions. SCF (Skp1/Cul1/F-box protein) complexes are modular ubiquitin ligases. We show here that Dia2 is itself targeted for ubiquitin-mediated proteolysis and that activation of the S-phase checkpoint pathway inhibits Dia2 protein degradation. S-phase checkpoint mutants fail to stabilize Dia2 in response to replication stress. Deletion of DIA2 from these checkpoint mutants exacerbates their sensitivity to hydroxyurea, suggesting that stabilization of Dia2 contributes to the replication stress response. Unlike the case for other F-box proteins, deletion of the F-box domain in Dia2 does not stabilize the protein. Rather, an N-terminal domain that is also required for nuclear localization is necessary for degradation. When a strong nuclear localization signal (NLS) is added to dia2 mutants lacking this domain, the Dia2 protein is both stable and nuclear. Together, our results suggest that Dia2 protein turnover does not involve an autocatalytic mechanism and that Dia2 proteolysis is inhibited by activation of the replication stress response.