Heike Laman

Modulation of p27Kip1 levels by the cyclin encoded by Kaposi's sarcoma-associated herpesvirus

DNA tumour viruses have evolved a number of mechanisms by which they deregulate normal cellular growth control. We have recently described the properties of a cyclin encoded by human herpesvirus 8 (also known as Kaposis sarcoma‐associated herpesvirus) which is able to resist the actions of p16Ink4a, p21Cip1 and p27Kip1 cdk inhibitors. Here we investigate the mechanism involved in the subversion of a G1 blockade imposed by overexpression of p27Kip1. We demonstrate that binding of K cyclin to cdk6 expands the substrate repertoire of this cdk to include a number of substrates phosphorylated by cyclin–cdk2 complexes but not cyclin D1–cdk6. Included amongst these substrates is p27Kip1 which is phosphorylated on Thr187. Expression of K cyclin in mammalian cells leads to p27Kip1 downregulation, this being consistent with previous studies indicating that phosphorylation of p27Kip1 on Thr187 triggers its downregulation. K cyclin expression is not able to prevent a G1 arrest imposed by p27Kip1 in which Thr187 is mutated to non‐phosphorylatable Ala. These results imply that K cyclin is able to bypass a p27Kip1‐imposed G1 arrest by facilitating phosphorylation and downregulation of p27Kip1 to enable activation of endogenous cyclin–cdk2 complexes. The extension of the substrate repertoire of cdk6 by K cyclin is likely to contribute to the deregulation of cellular growth by this herpesvirus‐encoded cyclin.

The Parkinson's disease–linked proteins Fbxo7 and Parkin interact to mediate mitophagy

Compelling evidence indicates that two autosomal recessive Parkinsons disease genes, PINK1 (PARK6) and Parkin (PARK2), cooperate to mediate the autophagic clearance of damaged mitochondria (mitophagy). Mutations in the F-box domain–containing protein Fbxo7 (encoded by PARK15) also cause early-onset autosomal recessive Parkinsons disease, by an unknown mechanism. Here we show that Fbxo7 participates in mitochondrial maintenance through direct interaction with PINK1 and Parkin and acts in Parkin-mediated mitophagy. Cells with reduced Fbxo7 expression showed deficiencies in translocation of Parkin to mitochondria, ubiquitination of mitofusin 1 and mitophagy. In Drosophila, ectopic overexpression of Fbxo7 rescued loss of Parkin, supporting a functional relationship between the two proteins. Parkinsons disease–causing mutations in Fbxo7 interfered with this process, emphasizing the importance of mitochondrial dysfunction in Parkinsons disease pathogenesis.

Viral cyclin-cyclin-dependent kinase 6 complexes initiate nuclear DNA replication.

ABSTRACT The cyclins encoded by Kaposi sarcoma-associated herpesvirus and herpesvirus saimiri are homologs of human D-type cyclins. However, when complexed to cdk6, they have several activities that distinguish them from D-type cyclin-cdk6 complexes, including resistance to cyclin-dependent kinase inhibitors and an enhanced substrate range. We find that viral cyclins interact with and phosphorylate proteins involved in replication initiation. Using mammalian in vitro replication systems, we show that viral cyclin-cdk6 complexes can directly trigger the initiation of DNA synthesis in isolated late-G1-phase nuclei. Viral cyclin-cdk6 complexes share this capacity with cyclin A-cdk2, demonstrating that in addition to functioning as G1-phase cyclin-cdk complexes, they function as S-phase cyclin-cdk complexes.

Small-molecule mimics of an alpha-helix for efficient transport of proteins into cells.

We designed and synthesized small-molecule mimics of an alpha-helical peptide protein transduction domain (PTD). These small-molecule carriers, which we termed SMoCs, are easily coupled to biomolecules, and efficiently deliver dye molecules and recombinant proteins into a variety of cell types. We designed the SMoCs using molecular modeling techniques. As an example of a protein cargo, we applied this new technology to the internalization of the DNA replication licensing repressor geminin, in vitro, providing evidence that extracellularly delivered SMoC-geminin can have an antiproliferative effect on human cancer cells. Uptake of SMoC-geminin was inhibited at 4 °C and by chlorpromazine, a compound that induces misassembly of clathrin-coated pits at the cell surface. Thus the mechanism of uptake is likely to be clathrin-mediated endocytosis.

Transforming activity of Fbxo7 is mediated specifically through regulation of cyclin D/cdk6

D cyclins (D1, D2 and D3) and their catalytic subunits (cyclin‐dependent kinases cdk4 and cdk6) have a facilitating, but nonessential, role in cell cycle entry. Tissue‐specific functions for D‐type cyclins and cdks have been reported; however, the biochemical properties of these kinases are indistinguishable. We report that an F box protein, Fbxo7, interacted with cellular and viral D cyclins and distinguished among the cdks that bind D‐type cyclins, specifically binding cdk6, in vitro and in vivo. Fbxo7 specifically regulated D cyclin/cdk6 complexes: Fbxo7 knockdown decreased cdk6 association with cyclin and its overexpression increased D cyclin/cdk6 activity and E2F activity. Fbxo7 interacted with p27, but its enhancement of cyclin D/cdk6 activity was p21/p27 independent. Fbxo7 overexpression transformed murine fibroblasts, rendering them tumorigenic in athymic nude mice. Transformed phenotypes were dependent on cdk6, as knockdown of cdk6 reversed them. Fbxo7 was highly expressed in epithelial tumors, but not in normal tissues, suggesting that it may have a proto‐oncogenic role in human cancers.

Disturbance of normal cell cycle progression enhances the establishment of transcriptional silencing in Saccharomyces cerevisiae.

Previous studies have indicated that mutation of RAP1 (rap1s) or of the HMR-E silencer ARS consensus element leads to metastable repression of HMR. A number of extragenic suppressor mutations (sds, suppressors of defective silencing) that increase the fraction of repressed cells in rap1s hmr delta A strains have been identified. Here we report the cloning of three SDS genes. SDS11 is identical to SWI6, a transcriptional regulator of genes required for DNA replication and of cyclin genes. SDS12 is identical to RNR1, which encodes a subunit of ribonucleotide reductase. SDS15 is identical to CIN8, whose product is required for spindle formation. We propose that mutations in these genes improve the establishment of silencing by interfering with normal cell cycle progression. In support of this idea, we show that exposure to hydroxyurea, which increases the length of S phase, also restores silencing in rap1s hmr delta A strains. Mutations in different cyclin genes (CLN3, CLB5, and CLB2) and two cell cycle transcriptional regulators (SWI4 and MBP1) also suppress the silencing defect at HMR. The effect of these cell cycle regulators is not specific to the rap1s or hmr delta A mutation, since swi6, swi4, and clb5 mutations also suppress mutations in SIR1, another gene implicated in the establishment of silencing. Several mutations also improve the efficiency of telomeric silencing in wild-type strains, further demonstrating that disturbance of the cell cycle has a general effect on position effect repression in Saccharomyces cerevisiae. We suggest several possible models to explain this phenomenon.

Crystal structure of a γ‐herpesvirus cyclin–cdk complex

Several γ‐herpesviruses encode proteins related to the mammalian cyclins, regulatory subunits of cyclin‐dependent kinases (cdks) essential for cell cycle progression. We report a 2.5 Å crystal structure of a full‐length oncogenic viral cyclin from γ‐herpesvirus 68 complexed with cdk2. The viral cyclin binds cdk2 with an orientation different from cyclin A and makes several novel interactions at the interface, yet it activates cdk2 by triggering conformational changes similar to cyclin A. Sequences within the viral cyclin N‐terminus lock part of the cdk2 T‐loop within the core of the complex. These sequences and others are conserved amongst the viral and cellular D‐type cyclins, suggesting that this structure has wider implications for other cyclin–cdk complexes. The observed resistance of this viral cyclin–cdk complex to inhibition by the p27Kip cdk inhibitor is explained by sequence and conformational variation in the cyclin rendering the p27Kip‐binding site on the cyclin subunit non‐functional.

Structure of a conserved dimerization domain within the F-box protein Fbxo7 and the PI31 proteasome inhibitor.

F-box proteins are the substrate-recognition components of the Skp1-Cul1-F box protein (SCF) E3 ubiquitin ligases. Here we report a structural relationship between Fbxo7, a component of the SCFFbxo7 E3 ligase, and the proteasome inhibitor PI31. SCFFbxo7 is known to catalyze the ubiquitination of hepatoma-up-regulated protein (HURP) and the inhibitor of apoptosis (IAP) protein but also functions as an activator of cyclin D-Cdk6 complexes. We identify PI31 as an Fbxo7·Skp1 binding partner and show that this interaction requires an N-terminal domain present in both proteins that we term the FP (Fbxo7/PI31) domain. The crystal structure of the PI31 FP domain reveals a novel α/β-fold. Biophysical and mutational analyses are used to map regions of the PI31 FP domain mediating homodimerization and required for heterodimerization with Fbxo7·Skp1. Equivalent mutations in Fbxo7 ablate interaction with PI31 and also block Fbxo7 homodimerization. Knockdown of Fbxo7 does not affect PI31 levels arguing against PI31 being a substrate for SCFFbxo7. We present a model for FP domain-mediated dimerization of SCFFbxo7 and PI31.

Beyond ubiquitination: the atypical functions of Fbxo7 and other F-box proteins.

F-box proteins (FBPs) are substrate-recruiting subunits of Skp1-cullin1-FBP (SCF)-type E3 ubiquitin ligases. To date, 69 FBPs have been identified in humans, but ubiquitinated substrates have only been identified for a few, with the majority of FBPs remaining ‘orphans’. In recent years, a growing body of work has identified non-canonical, SCF-independent roles for about 12% of the human FBPs. These atypical FBPs affect processes as diverse as transcription, cell cycle regulation, mitochondrial dynamics and intracellular trafficking. Here, we provide a general review of FBPs, with a particular emphasis on these expanded functions. We review Fbxo7 as an exemplar of this special group as it has well-defined roles in both SCF and non-SCF complexes. We review its function as a cell cycle regulator, via its ability to stabilize p27 protein and Cdk6 complexes, and as a proteasome regulator, owing to its high affinity binding to PI31. We also highlight recent advances in our understanding of Fbxo7 function in Parkinsons disease, where it functions in the regulation of mitophagy with PINK1 and Parkin. We postulate that a few extraordinary FBPs act as platforms that seamlessly segue their canonical and non-canonical functions to integrate different cellular pathways and link their regulation.

Identification of a nitrogen-regulated promoter controlling expression of Klebsiella pneumoniae urease genes.

Synthesis of urease by Klebsiella species is known to be induced when the nitrogen source of the growth medium is limiting, suggesting that urease gene expression is controlled by the nitrogen regulatory (ntr) system. This study showed that K. pneumoniae with mutations in either ntrA or ntrC, two integral components of the ntr system, were phenotypically urease‐negative. These mutants could be complemented back to a urease positive phenotype with recombinant plasmids encoding the corresponding ntr gene. A series of ure–lacZYA transcriptional fusions, in conjunction with primer extension analysis, identified a DNA region that encoded a nitrogen‐regulated promoter. This promoter region controlled transcription of ureD, the first gene in the Klebsiella pneumoniae urease gene cluster, and ureA, a gene that resides immediately downstream of ureD. A high level of transcription from the ureD promoter required NAC, a recently characterized member of the nitrogen regulatory cascade. NAC is a Lys R‐like transcriptional regulator that can act at σ;70 promoters; expression from nac itself is dependent upon NTRA. Therefore, expression of K. pneumoniae urease was dependent upon the nitrogen regulatory cascade, and transcription of at least two urease genes was from a promoter that was positively regulated by NAC.